EA045896B1 - METHODS FOR TREATING MULTIPLE MYELOMA AND THE USE OF RELATED BIOMARKERS 4-(4-(4-(((2-(2,6-DIOXOPIPERIDIN-3-YL)-1-OXOISOINDOLIN-4-YL)OXY)METHYL)BENZYL)PIPERAZINE-1 -IL)-3-FLUOROBENZONITRILE - Google Patents

Description

This application claims priority to U.S. Provisional Patent Application No. 62/675,732, filed May 23, 2018, and U.S. Provisional Patent Application No. 62/737,772, filed September 27, 2018, both of which are incorporated herein by reference in their entirety. .

1. Technical field

In some embodiments, provided herein are methods of using certain biomarkers, such as CRBN, Aiolos (IKZF3), Ikaros (IKZF1), zinc finger protein 91 (ZFP91), c-Myc, interferon regulatory factor 4 (IRF4), immune markers tumor (soluble CD25, cytokines; tumor infiltrating lymphocytes (TIL); T cell activation, T cell receptor clonality), circulating tumor cells (CTC), soluble BCMA (sBCMA), apoptotic markers (cleaved caspase-1, cleaved caspase-1 7, cleaved caspase-3, cleaved PARP, survivin, BCL-2-like protein 11 (BIM), TUNEL, free light chain (FLC)) and cell cycle markers (p21, p27, pRb1) in predicting and monitoring clinical sensitivity and therapeutic response to 4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1oxoisoindolin-4-yl)oxy)methyl)benzyl)piperazin-1-yl)-3-fluorobenzonitrile or an enantiomer, mixture of enantiomers, tautomer or pharmaceutically acceptable salt thereof, as well as methods of treating, preventing or controlling multiple myeloma using them. Also described herein is 4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)benzyl)piperazin-1 -yl) 3-fluorobenzonitrile or an enantiomer, mixture of enantiomers, tautomer or pharmaceutically acceptable salt thereof for use in methods of treating, preventing or controlling multiple myeloma. In certain embodiments of the invention, methods are also provided herein for identifying a patient who is likely to be susceptible, predicting the patient's susceptibility, determining dosage, or determining the effectiveness of a compound in treating diseases.

2. State of the art

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow. Typically, plasma cells produce antibodies and play a key role in the _functioning of the immune system. However, uncontrolled growth of these cells leads to bone fractures and bone pain, anemia, infection and other complications. Multiple myeloma is the second most common hematologic malignancy, although the exact causes of multiple myeloma remain unknown. Multiple myeloma causes high levels of proteins in the blood, urine, and organs, including, but not limited to, M protein and other immunoglobulins (antibodies), albumin, and beta-2 microglobulin, except in some patients (estimated at 1 to 5 %) whose myeloma cells do not secrete these proteins (so-called non-secretory myeloma). M protein, short for monoclonal protein, also known as paraprotein, is a particularly abnormal protein produced by myeloma plasma cells and can be found in the blood or urine of almost all patients with multiple myeloma, except patients who have nonsecretory myeloma, or whose myeloma cells produce heavy chain immunoglobulin light chains.

Skeletal symptoms, including bone pain, are among the most clinically significant symptoms of multiple myeloma. Malignant plasma cells release osteoclast-stimulating factors (including IL-1, IL-6, and TNF), which cause calcium to be leached from bone, thereby causing lytic lesions; Another symptom is hypercalcemia. Osteoclast-stimulating factors, also called cytokines, can prevent apoptosis or death of myeloma cells. Fifty percent of patients have radiographically detectable skeletal lesions associated with myeloma at the time of diagnosis. Other common clinical symptoms of multiple myeloma include polyneuropathy, anemia, hyperviscosity, infections and renal failure.

Current treatment for multiple myeloma may involve one or more of surgery, stem cell transplant, chemotherapy, immunotherapy, and/or radiation therapy to destroy multiple myeloma cells in a patient. All modern therapeutic approaches have significant disadvantages for the patient.

In the last decade, new therapeutic agents, particularly immunomodulatory drugs such as lenalidomide and pomalidomide, have significantly improved response rates, prolonged progression-free survival (PFS), and overall survival (OS) in patients with multiple myeloma. However, persistent levels of residual disease that are below the sensitivity of bone marrow (BM) morphology, protein immunofixation electrophoresis, and light chain quantitation exist in many patients with multiple myeloma, even after these patients have achieved complete remission (CR). , and can ultimately cause relapse of the disease.

There is a significant need for safe and effective compounds and methods for treating, preventing and controlling multiple myeloma, including for patients whose multiple myeloma is newly diagnosed or refractory to standard treatments, while reducing or avoiding toxicity and/or side effects. associated with traditional therapy.

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3. Essence of the invention

In one aspect, this document provides a method for identifying a subject having a cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

- 2 045896 (b) introducing a therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein are methods for identifying a subject having a cancer that is likely susceptible to a therapeutic compound wherein the level of a biomarker in a sample is higher than a control level of the biomarker. In other embodiments, provided herein are methods for identifying a subject having a cancer that is likely to be susceptible to a therapeutic compound wherein the level of a biomarker in the sample is lower than a control level of the biomarker.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of biomarker in the sample;

(c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering a therapeutically effective amount of a therapeutic compound to a subject who has been diagnosed as likely to be susceptible to the therapeutic compound;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of biomarker in the sample;

(c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering a therapeutically effective amount of a therapeutic compound to a subject who has been diagnosed as likely to be susceptible to the therapeutic compound;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof. In another version

- 3 045896 Presented herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of biomarker in the sample;

(c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering a therapeutically effective amount of a therapeutic compound to a subject who has been diagnosed as likely to be susceptible to the therapeutic compound;

wherein the therapeutic compound is Compound 3 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein are methods of treating cancer wherein the level of a biomarker in a sample is higher than a control level of the biomarker. In other embodiments, provided herein are methods of treating cancer wherein the level of a biomarker in a sample is lower than a control level of the biomarker. Also provided herein is Compound 1, Compound 2 and/or Compound 3 for use in a method of treating cancer as described above.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 1 or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 3

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or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein are methods for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound where the level of a biomarker in a sample is higher than a control level of the biomarker. In other embodiments, provided herein are methods for predicting the susceptibility of a subject having or suspected of having cancer that is likely to be susceptible to a therapeutic compound where the level of a biomarker in a sample is lower than a control level of the biomarker.

In another aspect, provided herein is a method for monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample; and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control

- 5,045,896 sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein are methods for monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, wherein an increased level of a biomarker compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject. In some embodiments, provided herein are methods of monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, wherein a reduced level of a biomarker compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject.

In some embodiments, methods of identifying a subject having cancer that is likely to be susceptible to a therapeutic compound further comprises administering a therapeutically effective amount of the therapeutic compound to the subject who is diagnosed as likely to be susceptible to the therapeutic compound. In further embodiments, methods of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound further comprises administering a therapeutically effective amount of the therapeutic compound to the subject who is diagnosed as likely to be susceptible to the therapeutic compound.

In some embodiments of any of the methods provided herein, a control sample is obtained from the subject prior to administration of the therapeutic compound to the subject, and wherein the control sample is obtained from the same source as the sample.

In some embodiments of any of the methods provided herein, the control sample is obtained from a healthy subject who does not have cancer, and wherein the control sample is obtained from the same source as the sample.

In some embodiments of any of the methods provided herein, the control sample is obtained from a subject receiving an anti-cancer compound that is not the specified therapeutic compound, and wherein the control sample is obtained from the same source.

- 6 045896 as the sample. In specific embodiments, the control sample is obtained from a subject receiving an anti-cancer compound that is not a therapeutic compound, wherein the control sample is obtained from the same source as the sample, and the anti-cancer compound is selected from the group consisting of lenalidomide, pomalidomide, or derivatives thereof.

In some embodiments of the methods provided herein, the cancer is multiple myeloma (MM). In some specific embodiments, the MM is relapsed, refractory, or resistant to conventional therapy. In one embodiment, the MM is lenalidomide-resistant MM. In another embodiment, the MM is pomalidomide-resistant MM. In some embodiments, the MM is newly diagnosed MM. In some embodiments, the MM is a transplantable MM. In some embodiments, the MM is non-transplantable MM.

In certain embodiments of the methods provided herein, the biomarker is cereblon (CRBN). In some embodiments of the methods provided herein, the biomarker is a protein associated with CRBN.

In some embodiments of the methods provided herein, the biomarker is involved in the apoptotic cascade of reactions. In yet another embodiment of the methods provided herein, the biomarker participates in a cascade of cell cycle reactions. In some embodiments of the methods provided herein, the biomarker is involved in T cell activation. In some embodiments of the methods provided herein, the biomarker is circulating tumor cells (CTCs).

In some embodiments of the methods provided herein, the biomarker is a tumor infiltrating lymphocyte (TIL).

In some specific embodiments, the biomarker is a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC, and IRF4. In some specific embodiments, the biomarker is IKZF1. In another embodiment, the biomarker is IKZF3. In yet another embodiment, the biomarker is ZFP91. In yet another embodiment, the biomarker is c-MYC. In some embodiments, the biomarker is IRF4.

In other embodiments, the biomarker is involved in apoptosis and is selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7) , cleaved PARP, survivin, BCL-2-like protein 11 (BIM) and free light chain in serum. In some embodiments, the biomarker is cleaved caspase-3 (p-caspase-3). In some embodiments, the biomarker is cleaved caspase-1 (p-caspase-1). In yet other embodiments, the biomarker is cleaved caspase-7 (p-caspase-7). In another embodiment, the biomarker is cleaved PARP. In yet another embodiment, the biomarker is survivin. In some embodiments, the biomarker is survivin. In other embodiments, the biomarker is BCL-2-like protein 11 (BIM). In some embodiments, the biomarker is a serum free light chain (sFLC).

In yet other embodiments, the biomarker is involved in apoptosis and is measured by terminal deoxynucleotidyl transferase dUTP single-strand end labeling (TUNEL). In some embodiments, the biomarker is involved in apoptosis and is measured by Annexin-V and 7-AAD. In still other embodiments, the biomarker is involved in apoptosis and is measured using Annexin-V and propidium iodide (PI)

In other embodiments of the methods provided herein, the biomarker is involved in the cell cycle and is selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27), and retinoblastoma protein (pRb1). In some embodiments, the biomarker is p21. In some embodiments, the biomarker is p27. In yet another embodiment, the biomarker is pRb1.

In other embodiments of the methods provided herein, the biomarker is involved in T cell activation and is selected from the group consisting of interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy), and T clonality -cell receptor (TCR). In other specific embodiments, the biomarker is IL-2. In other embodiments, the biomarker is TNFa. In some embodiments, the biomarker is IFNy. In some embodiments, the biomarker is T cell receptor (TCR) clonality. In a specific embodiment, the biomarker is T cell receptor (TCR) clonality, and the biomarker is measured by sequencing the TCR DNA. In some specific embodiments, the biomarker is involved in T cell activation, and the level of the biomarker is measured histologically.

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In some embodiments of the methods provided herein, the level of the biomarker is higher than the control level.

In other embodiments of the methods provided herein, the level of the biomarker is below the control level.

In some embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In other embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the responsiveness of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of biomarker in the sample; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control level of the biomarker;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable

- 8 045896 salt.

In another embodiment, provided herein is a method for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof. In yet another embodiment, provided herein is a method for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level;

in which the therapeutic compound is Compound 3 o

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In specific embodiments of methods for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, the biomarker is CRBN. In some embodiments of methods for predicting the susceptibility of a subject having multiple myeloma to a treatment compound, the methods include diagnosing the subject as likely to be responsive to the treatment compound if a level of the biomarker is detected in the sample and is lower than in the control sample. In some embodiments of methods for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, the methods include diagnosing the subject as likely to be responsive to the therapeutic compound if a biomarker is detected in the sample. In some specific embodiments, the MM is relapsed, refractory, or resistant to conventional therapy. In one embodiment, the MM is lenalidomide-resistant MM. In another embodiment, the MM is pomalidomide-resistant MM.

In some embodiments of any of the methods provided herein, the level of the biomarker is measured by determining the level of the biomarker protein. In other embodiments of any of the methods provided herein, the level of a biomarker is measured by determining the level of mRNA of the biomarker. In still further embodiments of any of the methods provided herein, the level of the biomarker is measured by determining the level of the biomarker cDNA. In some embodiments of any of the methods provided herein, the biomarker is determined by DNA sequencing. In other embodiments of any of the methods provided herein, the biomarker is determined by RNA sequencing (RNA-seq).

In some embodiments, the level of the biomarker is measured by determining the level of the biomarker protein, comprising contacting proteins in the sample with a first antibody that immunospecifically binds to the biomarker protein. In other embodiments, the level of the biomarker is measured by determining the level of the biomarker protein, comprising contacting proteins in the sample with a first antibody that immunospecifically binds to the biomarker protein, further comprising:

(a) contacting a biomarker protein bound to the first antibody with a second antibody having a detectable label, wherein the second antibody immunospecifically binds to the biomarker protein, and wherein the second antibody immunospecifically binds to a different epitope on the biomarker protein than the first antibody;

(b) detecting the presence of a second antibody associated with the biomarker protein; and (c) determining the amount of biomarker protein based on the amount of detectable label on the second antibody.

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In yet another embodiment, the level of the biomarker is measured by determining the level of the biomarker protein, comprising contacting proteins in the sample with a first antibody that immunospecifically binds to the biomarker protein, further comprising:

(a) contacting a first antibody bound to the biomarker protein with a second antibody having a detectable label, wherein the second antibody immunospecifically binds to the first antibody;

(b) detecting the presence of a second antibody bound to the first antibody; and (c) determining the amount of biomarker protein based on the amount of detectable label on the second antibody.

In certain embodiments, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from a subject at different points in time; and (c) determining the level of a biomarker in one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from the subject at different time points and (c) determining the level of the biomarker in the one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from the subject at different time points and (c) determining the level of the biomarker in the one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments of methods for determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, the biomarker is selected from the group consisting of IKZF1 and IKZF3. In some specific embodiments of methods for determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, the biomarker is IKZF1. In yet another specific embodiment of methods for determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, the biomarker is IKZF3.

In some embodiments, methods of treating cancer further include administering a therapeutically effective amount of a second active agent or maintenance therapy. In certain embodiments, methods of identifying a subject having cancer that is likely to be susceptible to a therapeutic compound further comprises administering the therapeutic

- 10,045,896 a therapeutically effective amount of a therapeutic compound to a subject who is diagnosed as likely to be susceptible to the therapeutic compound, and further comprises administering a therapeutically effective amount of a second active agent or maintenance therapy. In further embodiments, methods for predicting the responsiveness of a subject having or suspected of having cancer to a therapeutic compound further comprises administering a therapeutically effective amount of the therapeutic compound to the subject who has been diagnosed as likely to be susceptible to the therapeutic compound, and further comprising administering a therapeutically effective amount of a second active compound. agent or maintenance therapy. In specific embodiments, the second active agent is selected from the group consisting of high molecule compounds, small molecule compounds, or cell therapies, and the second active agent is optionally selected from the group consisting of melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin, bendamustine, proteasome inhibitor, histone deacetylase inhibitor, BET, BCL2 inhibitor, MCL-1 inhibitor, corticosteroid, dexamethasone, antibody, checkpoint inhibitor and CAR cells.

In some embodiments provided herein, the compounds provided herein can be used in any method of treating, preventing, and/or monitoring any of the diseases described herein.

4. Brief description of graphic materials

In fig. 1 shows that Compound 2 and Compound 3 degrade Aiolos protein in a time and concentration dependent manner. DF15 cells expressing Enhanced ProLabel (ePL) Aiolos were incubated with Compound 2 (triangle) or Compound 3 (square) for 45, 90 min, or 3 h at the indicated concentrations. Cell extracts were then prepared and Aiolos-ePL protein was quantified using the ePL luminescence assay.

In fig. Figure 2 shows that Compound 2 disrupts Aiolos and Ikaros even in pomalidomide-resistant cells and acts synergistically with dexamethasone to disrupt IRF4 and c-MYC. Pomalidomide-sensitive (OPM2) or resistant (OPM2-P1) cells were treated with placebo control (DMSO), pomalidomide, or Compound 2 either alone or in combination with 10 or 100 nM dexamethasone for 72 h. CRL4 ^crbn E3 ubiquitin ligase substrates were measured Aiolos and Ikaros and their downstream effectors c-Myc and IRF4. Tubulin is a load control.

In fig. Figure 3 shows that Compound 2 degrades Aiolos and Ikaros even in pomalidomide-resistant cells and acts synergistically with dexamethasone to induce apoptosis. Pomalidomide-sensitive (OPM2) or resistant (OPM2-P1) cells were treated with placebo control (DMSO), pomalidomide, or Compound 2 either alone or in combination with 10 or 100 nM dexamethasone for 72 h. CRL4 ^crbn E3 ubiquitin ligase substrates were measured Aiolos and Ikaros, as well as induction of the apoptosis cascade proteins BIM, cleaved PARP, cleaved caspase-3 and cleaved caspase-7. Tubulin is a load control.

In fig. Figure 4 shows that Compound 2 induced the destruction of Ikaros, Aiolos, ZFP91, c-Myc and IRF4, which correlates with the induction of the apoptosis protein cleaved caspase 3 and the cell cycle arrest protein p21. Immunoblot analysis of parental OPM2 cells incubated with Compound 2 at the indicated concentrations is shown. Actin controls load.

In fig. 5A shows that CRBN is required for Compound 2-mediated degradation of Ikaros and Aiolos. Shown are Cereblon, Ikaros, and Aiolos immunoblots of human CRBN ^wt DF15R and DF15R cell extracts that were incubated with DMSO, pomalidomide, or Compound 2 at 0.1 μM for 4 h. Tubulin is a loading control.

In fig. 5B shows that Ikaros is required for Compound 2-mediated suppression of c-Myc and IRF4, as well as the induction of apoptosis. Immunoblots of extracts from wild-type OPM2 cells or OPM2 cells overexpressing stabilized Ikaros, Aiolos, or ZFP91 mutants are shown. Cells were incubated with DMSO or the indicated concentration of Compound 2 for five days.

In fig. 6A and 6B show that Compound 2 degrades Ikaros and Aiolos in a concentration-dependent manner, which induces apoptosis and cell cycle arrest in pomalidomide-resistant multiple myeloma cells. Immunoblot analysis of pomalidomide-resistant MM cells (H9291051) incubated with Compound 2 for (Fig. 6A) 4 h or (Fig. 6B) 72 h is shown.

In fig. 7A and FIG. 7B shows that Compound 2 causes long-term destruction of Aiolos and Ikaros. Destruction of Aiolos and Ikaros is shown as a percentage of dimethyl sulfoxide (DMSO) control in cell models (Fig. 7A) H929-1051 and (Fig. 7B) OPM2-P10 after a single continuous exposure to Compound 2. These data are from a single experiment with a single sample. to the experimental point.

In fig. 8 shows that Compound 2 induced long-term degradation of Aiolos and Ikaros after 15 min of exposure. Compound 2 was incubated for 15 min with H929-1051 cells and degradation of Aiolos (left graph) and Ikaros (right graph) was measured. Data are from one experiment with one sample per experimental point. The y-axis represents the amount of Aiolos or Ikaros expressed as a percentage of the corresponding protein level in the dimethyl sulfoxide (DMSO) control; The x-axis shows the time in hours.

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In fig. 9 shows that recovery of Aiolos levels after washout after 6 hours of exposure to Compound 2 required almost 160 hours. Compound 2 was incubated for 6 hours with H929-1051 cells and Aiolos degradation was measured. These data are from one experiment with one sample per experimental point. The vertical dotted blue line segments the data during Compound 2 treatment (to the left of the vertical line) and after washout (to the right of the vertical line). Results are shown for Aiolos levels in OPM2-P10 cells (left) and H9291051 cells (right) for a total of 160 h, including a subsequent 6-h incubation with 0.01 (red) or 0.1 μM Compound 2 (blue).

In fig. 10 shows that chronic, rather than transient, exposure to Compound 2 induced apoptosis in multiple myeloma cells. The induction of apoptosis upon treatment with Compound 2 is shown in a model of lenalidomide-resistant MM cells (H929-1051 [top row] and a model of pomalidomide-resistant MM cells ORM2-P10 [bottom row]). These data are from one experiment with one sample per experimental point.

In fig. 11A and 11B show that Compound 2, but not pomalidomide, exhibits antiproliferative activity in multiple myeloma cell lines resistant to lenalidomide and pomalidomide, including those that have low levels of CRBN protein. Antiproliferative effects have been demonstrated on acquired-resistant human multiple myeloma cell lines with low CRBN. Cell lines were treated (Fig. 11A) with pomalidomide or (Fig. 11B) Compound 2 for 5 days, and IC ₅₀ values were assessed using an ATP assay (CellTiter-Glo). The percentage of control was calculated by subtracting the background and normalizing to the DMSO control (100% of control).

In fig. 12 shows that dexamethasone (Dex) + Compound 2 induces apoptosis. Induction of apoptosis after 72 hours of treatment with dexamethasone alone or in combination with Compound 2, lenalidomide or pomalidomide in a lenalidomide-resistant multiple myeloma cell line (H929-1051) was measured using caspase-Glo 3/7.

In fig. 13 shows that short daily exposures to Compound 2 of up to three days cause Ikaros degradation in CD34+ cells. Degradation of Ikaros after short daily exposure to Compound 2 was measured by flow cytometry. CD34+ cells obtained from the bone marrow of a healthy donor were exposed to Compound 2 for 2, 4 and 6 hours starting on day 14 for 1 (day 14), 2 (day 15) or 3 (day 16) consecutive days . The percentage of Ikaros content (normalized to the DMSO control) is presented. At the end of the exposure period, Compound 2 was removed and the cells were incubated in the absence of Compound 2 (recovery period). Ikaros was measured by flow cytometry during recovery on days 19, 21, and 23. Data are presented as the average of results from two donors.

In fig. Figure 14 shows the breakdown and recovery of Ikaros after three consecutive days of 6-hour exposure to Compound 2. CD34+ cells derived from bone marrow from healthy donors were exposed to Compound 2 on each of three consecutive days, starting on day 10, for 6 hours. Shown percentage inhibition of Ikaros compared to DMSO. Cultures were incubated in the absence of Compound 2 from day 13 to day 22. Ikaros was measured by flow cytometry after completion of each exposure and every other day during recovery.

In fig. 15 shows the breakdown and recovery of Ikaros after five consecutive days of 6 hour exposure to Compound 2. Bone marrow-derived CD34+ cells were exposed to Compound 2 on each of five consecutive days starting on day 10. The percentage inhibition of Ikaros compared to the DMSO control is shown. Cultures were incubated in the absence of Compound 2 from 15 to 22 days. Ikaros was measured by flow cytometry after completion of each exposure (days 10 to 14) and every other day during recovery (days 17, 20, and 22).

In fig. 16A and 16B show the treatment pattern and correlation between Ikaros inhibition (dashed line) and neutrophil differentiation (solid line) following exposure to Compound 2 (FIG. 16A). Bone marrow-derived CD34+ cells from healthy donors were cultured ex vivo and stimulated to become mature neutrophils at stage IV (Fig. 16B) Cultures were exposed to various concentrations of Compound 2 for 6 hours on each of three days (days 10, 11 and 12) and then cultured without further exposure to Compound 2 until day 22. The percentage inhibition of Ikaros compared to DMSO control (circle) after exposure to 1 nM (square), 10 nM (triangle), and 100 nM (inverted triangle) of Compound 2 is shown on the right Y-axis (dashed lines). The percentage of Stage IV cells is shown on the left Y-axis (solid lines). The shaded area shows 3 days of exposure to Compound 2.

In fig. 17A and 17B show that Compound 2 directly activated human peripheral blood mononuclear cells (PBMCs) to lyse K562 erythromyelocytic leukemia cells in a concentration-dependent manner. (Figure 17A) Representative sorting plots of fluorescently activated K562 cells co-cultured with human PBMCs that were pre-incubated with Compound 2 or DMSO. (Figure 17B) Apoptotic response in K562 cells after co-culture with PBMCs treated with Compound 2. Apoptosis was measured by PI and Annexin V staining in K562 cells after 24 hours of co-culture with PBMCs that would

- 12 045896 were treated with Compound 2 for 72 hours. Data are presented as the mean with error bars representing standard errors of the mean.

In fig. 18A and 18B show that immune cells are directly activated by Compound 2 to kill lenalidomide-sensitive and lenalidomide-resistant multiple myeloma cell lines. (Fig. 18A) Apoptotic response in NCI-H929 cells after 24 hours co-culture with PBMCs pretreated with Compound 2. (Fig. 18B) Apoptosis response in H929-1051 cells after 24 hours co-culture with PBMCs pretreated with Compound 2.

In fig. 19A-19D show that compound-primed immune cells exhibit enhanced tumor cell killing when multiple myeloma cells are pretreated with lenalidomide, pomalidomide, or Compound 2 prior to coculture. Compound-primed immune cells exhibit increased tumor cell killing in a co-culture model (right) in (FIG. 19A) H929, (FIG. 19B) H929-1051, (FIG. 19C) OPM2, and (FIG. 19D) OPM2 P10 cells. cells compared to single MM cultures (left) and little effect on PBMC viability (middle).

In fig. 20 shows that Compound 2 is more effective at inducing interleukin-2 production from PBMCs stimulated with anti-CD3 beads than lenalidomide or pomalidomide at 72 hours. Induction of interleukin-2 (IL-2) production by PBMCs stimulated with anti-CD3 beads. CD3 antibodies, after treatment with Compound 2, lenalidomide (Len) or pomalidomide (Pom) for 72 hours.

In fig. 21A-21C show that Compound 2 is a potent inducer of effector cytokines. Measurement of induction of effector cytokines upon treatment with Compound 2, pomalidomide (Pom) or lenalidomide (Len) for 24 hours (Fig. 21A) IL-2, (Fig. 21B) IFN-γ and (Fig. 21C) TNF-α. Data presented are averages from three or four donors and are presented as fold change compared to the DMSO control; Error bars represent standard error of the mean (SEM).

In fig. 22A-22C show that Compound 2 induces Ikaros degradation in CD4+ T cells. Measurement of Ikaros degradation in CD4+ T cells following treatment with Compound 2 (diamond), lenalidomide (Len; circle) or pomalidomide (Pom; square) for (FIG. 22A) 24 hours, (FIG. 22B) 48 hours, or (FIG. 22C) 72 hours Data presented are averages from two donors and are presented as MFI normalized to the DMSO control.

In fig. 23A-23C show that dexamethasone (Dex) in combination with Compound 2 reduces the production of interleukin-2 (IL-2) by PBMC stimulated with anti-CD3 antibodies. Interleukin-2 production by PBMCs stimulated with anti-CD3 antibodies following (Fig. 23A) lenalidomide (Len) alone or with dexamethasone, (Fig. 23B) pomalidomide (Pom) alone or with dexamethasone, or (Fig. 23C) Compound 2 alone or with dexamethasone.

In fig. 24A and 24B show that Compound 2 induces destruction of Aiolos in CD3+ T cells from the peripheral blood of patients with relapsed/refractory multiple myeloma following (FIG. 24A) a once-daily regimen 1-10, 15-24 /28 (Fig. 24B) or twice a day schemes (2 times a day) 1-3, 15-17/28.

In fig. 25A and 25B show that Compound 2 induces Ikaros degradation in CD3+ T cells from the peripheral blood of patients with relapsed/refractory multiple myeloma following (FIG. 25A) a once-daily regimen 1-10, 15-24 /28 (Fig. 25B) or twice a day schemes (2 times a day) 1-3, 15-17/28.

In fig. 26A-26E show the effect of Compound 2 on biomarker expression in CD138+ plasma cells from patients with relapsed/refractory multiple myeloma. (FIG. 26A) Aiolos, (FIG. 26B) Ikaros, (FIG. 26C) ZFP91, (FIG. 26D) c-Myc, and (FIG. 26E) IRF4.

In fig. 27 shows the effect of Compound 2 on soluble BCMA (sBCMA) expression in patients with relapsed/refractory multiple myeloma.

In fig. 28 shows the effect of Compound 2 on serum free light chain (sFLC) in patients with relapsed/refractory multiple myeloma.

5. Detailed description of the invention

The methods provided herein are based in part on the discovery that altered levels, e.g., increased levels and/or decreased levels of certain molecules (e.g., mRNA, cDNA, or proteins) or malignant cells (e.g., circulating tumor cells (CTCs) ) in a biological sample can be used to predict the susceptibility of a subject having or suspected of having MM to a therapeutic compound (e.g., Compound 1, Compound 2, Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof) .

5.1 Definitions

As used herein, the terms containing and including may be used interchangeably. The terms containing and including should be interpreted to indicate the presence of

- 13 045896 new features or components, as specified, but not excluding the presence or addition of one or more features or components, or groups thereof. Additionally, the terms containing and including are intended to include examples covered by the term consisting of. Therefore, the term consisting of may be used in place of the terms containing and including to provide more specific embodiments of the invention.

The term consisting of means that an item has at least 90, 95, 97, 98 or 99% of the stated features or components of which it is composed. In another embodiment of the invention, the term consisting of excludes from the scope of any subsequent discussion any other features or components other than those that are not essential to achieving the technical effect.

As used herein, the term or should be interpreted to include or to mean any one or any combination. Therefore, A, B or C means any of the following: A; IN; WITH; A and B; A and C; B and C; A, B and C. An exception to this definition will only occur if the combination of elements, functions, steps or activities is in some way mutually exclusive.

In general, the technical description of one embodiment may be combined with that disclosed in any other embodiments of the invention presented herein.

As used herein, the term cancer includes, but is not limited to, solid cancer and hematologic cancer. The terms cancer and cancerous refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer can be cancer of hematopoietic and lymphoid tissue. Hematopoietic malignancy is a cancer that affects the blood, bone marrow, lymph, and lymphatic system.

As used herein, the term multiple myeloma refers to hematological conditions characterized by malignant plasma cells and includes the following disorders: monoclonal gammopathy of unknown origin (MGUS); low-risk, intermediate-risk and high-risk multiple myeloma; newly diagnosed multiple myeloma (including newly diagnosed low-risk, intermediate-risk and high-risk multiple myeloma); multiple myeloma with and without the possibility of transplantation; smoldering (slowly progressive) multiple myeloma (including low-risk, intermediate-risk and high-risk smoldering multiple myeloma); active multiple myeloma; solitary plasmacytoma; extramedullary plasmacytoma; plasma cell leukemia; multiple myeloma of the central nervous system; light chain myeloma; nonsecretory myeloma; immunoglobulin D myeloma; and immunoglobulin E myeloma; and multiple myeloma characterized by genetic abnormalities such as cyclin D translocation (eg, t(411;14)(q13;q32); t(6;14)(p21;32); t(12;14)(p13;q32) ) or t(6,20)); MMSET translocations (e.g. t(4;14)(p16;q32)); MAF translocations (e.g. t(14;16)(q32;q32); t(20;22); t(16; 22)(q11;q13); or t(14;20)(q32;q11)); or other chromosomal factors (eg, deletion 17p13, or chromosome 13; del(17/17p), non-hyperploidy and gain(1q)).

As used herein and unless otherwise noted, the terms treat, treat, and treatment refer to the alleviation or reduction in severity of a symptom associated with the disease or condition being treated, such as multiple myeloma.

The term prevention applies to reducing the symptoms of a specific disease or disorder, such as multiple myeloma. In some embodiments, patients with a family history of multiple myeloma are candidates for preventive regimens. Generally, the term warning refers to administering the drug before the onset of symptoms, especially in patients with possible onset of multiple myeloma.

As used herein and unless otherwise specified, the term containment includes preventing the recurrence of a particular disease or disorder, such as multiple myeloma, in a patient who has suffered from it by prolonging the time that the patient who has suffered from that disease or disorder remains in remission, reducing patient mortality and/or maintaining a reduction in the severity or prevention of a symptom associated with a disease or condition being controlled.

As used herein, the term subject or patient means an animal, typically a mammal, including a human, such as a human patient.

As described herein, the term healthy subject means any person who does not have multiple myeloma. In some embodiments, the healthy subject has no pre-existing medical conditions.

As used herein and unless otherwise indicated, the terms a therapeutically effective amount and an effective amount of a compound refer to an amount sufficient to provide a therapeutic effect in the treatment, prevention and/or control of a disease, such as multiple myeloma, to delay or minimize one or more symptoms associated with the disease or disorder being treated. Terms therapeutically

- 14 045896 an effective amount or effective amount may include an amount that improves overall therapy, reduces or eliminates symptoms or causes of a disease or disorder, or enhances the therapeutic effectiveness of another therapeutic agent.

The term responsive or receptive, when used in relation to treatment, refers to the degree to which the treatment is effective in reducing or ameliorating the symptoms of the disease, such as MM, that is being treated. For example, the term enhanced susceptibility, when used with respect to treatment of a cell or subject, refers to an increase in the effectiveness of reducing or reducing disease symptoms compared to a control treatment (eg, the same cell or subject, or a different cell or subject) when measured using any methods known in the field. In some embodiments, the efficiency improvement is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

The term sensitivity or sensitive when applied to treatment with a compound is a relative term that refers to the degree of effectiveness of the compound in reducing or reducing the progression of the tumor or disease being treated. For example, the term increased sensitivity, when used in relation to treating a cell or tumor with a compound, refers to an increase of at least about 5% or more in the effectiveness of treating the tumor.

The term refractory or resistant refers to the circumstance when patients, even after intensive treatment, have residual cancer cells (eg, multiple myeloma cells) in their lymphatic system, blood and/or hematopoietic tissues (eg, bone marrow). In the context of multiple myeloma, the term refractory or resistant refers to the circumstance when patients, even after intensive treatment, have residual myeloma cells and/or reduced healthy cells in their bone marrow. It is understood that refractory disease is disease that does not respond to therapy (failure to achieve minimal response or development of progressive disease) or progresses within approximately 60 days after the last dose.

The term recurrent refers to a situation where patients who have had their cancer in remission after therapy experience a return of cancer cells (eg, multiple myeloma cells) to their lymphatic system, blood, and/or blood-forming tissues (eg, bone marrow) and a decrease in normal cells blood.

Improvement in cancer or cancer-related disease can be described as a complete response (CR) or partial response (PR). Complete response refers to the absence of clinically detectable disease with normalization of any abnormal results of previous radiographic studies, bone marrow and cerebrospinal fluid (CSF) studies, or abnormal results of monoclonal protein measurements. A partial response refers to a reduction of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the total measurable tumor burden ( i.e., the number of malignant cells present in the subject, or the measured volume of tumor masses, or the amount of abnormal monoclonal protein) in the absence of new lesions. The term treatment includes both complete and partial response.

In the context of cancer, inhibition can be assessed by inhibiting disease progression, inhibiting tumor growth, shrinking the primary tumor, alleviating tumor-associated symptoms, inhibiting factors secreted by the tumor, delaying the appearance of primary or secondary tumors, delaying the development of primary or secondary tumors, reducing the occurrence of primary or secondary tumors, slowing or reducing the severity of secondary effects of the disease, delaying tumor growth and regression of tumors, increasing time to progression (TTP), increasing progression-free survival (PFS), increasing overall survival (OS), among others. As used herein, OS refers to the time from the start of treatment to death from any cause. As used herein, TLT refers to the time from initiation of treatment to tumor progression; The VDP does not include deaths. In one embodiment, PFS means the time from the start of treatment to tumor progression or death. In one embodiment, PFS means the time from the first dose of a compound to the first occurrence of disease progression or death from any cause. In one embodiment, PFS rates will be calculated using Kaplan-Meier estimates. Event-free survival (EFS) refers to the time from the start of treatment until any occurrence of treatment failure, including disease progression, treatment interruption for any reason, or death. In one embodiment, overall response rate (ORR) means the proportion of patients who achieve a response to treatment. In one embodiment, ORR means the sum of the proportions of patients who achieve complete and partial responses. In one embodiment, ORR means the proportion of patients whose best response > partial response (PR), according to the IMWG uniform response criteria. In one embodiment, it is carried out

- 15 045896 duration of response (DO) is the time from achievement of response to relapse or disease progression. In one embodiment, the CR is the time from achieving response > partial response (PR) to disease relapse or progression. In one embodiment, the LO is the time from the first documentation of a response to the first documentation of disease progression or death. In one embodiment, the CR is the time from first documentation of response > partial response (PR) to first documentation of disease progression or death. In one embodiment, time to response (TTR) means the time from the first dose of a compound to the first documentation of a response. In one embodiment, TTR means the time from the first dose of a compound to the first documentation of a response > partial response (PR). As a last resort, complete inhibition is referred to herein as a warning or chemoprevention. As used herein, the term prevention includes either complete prevention of the onset of clinically obvious cancer or prevention of the onset of a preclinically obvious stage of cancer. Also intended to be covered by this definition is preventing transformation into malignant cells or arresting or reversing the progression of benign cells to malignant cells. This includes preventive treatment for individuals at increased risk of developing cancer.

In the context of multiple myeloma, response can be assessed using the International Myeloma Working Group (IMWG) consensus criteria for response and minimal residual disease assessment (Rajkumar et al., Blood, 2011, 117(18):4691-5; Kumar et al., Lancet Oncol., 2016,17(8):e328e346). The criteria can be assessed as follows (more information available in Lancet Oncol., 2016, 17(8):e328-e346).__________________________________________________________

Response Criteria MWG MRD Criteria (full response required as defined below) Sustainable MOB- negative MRD negativity by bone marrow (NGF or NGS, or both) and imaging, as defined below, was confirmed at minimum 1-year intervals. Subsequent assessments can be used to clarify the duration of negativity (eg, MRD negative after 5 years) MRD-negative on son flow cytometry Absence of phenotypically aberrant clonal plasma cells by NGF on bone marrow aspirates using the EuroFlow standard operating procedure for detection of MRD in multiple myeloma (or a validated equivalent method) with a minimum sensitivity of 1 in 10 ⁵ nucleated cells or better MRD negative based on sequencing Absence of clonal plasma cells by NGS on a bone marrow aspirate, in which the presence of a clone is defined as the presence of fewer than two identical sequencing reads obtained after DNA sequencing of bone marrow aspirates using the LymphoSIGHT platform (or a validated equivalent method) with a minimum sensitivity of 1 in ¹⁰⁵ cores cells or higher MRD-negative based on plus imaging MRD negativity as determined by NGF or NGS, plus disappearance of each area of increased uptake detected at baseline or prior PET/CT, or reduction of the SUV mediastinal blood pool to less than that of surrounding normal tissue IMWG Standard Response Criteria

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Strict complete answer Complete response as defined below, plus normal FLC ratio and absence of clonal cells on bone marrow biopsy by immunohistochemistry (κ/λ ratio <4:1 or >1:2 for patients with k and λ, respectively, after counting >100 plasma cells cells) Full answer Negative immunofixation in serum and urine and disappearance of any soft tissue plasmacytomas and <5% plasma cells in bone marrow aspirates Very good partial answer Serum and urine M protein detected by immunofixation but not electrophoresis or >90% decrease in serum and urine M protein levels <100 mg within 24 hours Partial answer Reduction of M-protein in serum >50% and decrease in M-protein in urine >90% or to <200 mg within 24 hours; If serum and urinary M protein are not measurable, >50% reduction in the difference between involved and uninvolved FLC levels is required instead of M protein criteria; If serum and urine M protein is unmeasurable and the facilitated serum-free assay is also unmeasurable, a >50% plasma cell reduction is required instead of M protein, provided the baseline percentage of bone marrow plasma cells was >30%. In addition to these criteria, size reduction (SPD) of soft tissue plasmacytomas >50% is also required if they are present at baseline Minimum answer A decrease in serum M-protein by >25% but <49% and a decrease in M-protein in 24-hour urine by 50-89%. In addition to the above criteria, size reduction (SPD) of soft tissue plasmacytomas >50% is also required if present at baseline

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Stable disease Not recommended for use as a response indicator; Disease persistence is best described by estimating the time to progression of the disease. Not meeting criteria for complete response, very good partial response, partial response, minimal response, or progressive disease Progressive disease Any one or more of the following criteria: An increase of 25% from the lowest value of the confirmed response in one or more of the following criteria: Serum M-protein (absolute increase should be >0.5 g/dL); Increase in serum M protein >1 g/dL if the lowest M component was >5 g/dL; M-protein in urine (absolute increase should be >200 mg/24 h); In patients without measurable levels of Mprotein in serum and urine, the difference between the levels of involved and non-involved FLC (absolute increase should be >10 mg/dL); In patients without measurable levels of M-protein in serum and urine and without measurable levels of involved FLC, the percentage of bone marrow plasma cells regardless of baseline (absolute increase should be >10%); Appearance of a new lesion, >50% increase from the nadir in SPD of >1 lesion, or >50% increase in the longest diameter of a previous lesion >1 cm short axis; >50% increase in circulating plasma cell count (minimum 200 cells per µl) if this is the only indicator of disease Clinical relapse Clinical relapse requires the presence of one or more of the following criteria:

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Direct indicators of increasing disease and/or end organ dysfunction (CRAB features) associated with an underlying clonal disorder of plasma cell proliferation. It is not used to calculate time to progression or survival-free survival. progression, but is listed as something that can be reported optionally or for use in clinical practice; Development of new soft tissue plasmacytomas or bone lesions (osteoporotic fractures are not considered progression); Definite increase in size of existing plasmacytomas or bone lesions. Defined enlargement is defined as an enlargement of 50% (and >1 cm) measured serially using the SPD of the lesion being measured; Hypercalcemia (> 11 mg/dL); A decrease in hemoglobin >2 g/dL not related to therapy or other conditions not related to myeloma; An increase in serum creatinine of 2 mg/dL or more since the start of therapy, which may be associated with myeloma; Hyperviscosity associated with serum paraprotein Relapse after complete response (only used if endpoint is disease-free survival) Any one or more of the following criteria: Reappearance of M protein in serum or urine by immunofixation or electrophoresis; Development of >5% plasma cells in bone marrow; Appearance of any other signs of progression (eg, new plasmacytoma, lytic bone lesion, or hypercalcemia, see above) Relapse after MRD- negative (only used if the end point is survival without diseases) Any one or more of the following criteria: Loss of MRD-negative (evidence of clonal plasma cells on NGF or NGS, or positive imaging for relapse of myeloma); Reappearance of M protein in serum or urine during immunofixation or electrophoresis; Development of >5% clonal plasma cells in the bone marrow; The appearance of any other signs of progression (eg, new plasmacytoma, lytic bone lesion, or hypercalcemia)

MR=minimal residual disease. NGF=npomo4HaM next generation cytometry. NGS=ceKeeHupoeaHue next generation. FLC=free light chain. M protein = myeloma protein. SPD=sum of the products of the maximum perpendicular diameters of the measured damage. Signs of CRAB=increased calcium levels, renal failure, anemia, lytic bone lesions. FCM=flow cytometry. SUVmax=maximum standardized absorbance value. ¹⁸ F-FDG PET=1^-fluorodeoxyglucose PET.

As used herein, induction therapy refers to the first treatment for a given disease, or the first treatment provided to induce complete remission of a disease, such as cancer. When used alone, it is generally accepted that induction therapy is the best

- 19 045896 with our available treatment. If residual cancer is found, patients are treated with a different chemotherapy called reinduction chemotherapy. If the patient is in complete remission after induction therapy, additional strengthening and/or maintenance therapy is then given to prolong remission or potentially cure the patient.

As used herein, the term maintenance therapy refers to the treatment of a disease after a first remission has been achieved. For example, cancer maintenance therapy is treatment given after the cancer has disappeared after initial therapy. Consolidation therapy may include radiation therapy, a stem cell transplant, or treatment with cancer drugs. Consolidation therapy is also called enhancement therapy and post-remission therapy.

As used herein, the term maintenance therapy refers to the treatment of a disease after remission or best response has been achieved to prevent or delay relapse. Maintenance therapy may include chemotherapy, hormonal therapy, or targeted therapy.

As used herein, the term reference level is intended to refer to a biomarker reference level used to estimate the biomarker level of interest in a sample from an individual. The control level may be a normal control level in a sample from a normal subject or a disease control level from a subject with a pathological condition. The normal reference level is the extent of biomarker expression in a subject or subjects without disease. A disease reference level is the extent of biomarker expression in a subject with a positive diagnosis of a disease or condition. The reference level may also be a stage-specific reference level. A stage-specific reference level refers to the level of a biomarker specific to that stage of disease or condition progression. The control level may also be the amount of biomarker expression before treatment or at other times during treatment. For example, the control level may be the level of biomarker expression in the bone marrow before treatment. In another example, the reference level may be the expression of a biomarker in the blood at a specific point during or after treatment.

The terms probable or likelihood usually refer to the increase in the likelihood of an event. The term likely, when used in relation to a patient's susceptibility, generally implies an increased likelihood that the patient will be responsive to the treatment compound. The term likely, when used in relation to patient responsiveness, can also generally refer to an increase in the level of biomarkers, such as mRNA or protein expression, that may indicate an increased likelihood that the patient will be responsive to the therapeutic compound.

The terms forecast or forecast as used herein generally mean to determine or determine in advance. When used, for example, to predict treatment response, the term prediction may mean that the likelihood of response or non-response to cancer treatment can be determined early on, before treatment begins, or before the treatment period has progressed significantly.

The terms monitor or monitor as used in this document generally refer to supervision, supervision, regulation, surveillance, tracking or control of activity. For example, the term monitoring the effectiveness of a compound refers to monitoring effectiveness in treating cancer in a patient or in a culture of tumor cells. Likewise, the term monitoring, when used in connection with a patient's adherence to a treatment regimen, either individually or in clinical trials, refers to tracking or confirming that the patient is actually taking the study medication as prescribed. Monitoring can be done, for example, by tracking the expression of mRNA or protein biomarkers.

As used herein, the terms T cell activation and activated T cells are intended to refer to the cellular activation of resting naïve T cells into effector T cells that are capable of causing tumor cell death. T cell activation can be initiated by the interaction of the T cell receptor (TKP)/CD3 complex with antigen. A typical activated T cell exhibits cellular responses that include, but are not limited to, cell proliferation, cytokine secretion, and/or effector function. In the context of the present application, activation of T cells can be caused by the administration of Compound 1, Compound 2 or Compound 3.

As used herein, the term T cell activation-associated cytokine refers to any of numerous factors that are secreted by activated T cells or whose secretion is increased in activated T cells compared to resting naïve T cells. Non-limiting examples of cytokines associated with T cell activation include IL-2, IFNy and TNFa.

As used herein, the term regulate refers to controlling the activity of a molecule or biological function, such as increasing or decreasing the activity or function.

A biological marker or biomarker is a substance whose detection indicates a specific biological condition, such as the presence of cancer. Typical biomarkers can be determined individually. It is clear that multiple biomarkers can be measured simultaneously. Usually

One of ordinary skill in the art will appreciate that a biomarker indicates a change in the level of mRNA expression that may correlate with response to treatment or the likelihood of a patient responding to treatment. The biomarker may be a nucleic acid such as mRNA or cDNA. Protein can also be a biomarker. A specific example of a biomarker is one or more tumor cells that circulate in the peripheral blood (ie, circulating tumor cells, CTCs). A biomarker can also be a change in the structure or sequence of a gene, resulting in a variant form caused by changes in single-base blocks of DNA or deletion, insertion or rearrangement of larger sections of genes or chromosomes.

An additional typical biomarker is one that indicates a change in the expression level of a polypeptide or protein that may correlate with response to treatment or the likelihood of a patient responding to treatment. The biomarker may be a polypeptide or protein, or a fragment thereof. The relative levels of specific proteins can be determined by methods known in the art. For example, antibody-based methods such as immunoblotting, enzyme-linked immunosorbent assay (ELISA), or other methods can be used.

The terms polypeptide and protein, used interchangeably herein, refer to a polymer of three or more amino acids in a sequential array linked by peptide bonds. The term polypeptide includes proteins, protein fragments, protein analogs, oligopeptides, and the like. As used herein, the term polypeptide can also refer to a peptide. The amino acids that make up the polypeptide may be obtained naturally or may be synthetic. The polypeptide can be purified from a biological sample. Polypeptide, protein or peptide also includes modified polypeptides, proteins and peptides, such as glycopolypeptides, glycoproteins or glycopeptides; or lipopolypeptides, lipoproteins or lipopeptides.

The terms antibody, immunoglobulin, or Ig, when used interchangeably herein, cover full assembly antibodies and antibody fragments that retain the ability to specifically bind an antigen. Antibodies provided herein include synthetic antibodies, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single chain Fv (scFv) (eg, including monospecific, bispecific etc.), camel antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above, but not limited to. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, antigen binding domains or molecules that contain an antigen binding site that immunospecifically binds to a CRBN antigen (e.g., one or more complementarity determining regions (CDRs) antibodies against CRBN). Immunoglobulins can consist of heavy and light chains. Antibodies provided herein may belong to any class (eg, IgG, IgE, IgM, IgD and IgA) or any subclass (eg, IgG1, IgG2, IgG3, IgG ₄ , IgA1 and IgA2) of the immunoglobulin molecule. In some embodiments, the anti-CRBN antibodies are fully human, such as fully human anti-CRBN monoclonal antibodies. In some embodiments, the antibodies provided herein are IgG antibodies or a subclass thereof (eg, human IgG1 or IgG4). In other embodiments, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, antigen binding domains or molecules that contain an antigen binding site that immunospecifically binds to Aiolos, Ikaros, c-MYC, IRF4, caspase-3, BCMA, free kappa light chains or free lambda light chains.

The terms immunoglobulin light chain or light chain refer to the small polypeptide subunit of an antibody. The light chain may be a lambda light chain or a kappa light chain. When a light chain is attached to a heavy chain, the light chains are called linked light chains. When the light chains are not attached to the heavy chain, they are called free light chains (FLCs). When free light chains can be detected in a patient's serum or plasma, they are referred to as free serum light chains or free plasma light chains.

It is further understood that the free light chains in whey may also be referred to as soluble free light chains. One skilled in the art will appreciate that the amount of free light chain in serum or plasma is proportional to the severity of the disease when the disease is multiple myeloma. It is also understood that free light chain levels or the ratio of free kappa light chain to free lambda light chain can be used to monitor or diagnose myeloma. For example, reducing pathologically high levels of free kappa light chain to levels of free lambda light chain in the serum of a patient with multiple myeloma being treated with Compound 2 can be used to monitor the effectiveness of treatment with Compound 2. Likewise, increasing the amount of free light chain in a patient being treated other compound, such as lenalidomide, may indicate resistance to

- 21 045896 treatment and may serve as a predictor of response to Compound 1, Compound 2 or Compound 3.

The terms antigen binding domain, antigen binding region, antigen binding fragment and similar terms refer to the part of an antibody that contains amino acid residues that interact with the antigen and give the binding agent its specificity and affinity for the antigen (eg, CDR). The antigen binding region can be derived from any animal species, such as rodents (eg rabbit, rat or hamster) and humans. In some embodiments, the antigen binding region is of human origin.

As used herein, the term epitope refers to a localized region on the surface of an antigen that is capable of binding to one or more antigen-binding regions of an antibody having antigenic or immunogenic activity in an animal, such as a mammal (eg, a human), and that is capable of eliciting an immune response. An immunogenic epitope is the portion of a polypeptide that elicits an antibody response in an animal. An antigenic epitope is the portion of a polypeptide to which an antibody immunospecifically binds, as determined by any method well known in the art, such as the immunoassays described herein. Antigenic epitopes do not necessarily have to be immunogenic. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. The region of the polypeptide that is included in the epitope may be contiguous amino acids of the polypeptide, or the epitope may consist of two or more non-contiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface structure of the antigen.

The terms cereblon or CRBN and similar terms refer to polypeptides (the terms polypeptides, peptides, and proteins are used interchangeably herein) containing the amino acid sequence of any CRBN, such as human CRBN protein (e.g., human CRBN isoform 1, GenBank accession number NP_057386 or isoform 2 human CRBN, GenBank accession number NP_001166953, each of which is incorporated herein by reference in its entirety), and similar polypeptides, including SNP variants thereof. CRBN-like polypeptides include allelic variants (e.g., SNP variants), splice variants, fragments, derivatives, substitution variants, deletion variants, insertion variants, fusion polypeptides, and interspecies homologues that, in some embodiments, retain CRBN activity and/or are sufficient to generate an immune response against CRBN.

As used herein, the term cereblon-bound protein or CAP refers to a protein that directly or indirectly interacts with or binds to cereblon (CRBN). For example, the term refers to any protein that directly binds to cereblon, as well as any protein that is an indirect downstream effector of the CRBN reaction cascade. An example of an ATS is a CRBN substrate, for example a protein substrate of the E3 ubiquitin ligase complex including CRBN, such as IKZF1, IKZF3, ZFP91, or their downstream effector proteins such as c-Myc or IRF4.

As used herein, the term cereblon modulator compound refers to a compound that binds to CRBN and modifies its activity or substrate specificity. For example, binding of the modulator compound cereblon can increase the degradation of IKZF1, IKZF3, ZFP91, IRF4, or c-MYC. Exemplary cereblon modulator compounds may be, but are not limited to, lenalidomide, pomalidomide, or the like.

As used herein, the term mutation refers to any change in the structure of a gene resulting in a variant (also called mutant) form. Gene mutations can be caused by the alternation of a single base in the DNA or by the deletion, insertion, or rearrangement of large sections of genes or chromosomes. In some embodiments, the mutation may affect the function or resulting protein. For example, a mutation of one DNA nucleotide (i.e., a point mutation) in the coding region of a protein can result in the formation of a codon that codes for a different amino acid (i.e., a missense mutation). This other amino acid may change the structure of the protein such that the compound or its derivatives cannot bind and/or inhibit the protein.

The term T cell receptor clonality (TCR) refers to the somatic reconfiguration of the germline T cell receptor genes to a unique configuration to allow the development of a clone of T cells with a T cell receptor specific for a given antigen. T cell receptor genes (alpha, beta, delta, and gamma) can be somatically rearranged to form heterodimeric T cell receptors on the cell surface.

Somatic rearrangements of the TCR gene can lead to the expansion of different clones (polyclonal) or to monoclonal expansion of a population of T cells with a single TCR rearrangement pattern. TCR clonality can be determined by standard methods in molecular biology, such as PCR, Southern blotting, or TCR sequencing (eg, next generation sequencing).

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The term soluble refers to forms of a biological molecule that are found in the extracellular space (such as serum) rather than on the cell surface. The term soluble B cell maturation antigen, soluble BCMA (sBCMA), soluble CD25, or soluble IL-2 receptor (sIL-2R) refers to soluble proteins, which are the released form of proteins and are present in blood serum.

As used herein, the term expressible or expression refers to the transcription of a gene to produce an RNA nucleic acid molecule at least partially complementary to a region of one of the two nucleic acid strands of the gene. The term expressible or expression as used herein also refers to the translation of an RNA molecule to produce a protein, polypeptide, or part thereof.

The term level refers to the amount, accumulation, or performance of a biomarker molecule. The level may be represented, for example, by the amount or rate of synthesis of messenger RNA (mRNA) encoded by a gene, the amount or rate of synthesis of a polypeptide or protein encoded by a gene, or the amount or rate of synthesis of a biological molecule accumulated in a cell or biological fluid. The term level refers to the absolute amount of a molecule in a sample or the relative amount of a molecule determined under steady-state or unsteady conditions.

An increase in mRNA levels typically occurs with a given treatment or condition. A decrease in mRNA level generally refers to a decrease in the level of mRNA expression in response to a given treatment or condition. In some situations, mRNA levels may remain unchanged for a given treatment or condition. The level of mRNA from a patient sample may be increased by drug treatment compared to untreated controls. This increase may be, for example, an increase of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1000%, about 5000% or more of a comparative control mRNA level. Alternatively, the level of mRNA may be decreased or expressed at a lower level in response to the administration of certain compounds or other agents. The reduced level of mRNA may, for example, be present at about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 1% or less of the comparative control mRNA level.

Similarly, the level of a polypeptide or protein biomarker in a patient sample may be increased by drug treatment compared to a control without treatment. This increase may be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200 %, about 300%, about 500%, about 1000%, about 5000% or more of the comparative control protein level. Alternatively, the level of the protein biomarker can be reduced in response to the administration of certain compounds or other agents. This reduction may be, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 1% or less of the comparative control level of the polypeptide or protein.

In addition, the level of CTCs in a patient sample may decrease in response to the administration of certain compounds or other agents. This reduction may, for example, be present at a level of about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, approximately 1% or less of the comparative control level of CTC.

TCR DNA sequence or TCR clonality may also be increased by drug treatment compared to untreated controls. Somatic rearrangements of the TCR gene can lead to the expansion of different clones (polyclonal) or to monoclonal expansion of a population of T cells with a single TCR rearrangement pattern. This increase in the number of specific clones may be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% , about 200%, about 300%, about 500%, about 1000%, about 5000% or more of the comparative control TCR clonality.

The terms definition, measurement, evaluation, evaluation and analysis used in this document generally refer to any form of measurement and include determining whether an item is present or not. These terms include quantitative and/or qualitative definitions. The rating can be relative or absolute. Assessing presence may involve determining how much something is present and whether it is present or absent.

The terms nucleic acid and polynucleotide are used interchangeably herein to describe a polymer of any length consisting of nucleotides, such as deoxyribonucleotides or ribonucleotides, or synthetically produced compounds that can hybridize with naturally occurring nucleic acids in a sequence-specific manner.

- 23 045896 activities in a manner similar to the hybridization of two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. As used herein in the context of a polynucleotide sequence, the term base (or base) is synonymous with nucleotides (or nucleotide), that is, the monomeric subunit of a polynucleotide. The terms nucleoside and nucleotide are intended to include those moieties that contain not only known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocyclic compounds. In addition, the terms nucleoside and nucleotide include those moieties that contain not only the common sugars ribose and deoxyribose, but also other sugars. Modified nucleosides or nucleotides also include modifications of the sugar moiety, for example, in which one or more hydroxyl groups are replaced by halogen atoms or aliphatic groups, or functionalized as ethers, amines, or the like. Analogues refer to molecules having structural features that are recognized in the literature as mimetics, derivatives having similar structures, or other similar terms, and include, for example, polynucleotides including non-natural nucleotides, nucleotide mimetics such as 2'-modified nucleosides, peptide nucleotides acids, oligomeric nucleoside phosphonates, and any polynucleotide to which substituent groups, such as protecting groups or linking moieties, have been added.

The term complementary refers to the specific binding of polynucleotides based on the sequences of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, for example, if they produce a given or detectable level of signal in a hybridization assay. Parts of polynucleotides are complementary to each other if they follow normal base-pairing principles, for example, A pairs with T (or U) and G pairs with C, although small regions (eg, less than about 3 bases) of mismatches, insertions, or remote sequence.

Sequence identity, or identity in the context of two nucleic acid sequences, refers to residues in two sequences that are the same when aligned for maximum fit within a specified comparison window, and can account for additions, deletions, and substitutions.

The term substantially identical or homologous in their various grammatical forms in the context of polynucleotides generally means that the polynucleotide includes a sequence that has the desired identity, for example, at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity and at least 95% identity compared to the reference sequence. Another sign that the nucleotide sequences are almost identical is that the two molecules hybridize to each other under stringent conditions.

The terms isolated and purified refer to the isolation of a substance (such as mRNA, DNA, or protein) so that the substance constitutes a significant portion of the sample in which it is found, that is, more than the portion of the substance that is normally found in its natural or unisolated state. Typically, a significant portion of the sample includes, for example, more than 1%, more than 2%, more than 5%, more than 10%, more than 20%, more than 50%, or more, typically up to about 90-100% of the sample. For example, a sample of isolated mRNA will typically contain at least about 1% of the total mRNA. Methods for purifying polynucleotides are well known in the art and include, for example, gel electrophoresis, ion exchange chromatography, affinity chromatography, flow sorting and density sedimentation.

As used herein, the term associated means direct or indirect attachment. In the context of chemical structures, connected (or connected) can refer to the existence of a chemical bond directly connecting two fragments or indirectly connecting two fragments (for example, through a linking group or any other intermediate part of the molecule). The chemical bond may be a covalent bond, ionic bond, coordination complex, hydrogen bond, van der Waals interactions, or hydrophobic stacking, or may exhibit characteristics of several types of chemical bonds. In some cases, bound includes embodiments in which the attachment is direct and embodiments in which the attachment is indirect.

As used herein, the term sample refers to a material or mixture of materials, typically, although not necessarily, in liquid form, containing one or more components of interest.

A biological sample as used herein refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid obtained, drawn or collected in vivo or in situ. A biological sample also includes samples from the field of biology

- 24 045896 human subject containing precancerous or cancerous cells or tissue. Such samples may include, but are not limited to, organs, tissues and cells isolated from a mammal. Examples of biological samples include, but are not limited to, cell lysate, cell culture, cell line, tissue, oral tissue, gastrointestinal tissue, organ, organelle, body fluid, blood sample, urine sample, skin sample, and the like. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMC, tissue biopsies including bone marrow biopsies, bone marrow aspirate, isolated bone marrow mononuclear cells, circulating tumor cells, and the like.

The term circulating tumor cells (CTCs) as used herein refers to multiple myeloma cells found in the peripheral blood that have separated from tumor cells in the bone marrow. In some embodiments, CTCs may serve as a biomarker of response and predictive of future disease progression. In other embodiments, the biomarker may be the mutational landscape of CTCs in MM.

As used herein, the term analyte refers to a known or unknown component of a sample.

As used herein, the term capture agent refers to an agent that binds mRNA or protein through an interaction sufficient to allow the agent to bind and concentrate the mRNA or protein from a heterogeneous mixture.

As used herein, the term probe refers to a capture agent that is directed to a specific biomarker sequence of a target mRNA. Accordingly, each probe in the probe set has a corresponding target mRNA biomarker. A probe/target mRNA duplex is a structure formed by hybridizing a probe with its target mRNA biomarker.

The term nucleic acid probe or oligonucleotide probe refers to a nucleic acid capable of binding to a target nucleic acid of complementary sequence, such as the mRNA biomarkers provided herein, typically through complementary pairing to form a hydrogen bond. The probe used herein may include natural (eg, A, G, C or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in the probe may be connected by a bond other than a phosphodiester bond, provided that this does not interfere with hybridization. One skilled in the art will appreciate that probes may bind to target sequences that are not fully complementary to the probe sequence, depending on the stringency of the hybridization conditions. The probes are preferably directly labeled with labels, for example chromophores, luminophores, chromogens, or indirectly labeled with biotin, to which the streptavidin complex can subsequently bind. By analyzing the presence or absence of a probe, the presence or absence of a target mRNA biomarker of interest can be detected.

The term stringent assay conditions refers to conditions that are compatible to produce binding pairs of nucleic acids, such as probes and target mRNAs, with sufficient complementarity to provide the desired level of specificity in the assay, but are generally incompatible to form binding pairs between binding partners with insufficient complementarity to provide the desired specificity. The term stringent assay conditions usually refers to a combination of hybridization and washing conditions.

A label or detectable fragment, when applied to a nucleic acid, refers to a composition that, when associated with a nucleic acid, makes the nucleic acid detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical or chemical methods. Examples of labels include, but are not limited to, radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, enzymes, biotin, digoxigenin, haptens, and the like. A labeled nucleic acid or oligonucleotide probe is typically a probe that is linked either covalently through a linker or chemical bond, or non-covalently through ionic bonds, van der Waals forces, electrostatic attraction, hydrophobic interactions or hydrogen bonds to the label such that the presence of nucleic acid acid or probe can be detected by detecting the presence of a label associated with the nucleic acid or probe.

The term polymerase chain reaction or PCR as used herein generally refers to a procedure in which small amounts of nucleic acid, RNA and/or DNA, are amplified, as described, for example, in US Pat. No. 4,683,195. Typically, sequence information should be available from the ends or outside the region of interest so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may match the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 1987,

- 25 045896

51:263-273; PCR Technology (Stockton Press, NY, Erlich, ed., 1989).

The term cycle number or C _T when used herein in relation to PCR methods refers to the PCR cycle number at which the level of fluorescence exceeds a given set threshold level. The C _T measurement can be used, for example, to approximate the levels of mRNA in the original sample. By measuring _CT , we often mean dC _T , or an estimate of the difference in CT when the _CT of one nucleic acid is subtracted from the _CT of another nucleic acid.

As used herein, the terms compound and therapeutic compound are used interchangeably and include Compound 1, Compound 2, Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

A tautomer as used herein refers to isomeric forms of a compound that are in equilibrium with each other. Concentrations of isomeric forms will depend on the environment in which the compound is found and may vary depending, for example, on whether the compound is a solid or in organic or aqueous solution. For example, in aqueous solution, pyrazoles can have the following isomeric forms, which are tautomers of each other:

As used herein and unless otherwise indicated, the term pharmaceutically acceptable salts includes amine salts such as, but not limited to, N-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, Nmethylglucamine, procaine, N -benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts such as, but not limited to, lithium, potassium and sodium; alkaline earth metal salts such as, but not limited to, barium, calcium and magnesium; transition metal salts such as, but not limited to, zinc; and salts of other metals such as, but not limited to, sodium hydrogen phosphate and disodium phosphate; and also include, but are not limited to, salts of mineral acids such as, but not limited to, hydrochlorides and sulfates; and salts of organic acids such as, but not limited to, acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, fumarates and organic sulfonates.

Unless specifically stated otherwise, when a compound can be expected to have alternative tautomeric, regioisomeric and/or stereoisomeric forms, all alternative isomers are embraced within the scope of the claimed subject matter. For example, where a compound may have one of two tautomeric forms, it is intended that both tautomers be included herein.

Thus, the compounds provided herein may be enantiomerically pure or be stereoisomeric or diastereomeric mixtures. As used herein and unless otherwise indicated, the term stereomerically pure or optically pure means a composition that contains one stereoisomer of a compound and substantially no other stereoisomers of the compound. For example, a stereomerically pure composition of a compound having a single chiral center will be substantially free of the opposite enantiomer of the compound. A stereopure composition of a compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of the other stereoisomers of the compound, more preferably more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight by weight of the other stereoisomers of the compound, even more preferably, more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, and most preferably, more than about 97% by weight of one stereoisomer of the compound, and less than about 3% by weight of other stereoisomers of the compound. As used herein, a stereomerically pure compound contains more than about 80% by weight of one stereoisomer of the compound, more preferably more than about 90% by weight of one stereoisomer of the compound, even more preferably more than about 95% by weight of one stereoisomer of a given compound, and most preferably, more than about 97% by weight of one stereoisomer of a given compound. As used herein and unless otherwise indicated, the term stereomerically enriched means a composition that contains more than about 60% by weight of one stereoisomer of a compound, preferably more than about 70% by weight, more preferably more than about 80% by weight one stereoisomer of a compound. As used herein and unless otherwise indicated, the term enantiomerically pure means a stereomerically pure composition of a compound having a single chiral center. Likewise, the term stereomerically enriched means a stereomerically enriched composition of a compound having a single chiral center. As used herein, mixtures of stereoisomers or diastereomers means a composition that contains more than one stereoisomer of a compound. Typical

- 26 045896 a mixture of stereomers of a compound contains about 50% by weight of one stereoisomer of the compound and about 50% by weight of other stereoisomers of the compound, or contains more than about 50% by weight of one stereoisomer of the compound and less than about 50% by weight of other stereoisomers of the same a compound, or contains more than about 45% by weight of one stereoisomer of the compound and less than about 55% by weight of the other stereoisomers of the compound, or contains more than about 40% by weight of one stereoisomer of the compound and less than about 60% by weight of the others stereoisomers of a given compound, or contains more than about 35% by weight of one stereoisomer of a given compound and less than about 65% by weight of other stereoisomers of a given compound.

It should be understood that the compounds presented herein may contain chiral centers. Such chiral centers may have either the (R) or (S) configuration, or may be a mixture of both. It should be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. Thus, one skilled in the art will appreciate that administering a compound in its (R) form is equivalent, in the case of compounds that undergo epimerization in vivo, to administering the compound in its (S) form.

Optically active (+) and (-), (R)- and (S)-, or (D)- and (b)-isomers can be prepared using chiral synthons or chiral reagents, or separated using conventional methods such as as chromatography with a chiral stationary phase.

In the description given herein, if there is any discrepancy between the chemical name and the chemical formula, the formula takes precedence.

It should also be noted that compounds may contain unnatural proportions of atomic isotopes on one or more atoms. For example, compounds may be labeled with radioactive isotopes, such as tritium ( ^3H ), iodine-125 ( ^125I ), cepa-35 ( ^35S ), or carbon-14 ( ^14C ), or may be enriched with isotopes , such as deuterium ( ^2H ), carbon-13 ( ^13C ) or nitrogen-15 ( ^15N ). As used herein, isotopologue or isotopologue means an isotopically enriched compound. The term isotopically enriched refers to an atom having an isotopic composition different from the natural isotopic composition of that atom. Isotopically enriched may also refer to a compound containing at least one atom having an isotopic composition different from the natural isotopic composition of that atom. The term isotopic composition refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, for example, therapeutic agents against cancer and inflammation, research reagents, for example, binding assay reagents, and diagnostic agents, for example, in vivo imaging agents. All isotopic embodiments of the compounds described herein, whether radioactive or not, are intended to be within the scope of these embodiments proposed herein. In some embodiments, compound isotopologues are provided, for example, the isotopologues are compounds enriched in deuterium, carbon-13, or nitrogen-15. In some embodiments, the isotopologues provided herein are deuterium-enriched compounds. In some embodiments, the isotopologues provided herein are deuterium-enriched compounds where deuteration occurs at a chiral center. In some embodiments, provided herein are isotopologues of compounds of Compound 1 where deuteration occurs at a chiral center. In some embodiments, isotopologues of Compound 2 are provided herein where deuteration occurs at a chiral center. In some embodiments, isotopologues of Compound 3 are provided herein where deuteration occurs at a chiral center.

It should be noted that if there is a discrepancy between a depicted structure and the name given to that structure, the depicted structure should be given more weight. In addition, unless the stereochemistry of a structure or part of a structure is indicated, for example, by bold or dotted lines, the structure or part of a structure should be interpreted as encompassing all of its stereoisomers.

As described herein, the term second active agent refers to any additional treatment that is biologically active. It is understood that the second active agent may be a hematopoietic growth factor, a cytokine, an anticancer agent, an antibiotic, a Cox-2 inhibitor, an immunomodulatory agent, an immunosuppressive agent, a corticosteroid, a therapeutic antibody that specifically binds to a cancer antigen, or a pharmacologically active mutant or derivative thereof.

The term maintenance agent refers to any substance that treats, prevents or inhibits an adverse event from treatment with Compound 1, Compound 2 or Compound 3, or an enantiomer or mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof. It is understood that the term maintenance therapy refers to any therapeutic agent that is primarily aimed at maintaining the patient's strength and/or comfort. Examples of supportive care include pain management therapy, intravenous fluids, and electrolyte support such as isotonic saline, glucose saline, or

- 27 045896 balanced crystalloid solutions, but not limited to them.

The term biological therapy refers to the administration of biological therapy, such as umbilical cord blood, stem cells, growth factors and the like.

The terms co-administration and in combination with include the administration of one or more therapeutic agents (for example, a compound provided herein and another anti-multiple myeloma agent, anti-cancer agent or maintenance therapy agent) simultaneously, in parallel or sequentially without any particular limitation on time. In one embodiment, the agents are present in the cell or body of the patient at the same time or exhibit their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or in a unit dosage form. In another embodiment, the therapeutic agents are in different compositions or different unit dosage forms.

As used herein, the terms immunospecifically binds, immunospecifically recognizes, specifically binds, and specifically recognizes are similar terms in the context of antibodies and refer to molecules that bind to an antigen/epitope in a manner understood by one skilled in the art. Antibodies that specifically bind to a target structure or its subunit do not cross-react with biological molecules that are not part of the family of target structures. In some embodiments, the antibody or antibody fragment binds to the selected antigen with a specific affinity greater than 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M or 10 ^-11 M, between 10 ^-8 M and 10 ^-11 M, between 10 ^-9 M and 10 ^-10 M, or between 10 ^-10 M and 10 ^-11 M. For example, a molecule (such as an antibody) that specifically binds to an antigen may bind to other peptides or polypeptides, typically more low affinity, as determined, for example, by immunoassays or other assays known in the art. In a particular embodiment, molecules that specifically bind to an antigen do not cross-react with other proteins.

As described herein, the term detectable label refers to the attachment of a specific label to an antibody to aid in the detection or isolation/purification of a protein. Examples of label types include, but are not limited to, radioisotope, fluorophore (eg, fluorescein isothiocyanate (FITC), phycoerythrin (PE)), chemiluminescence, reporter enzymes, and elemental particles (eg, gold particles). Detection may be direct or indirect. Optical techniques include microscopy (both confocal and non-confocal), imaging techniques, and non-imaging techniques. Electrochemical methods include voltammetry and amperometry methods. Radiofrequency techniques include multipolar resonance spectroscopy.

The term approximately or approximately means an acceptable error for a particular value, as determined by one skilled in the art, which depends in part on how the value is measured or determined. In some embodiments, the term about or about means within 1, 2, 3, or 4 standard deviations. In some embodiments, the term about or about means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.05% of set value or range.

The practice of the embodiments presented herein will use, unless otherwise indicated, conventional techniques of molecular biology, microbiology and immunology that are within the skill of those skilled in the art. Such methods are fully explained in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed. 1989); Glover, ed., DNA Cloning, Volumes I and II (1985); Gait, ed., Qligonucleotide Synthesis (1984); Hames & Higgins, eds., Nucleic Acid Hybridization (1984); Hames & Higgins, eds., Transcription and Translation (1984); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, N.Y., 2d ed. 1987); and Weir & Blackwell, eds., Handbook of Experimental Immunology, Volumes I-IV (1986).

5.2 Biomarkers and their uses

The methods provided herein are based in part on the discovery that detectable increases or decreases in certain biomarkers upon treatment with a compound are observed in subjects with MM who respond to a given treatment, for example, a compound such as Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, as described in section 5.7 below, and that the levels of these biomarkers can be used to predict the response of subjects to treatment. In some embodiments, biomarker levels predict response to Compound 1, Compound 2, or Compound 3. In some embodiments, the compound is as described herein. In some embodiments, the compound is Compound 1, Compound 2, or Compound 3. In one embodiment, the compound is Compound 1. In another embodiment, the compound is Compound 2. In yet another embodiment, the compound is Compound 3.

- 28 045896

In one aspect, the invention is directed to Compound 1, Compound 2 or Compound 3 for use in a method of treating, preventing and/or controlling diseases as provided herein.

As described in the examples in section 6 and shown in the figures, the levels of certain proteins, molecules, mRNA or cellular composition change in response to treatment with Compound 1, Compound 2 or Compound 3. These biomarkers include CRBN, IKZF1, IKZF3, ZFP91, c-Myc , IRF4, caspase-1, caspase-3, caspase-7, PARP, survivin, Bcl-2-like protein 11 (BIM), serum free light chain (sFLC), p21, p27, pRb1, IL-2, TNFa , IFNy, tumor-infiltrating lymphocytes (TILs), T-cell antigen receptor (TCR) clonality, and circulating tumor cells (CTCs). Additionally, the Examples and Figures indicate that the expression of certain proteins changes following treatment with Compound 1, Compound 2, or Compound 3, and may serve as biomarkers to predict treatment response and/or select patients who are likely to respond to treatment with Compound 1, Compound 2 or Compound 3. These biomarkers include CRBN. Thus, in some embodiments, the biomarker provided herein is selected from the group consisting of CRBN, IKZF1, IKZF3, ZFP91, c-Myc, IRF4, caspase-1, caspase-3, caspase-7, PaRp, survivin, Bcl-2-like protein 11 (BIM), serum free light chain (sFLC), p21, p27, pRb1, IL-2, TNFa, IFNy, tumor infiltrating lymphocytes (TIL), T cell antigen receptor (TCR) clonality and circulating tumor cells (CTCs). Each of the biomarkers presented herein includes different isoforms, phosphorylated forms, cleaved forms, modified forms and their splice variants. For example, caspase-3 includes a cleaved form of caspase-3, caspase-1 includes a cleaved form of caspase-1, caspase-7 includes a cleaved form of caspase-7, PARP includes cleaved PARP, and pRb1 includes phosphorylated pRb1. Thus, in some embodiments, the levels of isoforms, phosphorylated forms, cleaved forms, modified forms, and/or splice variants of these biomarkers increase or decrease in response to treatment with a compound, and thus the isoforms, phosphorylated forms, cleaved forms, modified forms and/or biomarker splice variants can be used to predict patient response.

IKAROS family zinc finger 1 (IKZF1, also known as Ikaros) and IKAROS family zinc finger 3 (IKZF3, also known as Aiolos) are hematopoietic transcription factors involved in the regulation of lymphocyte development. Expression of IKZF1 and IKZF3 is restricted to the fetal and adult hemolymphopoietic system and functions as regulators of lymphocyte differentiation. Regulation of gene expression includes Ikaros homodimers, Ikaros/Aiolos heterodimers, and Aiolos homodimers. Multiple isoforms of human Ikaros and Aiolos have been found in both normal and leukemic B cells. The non-DNA binding isoforms are primarily found in the cytoplasm and are thought to act as dominant negative factors. Overexpression of certain dominant negative isoforms has been associated with B cell malignancies such as acute lymphoblastic leukemia (ALL).

Caspases-1, -3 and -7 are members of the caspase family. Caspase-3 cleaves and activates caspase7, as well as caspase-6 and -9. Caspase 7 cleavage occurs when cell death is stimulated and induces apoptosis. Caspase-3 itself is cleaved by caspase-8, -9 and -10. The effector caspases, caspase-3, -6, and -7, proteolytically degrade host intracellular proteins to effect cell apoptosis. Caspase-3 has virtually no activity until it is cleaved by the initiator caspase after apoptotic signaling events have occurred. Similarly, procaspase-1 is converted to active caspase-1 upon cleavage, and caspase-1 is also involved in some forms of apoptosis. In addition, poly(ADP-ribose) polymerase (PARP) can be cleaved by caspases. For example, PARP is known to be cleaved by caspase-3 during apoptosis. PARP is a family of proteins involved in the regulation of various important cellular processes such as differentiation, proliferation and tumor transformation. PARP also regulates molecular processes involved in cell repair after DNA damage. Caspase activation can be inhibited by survivin, thereby preventing apoptosis. Another protein involved in promoting apoptosis is Bcl-2-like protein 11 (BIM). Apoptosis may also be promoted by serum free light chain (sFLC). Thus, in some embodiments, cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP, survivin, BIM, and Serum free light chain (sFLC) are biomarkers that indicate apoptosis.

In some embodiments of the methods provided herein, the biomarker is a protein that is directly or indirectly affected by cerblone (CRBN), for example, through protein-protein interactions (e.g., certain CRBN substrates or their downstream effectors) or through various cellular reaction cascades (for example, signal transduction pathways). In specific embodiments, the biomarker is a CRBN-associated protein (CAP). In some embodiments, the biomarker is the mRNA of a protein that is directly or indirectly affected by CRBN. In other embodiments, the biomarker is

- 29 045896 cDNA of a protein that is directly or indirectly affected by CRBN. There are at least two isoforms of the CRBN protein, which are 442 and 441 amino acids in length, respectively.

As described in the Examples, treatment with Compounds 1-3 reduces the levels of Aiolos, Ikaros, ZFP91, c-Myc and IRF4 (eg, FIG. 4). Therefore, determining the level of proteins associated with CRBN may be useful for monitoring efficacy, predicting response, identifying a subject with cancer that is likely to be susceptible, treating cancer, identifying a subject with multiple myeloma that is likely to be susceptible to a therapeutic compound, and determining or adjusting the dose for treating a subject having multiple myeloma with a therapeutic compound. Therefore, in some embodiments, the biomarker is a CRBN-bound protein and the treatment compound is Compound 1. In other embodiments, the biomarker is a CRBN-linked protein and the treatment compound is Compound 2. In other embodiments, the biomarker is is a CRBN-associated protein, and the therapeutic compound is Compound 3. In some embodiments, the biomarker is a CRBN-associated protein selected from the group consisting of Aiolos, Ikaros, ZFP91, c-Myc, and IRF4. In some specific embodiments, the biomarker is a CRBN-associated protein selected from the group consisting of Aiolos, Ikaros, ZFP91, c-Myc, and IRF4, and the treatment compound is Compound 1. In some specific embodiments, the biomarker is a CRBN-associated protein. A CRBN selected from the group consisting of Aiolos, Ikaros, ZFP91, c-Myc, and IRF4, and the therapeutic compound is Compound 2. In some specific embodiments, the biomarker is a CRBN-associated protein selected from the group consisting of Aiolos, Ikaros, ZFP91, c-Myc and IRF4, and the therapeutic compound is Compound 3.

In some embodiments, the biomarker is a zinc finger transcription factor member of the IKAROS family, such as IKZF1 or IKZF3. In a specific embodiment, the biomarker is IKZF1 and the treatment compound is Compound 1. In another specific embodiment, the biomarker is IKZF1 and the treatment compound is Compound 2. In another specific embodiment, the biomarker is IKZF1 and the treatment compound is Compound 3. In a particular embodiment, In another specific embodiment, the biomarker is IKZF3 and the therapeutic compound is Compound 1. In another specific embodiment, the biomarker is IKZF3 and the therapeutic compound is Compound 2. In another specific embodiment, the biomarker is IKZF3 and the therapeutic compound is Compound 3. In a specific embodiment, the biomarker is ZFP91 and the treatment compound is Compound 1. In another specific embodiment, the biomarker is I ZFP91 and the treatment compound is Compound 2. In another specific embodiment, the biomarker is ZFP91 and the treatment compound is Compound 3. In yet another specific embodiment, the biomarker is c-Myc and the treatment compound is Compound 1. In another specific embodiment, the biomarker is c-Myc and the treatment compound is Compound 2. In another specific embodiment, the biomarker is c-Myc and the treatment compound is Compound 3. In another specific embodiment, in an embodiment, the biomarker is IRF4 and the treatment compound is Compound 1. In another specific embodiment, the biomarker is IRF4 and the treatment compound is Compound 2. In another specific embodiment, the biomarker is IRF4 and the treatment compound is Compound 3. In other embodiments, the biomarker is is a binding partner, downstream effector, or factor in a cellular cascade of reactions influenced by IKZF1, IKZF3, ZFP91, c-Myc, or IRF4.

As described in the Examples, the levels of apoptotic cascade proteins such as caspase-1, caspase-3, caspase-7, PARP, survivin, BIM and sFLC are altered upon treatment with Compounds 1-3. For example, treatment with Compounds 1-3 can increase the levels of β-caspase-3, β-caspase-1, β-caspase-7, cleaved PARP, BIM and sFLC in multiple myeloma cells, thereby indicating apoptosis. Treatment with Compounds 1-3 may also reduce survivin levels, thereby indicating apoptosis. Detection of apoptosis may be useful for monitoring efficacy, predicting response, identifying a subject having cancer that is likely to be susceptible, treating the cancer, identifying a subject with multiple myeloma that is likely to be susceptible to a therapeutic compound, and determining or adjusting a dose for treating a subject having multiple myeloma, therapeutic compound. Therefore, in some embodiments, the biomarker is involved in the apoptotic cascade. In some embodiments, the biomarker is involved in the apoptosis cascade and the treatment compound is Compound 1. In some embodiments, the biomarker is involved in the apoptosis cascade and the treatment compound is Compound 2. In some embodiments, the biomarker is involved in the apoptosis cascade and the therapeutic compound is Compound 3.

In specific embodiments, the biomarker is involved in the apoptosis cascade and is selected from the group consisting of cleaved caspase 1 (β-caspase 1), cleaved caspase 3 (β-caspase 3),

- 30 045896 cleaved caspase 7 (p-caspase 7), cleaved PARP, survivin, BCL-2-like protein 11 (BIM) and free light chain in serum. Thus, in some embodiments, the biomarker is involved in the apoptosis cascade and is selected from the group consisting of cleaved caspase 1 (β-caspase 1), cleaved caspase 3 (β-caspase 3), cleaved caspase 7 (β-caspase 7), cleaved PARP, survivin, BCL-2-like protein 11 (BIM), and free chain in serum, and the therapeutic compound is Compound 1. In some embodiments, the biomarker is involved in the apoptosis cascade and is selected from the group consisting of cleaved caspase 1 ( β-caspase 1), cleaved caspase 3 (β-caspase 3), cleaved caspase 7 (β-caspase 7), cleaved PARP, survivin, BCL-2-like protein 11 (BIM) and free chain in serum, and therapeutic the compound is Compound 2. In some embodiments, the biomarker is involved in the apoptosis cascade and is selected from the group consisting of cleaved caspase 1 (β-caspase 1), cleaved caspase 3 (β-caspase 3), cleaved caspase 7 (β-caspase 7 ), cleaved PARP, survivin, BCL-2-like protein 11 (BIM), and free chain in serum, and the treatment compound is Compound 3. In specific embodiments, levels of caspase-3, including cleaved caspase-3, are the biomarker and the treatment compound is is Compound 1. In some embodiments, levels of caspase-3, including cleaved caspase-3, are a biomarker, and the therapeutic compound is Compound 2. In some embodiments, levels of caspase-3, including cleaved caspase-3, are a biomarker, and the therapeutic compound is Compound 3. In some embodiments, levels of caspase-1, including cleaved caspase-1, are a biomarker, and the therapeutic compound is Compound 1. In some embodiments, levels of caspase-1, including cleaved caspase-1, are a biomarker and the treatment compound is Compound 2. In some embodiments, the levels of caspase-1, including cleaved caspase-1, are the biomarker, and the treatment compound is Compound 3. In some embodiments, the levels of caspase-7, including cleaved caspase-7, is a biomarker and the treatment compound is Compound 1. In some embodiments, the levels of caspase-7, including cleaved caspase-7, are the biomarker and the treatment compound is Compound 2. In some embodiments, the levels of caspase-7, including cleaved caspase-7 7 is a biomarker, and the treatment compound is Compound 3. In some embodiments, PARP levels, including cleaved PARP, are a biomarker, and the treatment compound is Compound 1. In some embodiments, PARP levels, including cleaved PARP, are a biomarker, and the treatment compound is Compound 2. In some embodiments, PARP levels, including cleaved PARP, are a biomarker and the treatment compound is Compound 3. In some embodiments, survivin levels are a biomarker and the treatment compound is Compound 1. In some embodiments, the treatment compound is Compound 3. survivin levels are a biomarker and the treatment compound is Compound 2. In some embodiments, survivin levels are a biomarker and the treatment compound is Compound 3. In some embodiments, BIM levels are a biomarker and the treatment compound is Compound 1. In some embodiments implementation, BIM levels are a biomarker and the treatment compound is Compound 2. In some embodiments, BIM levels are a biomarker and the treatment compound is Compound 3. In addition, changes in serum free light chain (sFLC) and CTC levels vary depending from medicinal Compounds 1-3. For example, treatment with Compounds 1-3 may reduce the amount of sFLC found in blood or serum/plasma. Therefore, in some embodiments, the sFLC is a biomarker and the treatment compound is Compound 1. In some embodiments, the sFLC is a biomarker and the treatment compound is Compound 2. In some embodiments, the sFLC is a biomarker and the treatment compound is Compound 3. Similarly, treatment with Compounds 1-3 can reduce the number of CTCs found in peripheral blood. Therefore, in some embodiments, peripheral blood CTC levels are a biomarker and the treatment compound is Compound 1. In some embodiments, peripheral blood CTC levels are a biomarker and the treatment compound is Compound 2. In some embodiments, peripheral blood CTC levels are a biomarker and the treatment compound is Compound 2. In some embodiments, peripheral blood CTC levels are a biomarker and the treatment compound is Compound 2. blood are a biomarker, and the treatment compound is Compound 3. Treatment with Compounds 1 - 3 can also reduce the amount of soluble BCMA (sBCMA) found in the serum of patients with multiple myeloma. Therefore, in some embodiments, sBCMA levels are a biomarker and the treatment compound is Compound 1. In some embodiments, sBCMA levels are a biomarker and the treatment compound is Compound 2. In some embodiments, sBCMA levels are a biomarker and the treatment compound is Connection 3.

Terminal deoxynucleotidyl transferase dUTP single-strand end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling 3'-hydroxyl

- 31 045896 ends in double-stranded DNA breaks formed during apoptosis. This is a common method known in the art (Darzynkiewicz et al., Methods, 2008, 44(3): 250-254). Thus, in some embodiments, the biomarker is a TUNEL detection of apoptosis and the treatment compound is Compound 1. In some embodiments, the biomarker is a TUNEL detection of apoptosis and the treatment compound is Compound 2. In some embodiments, the biomarker is a TUNEL detection apoptosis using TUNEL, and the therapeutic compound is Compound 3.

Apoptosis can also be measured using annexin-V and 7-AAD or annexin-V and propidium iodide (PI). Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that bind to phosphatidylserine (PS) in a calcium-dependent manner. PS is usually found only at the intracellular surface of the plasma membrane in healthy cells, but during early apoptosis the membrane asymmetry is lost and PS moves to the outer surface. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. Annexin V binding alone cannot distinguish between apoptotic and necrotic cells. To help distinguish between necrotic and apoptotic cells, 7-aminoactinomycin D (7-AAD) or PI solution can be used. Early apoptotic cells will exclude 7-AAD and PI, while late apoptotic and necrotic cells will stain positive due to the passage of these dyes into the nucleus where they bind to DNA. Thus, in some embodiments, the biomarker is an annexin V/7-AAD detection of apoptosis and the treatment compound is Compound 1. In some embodiments, the biomarker is an annexin V/7-AAD detection of apoptosis and the treatment compound is Compound 2 In some embodiments, the biomarker is an annexin V/7-AAD detection of apoptosis and the treatment compound is Compound 3. In other embodiments, the biomarker is an annexin V/PI detection of apoptosis and the treatment compound is Compound 1. In yet another In one embodiment, the biomarker is an annexin V/PI detection of apoptosis and the treatment compound is Compound 2. In some embodiments, the biomarker is an annexin V/PI detection of apoptosis and the treatment compound is Compound 3.

In some embodiments, detection of modulation of the immune response may assist in monitoring efficacy, predicting response, identifying a subject having cancer that is likely to be susceptible, treating the cancer, identifying a subject having multiple myeloma that is likely to be susceptible to a therapeutic compound, and determining or adjusting dosage for treating a subject having multiple myeloma with a therapeutic compound. For example, the examples in section 6 below describe enhanced T cell-mediated lysis of tumor cells in a co-culture model (Example 11), activation of effector T cells (Example 12), and activation and proliferation of T cells (Example 16) by therapeutic Compounds 1-3. Therefore, in some embodiments, T cell activation may be a biomarker for treatment with Compound 1. In some embodiments, T cell activation may be a biomarker for treatment with Compound 2. In some embodiments, T cell activation may be a biomarker for treatment with Compound 3.

In some specific embodiments, T cell activation may be a biomarker for treatment with a therapeutic compound, and the biomarker is selected from the group consisting of interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (ZFNy), and T clonality -cell receptor (TCR). For example, in some embodiments, the biomarker is T Cell Receptor (TCR) clonality following administration of Compounds 1-3. In some specific embodiments, TCR clonality may be a biomarker and the treatment compound is Compound 1. In some specific embodiments, TCR clonality may be a biomarker and the treatment compound is Compound 2. In some specific embodiments, TCR clonality may be a biomarker and the therapeutic compound is Compound 3.

In addition, cytokine release is central to many aspects of T cell function. For example, IL-2 is a potent T cell growth factor that is required for the long-term proliferation of activated T cells. Secretion of other cytokines, such as TNFa and IFNy, may facilitate the function of effector T cells to kill tumor cells. Therefore, cytokine detection may indicate T cell activation. In some embodiments, the biomarker is involved in T cell activation, the biomarker is IL-2, and the treatment compound is Compound 1. In some embodiments, the biomarker is involved in T cell activation, the biomarker is IL-2, and the treatment compound is is Compound 2. In some embodiments, the biomarker is involved in T cell activation, the biomarker is IL-2, and the treatment compound is Compound 3. In others

- 32 045896 embodiments, the biomarker is involved in T cell activation, the biomarker is TNFa, and the treatment compound is Compound 1. In some embodiments, the biomarker is involved in T cell activation, the biomarker is TNFa, and the treatment compound is Compound 2. In some embodiments, the biomarker is involved in T cell activation, the biomarker is TNFa, and the treatment compound is Compound 2. In some embodiments, implementation, the biomarker is involved in T cell activation, the biomarker is TNFa, and the treatment compound is Compound 3. In yet another embodiment, the biomarker is involved in T cell activation, the biomarker is IFNy, and the treatment compound is Compound 1. In some embodiments, the biomarker is involved in T cell activation, the biomarker is IFNy, and the therapeutic compound is Compound 2. In some embodiments, the biomarker is involved in T cell activation, the biomarker is IFNy, and the therapeutic compound is Compound 3.

Circulating tumor cells (CTCs) are prognostic factors in multiple myeloma, and characterization of CTCs, including identification of relevant mutations, from peripheral blood can indicate disease severity and may predict response to treatment (Mishima et al., Cell Rep., 2017, 19 (1):218-224; Lohr et al., Sci Transl Med., 2016, ;8(363):363ra147). Therefore, in some embodiments, CTCs in peripheral blood may be a biomarker and the treatment compound is Compound 1, Compound 2, or Compound 3. In some specific embodiments, CTCs in peripheral blood may be a biomarker and the treatment compound is Compound 1. B In some specific embodiments, the CTCs in the peripheral blood may be a biomarker and the treatment compound is Compound 2. In some specific embodiments, the CTCs in the peripheral blood may be a biomarker and the treatment compound is Compound 3. In other embodiments, the mutational profile of the CTCs may be a biomarker to predict response to treatment with Compound 1, Compound 2, or Compound 3. In some specific embodiments, the CTC mutational profile may be a biomarker and the treatment compound is Compound 1. In some specific embodiments, the CTC mutational profile may be a biomarker and the treatment compound is is Compound 2. In some specific embodiments, the CTC mutational profile may be a biomarker and the therapeutic compound is Compound 3.

Inhibiting cell cycle progression in a cancer cell may be an effective means of preventing cancer progression. As described in Example 6, Compound 2 can induce G1 cell cycle arrest and apoptosis in multiple myeloma cells. Detecting members of the cell cycle pathway may be important for monitoring efficacy, predicting response, identifying a subject having a cancer that is likely to be susceptible, treating the cancer, identifying a subject having multiple myeloma that is likely to be susceptible to a therapeutic compound, and determining or adjusting dosage for treating a subject having multiple myeloma with a therapeutic compound. Therefore, in some embodiments, the biomarker participates in a cascade of cell cycle reactions. In some specific embodiments, the biomarker participates in a cell cycle cascade and the therapeutic compound is Compound 1. In other specific embodiments, the biomarker participates in a cell cycle cascade and the therapeutic compound is Compound 2. In other embodiments, the biomarker participates in the cascade cell cycle reactions, and the therapeutic compound is Compound 3.

In some embodiments, the biomarker is involved in the cell cycle cascade and is selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27), and retinoblastoma protein (pRb1). In some embodiments, the biomarker is selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27), and retinoblastoma protein (pRb1), and the treatment compound is Compound 1. In other embodiments, the biomarker is selected from the group, consisting of a cyclin-dependent kinase inhibitor 1 (p21), a cyclin-dependent kinase inhibitor 1B (p27) and a retinoblastoma protein (pRb1), and the therapeutic compound is Compound 2. In other embodiments, the biomarker is selected from the group consisting of a cyclin-dependent kinase inhibitor 1 (p21) , cyclin-dependent kinase 1B inhibitor (p27) and retinoblastoma protein (pRb1), and the treatment compound is Compound 3. In certain embodiments, the biomarker is p21 and the treatment compound is Compound 1. In other embodiments, the biomarker is p21, and the treatment compound is Compound 2. In yet other embodiments, the biomarker is p21 and the treatment compound is Compound 3. In some embodiments, the biomarker is p27 and the treatment compound is Compound 1. In other embodiments, the biomarker is p27 and the treatment compound is Compound 2. In still other embodiments, the biomarker is p27 and the treatment compound is Compound 3. In certain embodiments, the biomarker is pRb1 and the treatment compound is

- 33 045896 is Compound 1. In other embodiments, the biomarker is pRbl and the treatment compound is Compound 2. In still other embodiments, the biomarker is pRb1 and the treatment compound is the compound, the biomarker is pRb1 and the treatment compound is Connection 3.

As described in the Examples in Section 6, therapeutic compounds can inhibit the proliferation of multiple myeloma cells with acquired resistance to Cereblon modulators, such as lenalidomide and pomalidomide, even if CRBN levels in the cells are reduced but detectable (Example 8). Therefore, in some embodiments, reduced CRBN levels are a biomarker for diagnosing a subject that is likely to be susceptible to a therapeutic compound, and the therapeutic compound is Compound 1. In other embodiments, reduced CRBN levels are a biomarker for diagnosing a subject that is likely to be susceptible to a therapeutic compound. compound, and the treatment compound is Compound 2. In yet another embodiment, the reduced levels of CRBN are a biomarker for diagnosing a subject who is likely to be susceptible to the treatment compound, and the treatment compound is Compound 3. In some embodiments of methods for predicting the susceptibility of a subject having multiple myeloma, to a therapeutic compound, the methods include diagnosing a subject as likely susceptible to the therapeutic compound if a biomarker in the sample is detectable. In some specific embodiments, the MM is relapsed, refractory, or resistant to conventional therapy. In one embodiment, the MM is lenalidomide-resistant MM. In another embodiment, the MM is pomalidomide-resistant MM.

Biomarkers may also be useful in determining or adjusting dosage for treating a subject having multiple myeloma with a therapeutic compound. For example, detection of an increase in a biomarker, such as IKZF1, after treatment with a therapeutic compound may indicate that the subject requires more frequent dosing or treatment over an extended period. Therefore, in some embodiments, the biomarker is intended to determine or adjust the dose for treating a subject having multiple myeloma with a therapeutic compound and is selected from the group consisting of IKZF1 and IKZF3. In specific embodiments, the biomarker is selected from the group consisting of IKZF1 and IKZF3, and the treatment compound is Compound 1. In another specific embodiment, the biomarker is selected from the group consisting of IKZF1 and IKZF3, and the treatment compound is Compound 2. In yet another embodiment in a particular embodiment, the biomarker is selected from the group consisting of IKZF1 and IKZF3, and the treatment compound is Compound 3. In some embodiments, the biomarker is IKZF1 and the treatment compound is Compound 1. In some embodiments, the biomarker is IKZF1 and the treatment the compound is Compound 2. In some embodiments, the biomarker is IKZF1 and the treatment compound is Compound 3. In some embodiments, the biomarker is IKZF3 and the treatment compound is Compound 1. In other embodiments, the biomarker is IKZF3 and the treatment compound is the biomarker compound is IKZF3 and the treatment compound is Compound 2. In still other embodiments, the biomarker is IKZF3 and the treatment compound is Compound 3.

In some embodiments, the biomarker being measured includes a single biomarker. In some embodiments, the biomarker being measured includes one or more biomarkers. In some embodiments, the biomarkers measured include two biomarkers. In some embodiments, the biomarker being measured includes two or more biomarkers. In other embodiments, the biomarkers measured include three biomarkers. In some embodiments, the biomarker being measured includes three or more biomarkers. In some embodiments, the biomarkers measured include four biomarkers. In some embodiments, the biomarker being measured includes four or more biomarkers. In other embodiments, the biomarkers measured include five biomarkers. In some embodiments, the biomarker being measured includes five or more biomarkers. In other embodiments, the biomarkers measured include six biomarkers. In some embodiments, the biomarker being measured includes six or more biomarkers. In other embodiments, the biomarkers measured include seven biomarkers. In some embodiments, the biomarker being measured includes seven or more biomarkers. In some embodiments, the biomarkers measured include eight biomarkers. In some embodiments, the biomarker being measured includes eight or more biomarkers. In other embodiments, the biomarkers measured include nine biomarkers. In some embodiments, the biomarker being measured includes nine or more biomarkers. In some embodiments, the biomarker being measured includes ten or more biomarkers.

Also provided herein are methods of treating or controlling cancer using a biomarker, for example, Aiolos, Ikaros, CRBN, ZFP91, c-MYC, IRF4, p-caspase 1, p-caspase-3, p

- 34 045896 caspase 7, cleaved PARP, survivin, BIM, sFLC, p21, p27, pRB1, soluble BCMA, CTC, TIL, IL-2, IFNy, TNFa or TCR clonality as a predictive or prognostic factor for the compounds presented herein document. In some embodiments, provided herein are methods of screening or identifying patients with multiple myeloma for treatment with a compound using the level of one or more biomarkers provided herein, e.g., Aiolos, Ikaros, CRBN, ZFP91, c-MYC, IRF4, p- caspase 1, p-caspase-3, p-caspase 7, cleaved PARP, survivin, BIM, sFLC, p21, p27, pRB1, soluble BCMA, CTC, TIL, IL-2, IFNy, TNFa or TCR clonality as a predictive or prognostic factor . In some embodiments, provided herein are methods for selecting patients who have a higher rate of response to therapy using a compound provided herein using a biomarker (eg, Aiolos, Ikaros, CRBN, ZFP91, c-MYC, IRF4, p- caspase 1, p-caspase-3, p-caspase 7, cleaved PARP, survivin, BIM, sFLC, p21, p27, pRB1, soluble BCMA, CTC, TIL, IL-2, IFNy, TNFa or TCR clonality) as a predictor or prognostic factor. In some embodiments, the treatment compound is Compound 1, Compound 2, or Compound 3. In one embodiment, the treatment compound is Compound 1. In another embodiment, the compound is Compound 2. In another embodiment, the treatment compound is Compound 3.

In one aspect, this document provides a method for identifying a subject having a cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 1:

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof

In another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 3:

.o ⁰ or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect of the present invention, a method for identifying a subject having cancer that

- 35 045896 likely to be susceptible to the therapeutic compound that was administered to the subject includes:

(a) determining the level of a biomarker in a sample obtained from the subject; and (b) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; wherein the therapeutic compound is Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of biomarker in the sample; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker;

wherein the therapeutic compound is Compound 1:

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of biomarker in the sample; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker;

wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level;

wherein the therapeutic compound is Compound 3:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to a sample obtained from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level; wherein the therapeutic compound is Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

In another aspect, when a subject is diagnosed as likely to be susceptible to a therapeutic compound, the methods provided herein further include administering a therapeutically effective amount of the therapeutic compound to the subject.

Thus, in some embodiments, provided herein is a method of treating cancer, comprising:

- 36 045896 (a) obtaining a sample from a subject with cancer;

(b) determining the level of biomarker in the sample;

(c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 1:

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of biomarker in the sample;

(c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of biomarker in the sample;

(c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 3:

.o ⁰ or tautomer, isotopologue or pharmaceutically acceptable salt.

In some embodiments, provided herein is Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof for use in a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of biomarker in the sample;

(c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of the therapeutic compound.

In some embodiments of the various methods provided herein, the method includes or further includes the step of administering a therapeutic compound. In other embodiments, provided herein are methods for selectively treating a patient selected using the methods provided herein.

One embodiment of the methods provided herein involves treating a patient with multiple myeloma who is selected based on the biomarkers described herein, comprising administering to the patient Compound 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

Another embodiment of the methods provided herein further comprises a method of treating a patient with multiple myeloma that is selected based on the level of biomarkers described herein, comprising administering to the patient Compound 2 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

In yet another embodiment, the methods provided herein further include a method of treating a patient with multiple myeloma that is selected based on the level of bio

- 37 045896 markers described herein, comprising administering to a patient Compound 3 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

Another embodiment of the methods provided herein further includes a method for preventing multiple myeloma in a patient selected based on the level of biomarkers described herein, which includes administering to the patient a compound provided herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, the methods provided herein further include a method of treating multiple myeloma based on the level of biomarkers described herein, which includes administering to a patient a compound provided herein, for example, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

One embodiment of the methods presented herein further includes methods for inducing a therapeutic response assessed using the International Uniform Response Criteria for Multiple Myeloma (IURC) (see Durie BG, et al., Leukemia, 2006, 20(9): 146773 ) in a patient based on the level of biomarkers described herein, comprising administering an effective amount of a compound described herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof to the patient with multiple myeloma. In another embodiment, provided herein are methods of achieving strict complete remission, complete remission, or very good partial response as assessed by International Uniform Response Criteria for multiple myeloma (IURC) in a patient, comprising administering an effective amount of a compound described herein, e.g. , Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof to a patient suffering from multiple myeloma. In another embodiment, provided herein are methods of achieving an increase in overall survival, progression-free survival, event-free survival, time to disease progression, or disease-free survival in a patient, comprising administering an effective amount of a compound described herein, e.g., Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof to a patient suffering from multiple myeloma. In another embodiment, provided herein are methods of achieving an increase in overall survival in a patient, comprising administering an effective amount of a compound described herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically thereof. acceptable salt to a patient suffering from multiple myeloma. In another embodiment, provided herein are methods of achieving increased progression-free survival in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically thereof. acceptable salt to a patient suffering from multiple myeloma. In another embodiment, provided herein are methods of achieving increased event-free survival in a patient, comprising administering an effective amount of a compound described herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable compound thereof. salt to a patient suffering from multiple myeloma. In another embodiment, provided herein are methods of achieving an increase in time to disease progression in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt to a patient suffering from multiple myeloma. In another embodiment, provided herein are methods of achieving increased disease-free survival in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt to a patient suffering from multiple myeloma.

Also provided herein are methods further comprising treating patients previously treated for multiple myeloma who have not responded to standard therapeutic agents, as well as treatment-naïve patients. Also included are methods of treating patients who have undergone surgery for the treatment of multiple myeloma, as well as patients who have not undergone surgery. This document also proposes treatments for patients who have previously undergone organ transplantation, as well as patients who have not previously undergone such treatment.

In some embodiments, the methods provided herein additionally

- 38 045896 include the treatment of multiple myeloma that is relapsed, refractory or resistant. Methods provided herein include the prevention of multiple myeloma that is relapsed, refractory or refractory. The methods provided herein include the control of multiple myeloma that is relapsed, refractory or resistant. In some such embodiments, the myeloma is single-, double-, triple-, quadruple-, or quintuple-relapsed multiple myeloma. In one embodiment, the methods provided herein reduce, maintain or eliminate minimal residual disease (MRD). In one embodiment, the methods provided herein include the treatment, prevention or control of various types of multiple myeloma, such as monoclonal gammopathy of unknown origin (MGUS); low-risk, intermediate-risk and high-risk multiple myeloma; newly diagnosed multiple myeloma (including newly diagnosed low-risk, intermediate-risk and high-risk multiple myeloma); multiple myeloma with and without the possibility of transplantation; smoldering (slowly progressive) multiple myeloma (including low-risk, intermediate-risk and high-risk smoldering multiple myeloma); active multiple myeloma; solitary plasmacytoma; extramedullary plasmacytoma; plasma cell leukemia; multiple myeloma of the central nervous system; light chain myeloma; nonsecretory myeloma; immunoglobulin D myeloma; and immunoglobulin E myeloma, by administering a therapeutically effective amount of a compound described herein. In another embodiment, the methods provided herein include the treatment, prevention or control of multiple myeloma characterized by genetic abnormalities such as cyclin D translocation (e.g., t(411;14)(q13;q32); t(6;14) (p21;32); t(12;14)(p13;q32); or t(6;20);); MMSET translocations (e.g. t(4;14)(p16;q32)); MAF translocations (e.g. t(14;16)(q32;q32); t(20;22); t(16; 22)(q11;q13); or t(14;20)(q32;q11)); or other chromosomal factors (eg, deletion of 17p13, or chromosome 13; del(17/17p), non-hyperploidy and gain(1q)), by administering a therapeutically effective amount of a compound described herein. In one embodiment, these methods include administering a therapeutically effective amount of Compound 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof. In another embodiment, the methods include administering a therapeutically effective amount of Compound 2 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof. In another embodiment, the methods comprise administering a therapeutically effective amount of Compound 3 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

In one embodiment, high-risk multiple myeloma is multiple myeloma that has relapsed within 12 months of the first course of treatment. In yet another embodiment, high-risk multiple myeloma is multiple myeloma that is characterized by genetic abnormalities, for example, one or more of del(17/17p) and t(14; 16)(q32;q32).

In some such embodiments, the multiple myeloma is a newly diagnosed transplantable multiple myeloma. In another embodiment, the multiple myeloma is newly diagnosed multiple myeloma without the possibility of transplantation.

In yet other embodiments, multiple myeloma is characterized by early progression (eg, less than 12 months) after initial treatment. In yet other embodiments, multiple myeloma is characterized by early progression (eg, less than 12 months) after autologous stem cell transplantation. In another embodiment, multiple myeloma is refractory to pomalidomide. In some such embodiments, multiple myeloma is predicted to be refractory to pomalidomide (eg, by molecular characteristic). In another embodiment, the multiple myeloma is relapsed or refractory to 3 or more treatments and has been exposed to a proteasome inhibitor (eg, bortezomib, carfilzomib, ixazomib, oprosomib, or marizomib) and an immunomodulatory compound (eg, thalidomide, lenalidomide, and pomalidomide), or is double refractory to proteasome inhibitor and immunomodulatory compound. In still other embodiments, the multiple myeloma is relapsed or refractory to 3 or more treatments, including, for example, a CD38 monoclonal antibody (CD38 mAb, e.g., daratumumab or isatuximab), a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixazomib, or marizomib), and an immunomodulatory compound (eg, thalidomide, lenalidomide, and pomalidomide), or dual refractory to a proteasome inhibitor or immunomodulatory compound and a CD38 mAb. In subsequent other embodiments, the multiple myeloma is triple refractory, e.g., the multiple myeloma is refractory to a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixazomib, oprosomib, or marizomib) and an immunomodulatory compound (e.g., thalidomide, lenalidomide, and pomalidomide) and one other agent

- 39 045896 tive agent as described herein.

Some embodiments of the methods provided herein further include methods of treating, preventing and/or controlling multiple myeloma, including relapsed/refractory multiple myeloma in patients with impaired renal function or symptom thereof, which include administering a therapeutically effective amount of Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof to a patient with impaired renal function having relapsed/refractory multiple myeloma.

Some embodiments of the methods provided herein further include methods for treating, preventing, and/or controlling multiple myeloma, including relapsed or refractory multiple myeloma in frail patients or a symptom thereof, which includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof to a frail patient having multiple myeloma. In some such embodiments, the frail patient is characterized by failure of induction therapy or intolerance to dexamethasone treatment. In some such embodiments, the frail patient is of advanced age, such as over 65 years of age.

In some embodiments, the therapeutically or prophylactically effective amount of the compound is from about 0.01 to about 25 mg per day, from about 0.01 to about 10 mg per day, from about 0.01 to about 5 mg per day, from about 0 .01 to about 2 mg per day, from about 0.01 to about 1 mg per day, from about 0.01 to about 0.5 mg per day, from about 0.01 to about 0.25 mg per day, from about 0.1 to about 25 mg per day, about 0.1 to about 10 mg per day, about 0.1 to about 5 mg per day, about 0.1 to about 2 mg per day, about 0 .1 to about 1 mg per day, from about 0.1 to about 0.5 mg per day, from about 0.1 to about 0.25 mg per day, from about 0.5 to about 25 mg per day, from about 0.5 to about 10 mg per day, about 0.5 to about 5 mg per day, about 0.5 to about 2 mg per day, about 0.5 to about 1 mg per day, about 1 up to about 25 mg per day, from about 1 to about 10 mg per day, from about 1 to about 5 mg per day, from about 1 to about 2.5 mg per day, or from about 1 to about 2 mg per day. In one embodiment, a therapeutically or prophylactically effective amount of Compound 1, Compound 2, or Compound 3 is from about 0.1 mg per day to about 0.4 mg per day.

In some embodiments, the therapeutically or prophylactically effective amount is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8 , about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20 or about 25 mg per day. In some such embodiments, the therapeutically or prophylactically effective amount is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, or about 0.7 mg per day.

In one embodiment, the recommended daily dosage range of Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof under the conditions described herein is from about 0.1 to about 25 mg per day, preferably as a single dose once daily or in divided doses throughout the day. In other embodiments, dosage ranges are from about 0.1 to about 10 mg per day. Specific doses per day include 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg per day. More specific dosages per day include 0.1, 0.2, 0.3, 0.4, or 0.5 mg per day.

In a specific embodiment, the recommended starting dose may be 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, or 25 mg per day. In another embodiment, the recommended starting dose may be 0.1, 0.2, 0.3, 0.4 or 0.5 mg per day. The dose can be increased to 1, 2, 3, 4 or 5 mg per day.

In specific embodiments, the therapeutically or prophylactically effective amount is from about 0.001 to about 5 mg/kg/day, from about 0.001 to about 4 mg/kg/day, from about 0.001 to about 3 mg/kg/day, from about 0.001 to about 2 mg/kg/day, about 0.001 to about 1 mg/kg/day, 0.001 to about 0.05 mg/kg/day, about 0.001 to about 0.04 mg/kg/day, about 0.001 to about 0.03 mg/kg/day, about 0.001 to about 0.02 mg/kg/day, about 0.001 to about 0.01 mg/kg/day, or about 0.001 to about 0.005 mg/kg/day.

The administered dose may also be expressed in units other than mg/kg/day. For example, doses for parenteral administration may be expressed in mg/m/day. One skilled in the art will readily determine how to convert doses from mg/kg/day to mg/ ^m2 /day based on the subject's height or weight, or both (see www.fda.gov/cder/cancer/animalframe.htm) . For example, a dose of 1 mg/kg/day for a 65 kg person is approximately 38 mg/ ^m2 /day.

- 40 045896

In some embodiments, the patient to be treated with one of the methods provided herein has not received therapy against multiple myeloma prior to administration of Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt. In some embodiments, a patient to be treated with one of the methods provided herein has received therapy against multiple myeloma prior to administration of Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutical thereof. acceptable salt. In some embodiments, a patient being treated with one of the methods provided herein has developed drug resistance to multiple myeloma therapy. In other such embodiments, the patient has developed resistance to one, two, or three anti-multiple myeloma treatments selected from the group consisting of a CD38 monoclonal antibody (CD38 mAb, e.g., daratumumab or isatuximab), a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixazomib, or marizomib) and an immunomodulatory compound (eg thalidomide, lenalidomide and pomalidomide).

The methods proposed herein include treating a patient, regardless of the patient's age. In some embodiments, the subject is 18 years of age or older. In other embodiments, the subject's age is greater than 18, 25, 35, 40, 45, 50, 55, 60, 65, or 70 years. In other embodiments, the subject is at least 65 years of age. In other embodiments, the subject is no more than 65 years of age. In one embodiment, the subject is an elderly subject with multiple myeloma, such as a subject over 65 years of age. In one embodiment, the subject is an elderly subject with multiple myeloma, such as a subject over 75 years of age.

Depending on the condition of the disease being treated and the condition of the subject, Compound 1 or Compound 2 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, can be administered orally, parenterally (e.g., intramuscularly, intraperitoneal, intravenous, IV, intracisternal injection or infusion, subcutaneous injection or implant), by inhalation, nasal, vaginal, rectal, sublingual or topical (eg, transdermal or topical) route of administration. Compound 1, Compound 2 or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof may be formulated, alone or together, into a suitable unit dosage form with pharmaceutically acceptable excipients, carriers, adjuvants and liquid media suitable for each route of administration.

In one embodiment, Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, is administered orally. In another embodiment, Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, is administered parenterally. In yet another embodiment, Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof is administered intravenously.

Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, can be delivered in a single dose, such as, for example, a single bolus injection or oral tablets or pills; or over a specified period of time, for example, by continuous infusion over a specified time or in divided bolus doses over a specified time. The compounds described herein may be administered multiple times as necessary, for example, until the patient's disease is stabilized or regressed, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or absence thereof is determined by methods known in the art, such as assessment of the patient's symptoms, physical examination, imaging of the tumor by X-ray, CT, PET or MRI, and other conventional assessment methods.

Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, may be independently administered once daily (QD) or may be divided into multiple daily doses, such as for dosing twice daily (BID), three times daily (TID) and four times daily (QID). In addition, administration can be continuous (ie, daily for several days in a row or every day), intermittent, for example, in cycles (ie, including days, weeks or months of rest without drug). As used herein, the term daily means that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof is administered one or more times per day, for example, over a period of time. The term continuous means

- 41 045896 expects that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof is administered daily for a continuous period of at least from 7 days to 52 weeks. The term periodic or periodic as used herein means stopping and starting at regular or irregular intervals. For example, periodic administration of Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, is administration for one to six days per week, administration in cycles (e.g. , daily administration for two to eight consecutive weeks, followed by a rest period without administration for up to one week) or administration every other day. As used herein, the term cyclic means that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or a pharmaceutically acceptable salt thereof is administered daily or continuously, but with a rest period. In some such embodiments, administration is performed once daily for two to six days, followed by a rest period without administration for five to seven days.

In some embodiments, the frequency of administration ranges from about a daily dose to a monthly dose. In specific embodiments, administration is once daily, twice daily, three times daily, four times daily, once daily, twice weekly, once weekly, once every two weeks, once every three weeks or once every four weeks. In one embodiment, Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, is administered once daily. In another embodiment, Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, is administered twice daily. In yet another embodiment, Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, is administered three times daily. In yet another embodiment, Compound 1, Compound 2, or Compound 3 as provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or a pharmaceutically acceptable salt thereof, is administered four times daily.

In one embodiment, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered in a treatment cycle that includes an administration period of no more than 20 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered in a treatment cycle that includes an administration period of no more than 15 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered in a treatment cycle that includes an administration period of no more than 10 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered in a treatment cycle that includes an administration period of no more than 7 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered in a treatment cycle that includes an administration period of no more than 5 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered in a treatment cycle that includes an administration period of no more than 4 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered in a treatment cycle that includes an administration period of no more than 3 days, followed by a rest period.

In one embodiment, the treatment cycle includes an administration period of no more than 14 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of no more than 10 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of no more than 7 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of no more than 5 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of no more than 4 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of no more than 3 days, followed by a rest period.

In one embodiment, the rest period is from about 2 days to about 11 days. In one embodiment, the rest period is from about 2 days to about 10 days. In one embodiment, the rest period is approximately 2 days. In one embodiment, the rest period is approximately 3 days. In one embodiment, the rest period is approximately 4 days. In one embodiment, the rest period is approximately 5 days. In one embodiment, the rest period is approximately 6 days. In a different

- 42 045896 embodiment, the rest period is approximately 7 days. In another embodiment, the rest period is approximately 8 days. In another embodiment, the rest period is approximately 9 days. In another embodiment, the rest period is approximately 10 days. In another embodiment, the rest period is approximately 11 days.

In one embodiment, the treatment cycle includes an administration period of no more than 15 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of no more than 10 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of no more than 7 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of no more than 5 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of no more than 3 days, followed by a rest period of about 10 days to about 15 days. In one embodiment, the treatment cycle includes an administration period of no more than 3 days, followed by a rest period of about 3 days to about 15 days.

In one embodiment, the treatment cycle includes an administration period of no more than 15 days, followed by a rest period of 7 days. In one embodiment, the treatment cycle includes an administration period of no more than 10 days, followed by a rest period of 5 days. In one embodiment, the treatment cycle includes an administration period of no more than 10 days, followed by a rest period of 4 days. In one embodiment, the treatment cycle includes an administration period of no more than 10 days, followed by a rest period of 3 days. In one embodiment, the treatment cycle includes an administration period of no more than 10 days, followed by a rest period of 2 days. In one embodiment, the treatment cycle includes an administration period of no more than 7 days, followed by a rest period of 7 days. In one embodiment, the treatment cycle includes an administration period of no more than 5 days, followed by a rest period of 5 days. In one embodiment, the treatment cycle includes an administration period of no more than 3 days, followed by a rest period of 11 days. In another embodiment, the treatment cycle includes an administration period of no more than 5 days, followed by a rest period of 9 days. In another embodiment, the treatment cycle includes an administration period of no more than 5 days, followed by a rest period of 2 days. In another embodiment, the treatment cycle includes an administration period of no more than 3 days, followed by a rest period of 4 days.

In one embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-5 of a 28-day cycle. In another embodiment, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 on days 1-10 of a 28-day cycle. In one embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-21 of a 28-day cycle. In another embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-5 of a 7-day cycle. In another embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-7 of a 7-day cycle. In one embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-10 and days 15-24 of a 28-day cycle. In one embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-3 and days 15-18 of a 28-day cycle. In one embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-7 and days 15-21 of a 28-day cycle. In one embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-5 and days 15-19 of a 28-day cycle. In one embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-3 and days 15-17 of a 28-day cycle.

In one embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-14 of a 21-day cycle. In another embodiment, the treatment cycle includes administration of Compound 1, Compound 2 or Compound 3 on days 1-4 and 8-11 of a 21-day cycle. In one embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-5 and 812 of a 21-day cycle. In another embodiment, a treatment cycle comprises administering a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1-5 and 11-15 of a 21-day cycle. In another embodiment, a treatment cycle comprises administering a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1-5 and 8-12 and 15-19 of a 21-day cycle. In another embodiment, the treatment cycle includes administering a therapeutically effective

- 43 045896 effective amount of Compound 1, Compound 2 or Compound 3 on days 1-4 and 8-11 and 15-18 of a 21-day cycle. In another embodiment, a treatment cycle comprises administering a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1-4 and 8-10 and 15-17 of a 21-day cycle. In another embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-3 and 8-11 of a 21-day cycle. In another embodiment, a treatment cycle includes administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1-3 and 11-13 of a 21-day cycle.

Any treatment cycle described herein may be repeated for a number of cycles of at least 2, 3, 4, 5, 6, 7, 8 or more. In some cases, the treatment cycle described herein comprises from 1 to about 24 cycles, from about 2 to about 16 cycles, or from about 2 to about 4 cycles. In some cases, the treatment cycle described herein includes from 1 to about 4 cycles. In some embodiments, cycles 1-4 are all 28 day cycles. In some embodiments, a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 is administered over 1 to 13 cycles of 28 days (eg, over about 1 year). In some cases, cyclic therapy is not limited by the number of cycles, and therapy is continued until the disease progresses. Cycles in some cases may include varying lengths of administration periods and/or rest periods described herein.

In one embodiment, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 at dosage levels of about 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, 0.4 mg/day, 0.5 mg/day, 0.6 mg/day, 0.7 mg/day, 0.8 mg/day, 0.9 mg/day, 1.0 mg/day, 5.0 mg/day or 10 mg/day, when administered once daily. In one embodiment, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 at dosage levels of about 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, 0.4 mg/day, 0.5 mg/day, 0.6 mg/day, 0.7 mg/day, or 0.8 mg/day, administered once daily. In some such embodiments, the treatment cycle includes administering Compound 1, Compound 2, or Compound 3 times daily at a dosage level of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or 0.5 mg. , on days 1-10 of a 28-day cycle. In some such embodiments, the treatment cycle includes administering Compound 1, Compound 2, or Compound 3 times daily at a dosage level of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or 0.5 mg. , on days 1-10 and 15-24 of a 28-day cycle. In some such embodiments, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 times daily at a dosage level of about 0.1 mg on days 1-10 and 15-24 of a 28-day cycle. In other embodiments, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 twice daily at a dosage level of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or 0.5 mg, on days 1-3 of a 28-day cycle. In other embodiments, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 twice daily at a dosage level of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or 0.5 mg, on days 1-3 and 15-19 of a 28-day cycle. In other embodiments, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 twice daily at a dosage level of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or 0.5 mg, on days 1-3 and 15-17 of a 28-day cycle. In other embodiments, the treatment cycle includes administration of Compound 1, Compound 2, or Compound 3 twice daily at a dosage level of approximately 0.2 mg on days 1-3 and 15-17 of a 28-day cycle. In one such embodiment, the compound is administered on days 1 to 3 (morning and evening), day 14 (evening only), days 15 and 16 (morning and evening), and day 17 (morning only) in Cycle 1.

In some embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control biomarker level;

wherein the therapeutic compound is Compound 1:

O

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In other embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy, who is likely to be responsive to a therapeutic compound, including

- 44 045896 tea:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control biomarker level;

wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control biomarker level;

wherein the therapeutic compound is Compound 3:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control level of the biomarker; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for predicting the responsiveness of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control biomarker level;

wherein the therapeutic compound is Compound 1:

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control biomarker level;

wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

- 45 045896

In yet another embodiment, a method of predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control biomarker level;

wherein the therapeutic compound is Compound 3:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for predicting the responsiveness of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is lower than the control level of the biomarker; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

It is understood that the dosing schedule can be adjusted depending on the patient's response or lack thereof according to the biomarker level. For example, the dose may be adjusted if the patient is neutropenic or the patient is unresponsive. Therefore, in some embodiments, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from a subject at different points in time;

(c) determining the level of a biomarker in one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 1:

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from a subject at different points in time;

(c) determining the level of a biomarker in one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from a subject at different points in time;

(c) determining the level of a biomarker in one or more samples and thereby determining whether the dose is appropriate or requires adjustment; wherein the therapeutic compound is Compound 3:

- 46 045896

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, wherein the compound is administered to the subject, comprising:

(a) determining the level of a biomarker in one or more samples obtained from the subject, and thereby determining whether the dose is appropriate or requires adjustment; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, the various time points may include a control sample obtained prior to treatment with the therapeutic compound and one or more samples obtained from the subject at different times during treatment with the therapeutic compound. In other embodiments, the various time points may include a control sample obtained during treatment and one or more samples obtained later during treatment with the treatment compound. In yet another embodiment, the different time points may include a control sample taken before treatment and a sample taken after treatment.

Also provided herein are methods for predicting or monitoring a patient's responsiveness to a therapeutic compound or the effectiveness of a therapeutic compound using a biomarker (e.g., Aiolos, Ikaros, CRBN, ZFP91, c-MYC, IRF4, β-caspase 1, β-caspase-3, β -caspase 7, cleaved PARP, survivin, BIM, sFLC, p21, p27, pRB1, soluble BCMA, CTC, TIL, IL-2, IFNy, TNFa or TCR clonality). In some embodiments, provided herein are methods for predicting the susceptibility of a subject having or suspected of having cancer (eg, multiple myeloma) to a therapeutic compound using a predictive or prognostic factor such as Aiolos, Ikaros, CRBN, ZFP91, c-MYC , IRF4, p-caspase 1, p-caspase3, p-caspase 7, cleaved PARP, survivin, BIM, sFLC, p21, p27, pRB1, soluble BCMA, CTC, TIL, IL-2, IFNy, TNFa or TCR clonality. In some embodiments, provided herein are methods for monitoring the effectiveness of treatment of a cancer (e.g., multiple myeloma) in a subject with a therapeutic compound using a biomarker level (e.g., Aiolos, Ikaros, CRBN, ZFP91, c-MYC, IRF4, p-caspase 1 , p-caspase-3, p-caspase 7, cleaved PARP, survivin, BIM, sFLC, p21, p27, pRB1, soluble BCMA, CTC, TIL, IL-2, IFNy, TNFa or TCR clonality) as a prognostic or prognostic factor . In some embodiments, the treatment compound is Compound 1, Compound 2, or Compound 3. In one embodiment, the treatment compound is Compound 1. In another embodiment, the compound is Compound 2. In another embodiment, the treatment compound is Compound 3.

Thus, in some aspects, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 1:

O

or an enantiomer, mixture of enantiomers, tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In another aspect, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

- 47 045896 wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 3:

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject; and (b) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 1:

or an enantiomer, mixture of enantiomers, tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In yet another aspect, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample;

wherein the therapeutic compound is Compound 2:

or a tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In yet another aspect, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of the biomarker in the sample; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level

- 48 045896 the level of biomarker in the sample differs from the level of biomarker obtained from the control sample; wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In yet another aspect, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to a sample obtained from the subject;

(b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In some embodiments of the various methods provided herein, the level of the biomarker in the sample is higher than the level of the biomarker obtained from the control sample. In other embodiments of the various methods provided herein, the level of the biomarker in the sample is lower than the level of the biomarker obtained from the control sample.

In yet another aspect, provided herein is a method for monitoring the effectiveness of treating cancer in a subject with a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In yet another aspect, provided herein is a method for monitoring the effectiveness of treating cancer in a subject with a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject; wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In yet another aspect, provided herein is a method for monitoring the effectiveness of treating cancer in a subject with a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 3

- 49 045896

or a tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In yet another aspect, provided herein is a method for monitoring the effectiveness of treating cancer in a subject with a therapeutic compound that was administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject; and (b) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating the cancer in the subject;

wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3 or a tautomer, isotopologue, pharmaceutically acceptable salt or polymorph thereof.

In some embodiments, an increased level compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject. For example, increased p-caspase-3, p-caspase 1, p-caspase 7, cleaved PARP, BIM, TUNEL, Ah^^u^-V/T-AAD, Annexin-V/PI, p21, p27, free lung chains in serum, IL-2, TNFa, IFNy or TCR clonality relative to control indicates the effectiveness of the therapeutic compound in treating multiple myeloma in the subject. In other embodiments, a reduced level compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject. For example, a decrease in Aiolos (IKZF3), Ikaros (IKZF1), CTC, ZFP91, c-MYC, IRF4, phospho-Rbl compared to control indicates the effectiveness of the therapeutic compound in treating multiple myeloma in the subject.

It is assumed that the biomarker level will be relative to the reference level. Thus, in some embodiments, a reduced level compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject. For example, a decrease in p-caspase-3, p-caspase 1, p-caspase 7, cleaved PARP, BIM, TUNEL, Annexin-I-T-LLG), Annexin-V/PI, p21, p27, free light chain in serum, IL- 2, TNFa, IFNy or TCR clonality from a healthy patient compared to a control from a patient with multiple myeloma indicates the effectiveness of the therapeutic compound in treating multiple myeloma in the subject. In other embodiments, an increased level compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject. For example, an increase in Aiolos (IKZF3), Ikaros (IKZF1), CTC, ZFP91, c-MYC, IRF4, phospho-Rbl in a healthy subject compared to a control from a multiple myeloma patient indicates the effectiveness of the therapeutic compound in treating multiple myeloma in the subject.

As described herein, a method is provided for reducing, treating and/or preventing adverse or undesirable events associated with conventional therapy, including, but not limited to, surgery, chemotherapy, radiation therapy, biological therapy and immunotherapy. A compound provided herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt and other active ingredient thereof, can be administered to a patient identified using the biomarkers described herein before , during or after the occurrence of an adverse event associated with conventional therapy.

Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof, may also be combined or used in combination with other therapeutic agents useful for the treatment and/or prevention of multiple myeloma described in this document.

In some embodiments of the various methods presented herein, the method includes administering to a patient Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof in combination with one or more second active agents and optionally in combination with radiation therapy, blood transfusion or surgery.

As used herein, the term combination includes the use of more than one therapy (eg, one or more prophylactic and/or therapeutic agents). However, use of the term in combination does not limit the order in which therapeutic agents (eg, prophylactic and/or therapeutic agents) are administered to a patient suffering from a disease or disorder. The first therapy (e.g., a prophylactic or therapeutic agent, such as a compound provided herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof) may be administered before (e.g. , in 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks), at the same time

- 50 045896 immediately with or after (for example, after 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) of administering a second therapy (eg, prophylactic or therapeutic agent) to the subject. Triple therapy is also discussed in this document, as is quadruple therapy. In one embodiment, the second therapeutic agent is dexamethasone.

Administration of Compound 1, Compound 2, or Compound 3, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof, and one or more second active agents to a patient can be accomplished simultaneously or sequentially by one or different routes of administration. The suitability of a particular route of administration used for a particular active agent depends on the active agent itself (eg, whether it can be administered orally without degrading before entering the bloodstream).

The route of administration of Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof is independent of the route of administration of the second therapeutic agent. In one embodiment, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, is administered orally. In another embodiment, Compound 1,

Compound 2 or Compound 3 is administered intravenously. Thus, in these embodiments, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof is administered orally or intravenously, and the second therapy may be administered orally, parenterally, intraperitoneally, intravenously, intra-arterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, liposomal, inhalation, vaginal, intraocular, local delivery via catheter or stent, subcutaneous, intra-adiposal, intra-articular, intrathecal or slow-release dosage form. In one embodiment, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, and the second therapy are administered by the same route of administration, orally or IV. In another embodiment, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof is administered by one route of administration, e.g., IV administration, while the second agent (an anti-multiple myeloma agent) is administered in another way, for example orally.

In one embodiment, the second active agent is administered intravenously or subcutaneously and once or twice daily in an amount of from about 1 to about 1000 mg, from about 5 to about 500 mg, from about 10 to about 350 mg, or from about 50 to about 200 mg. The specific amount of the second active agent will depend on the particular agent used, the type of multiple myeloma being treated or controlled, the severity and stage of the disease, and the amount of Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutical thereof acceptable salt provided herein, and any optional additional active agents simultaneously administered to the patient.

The methods and compositions provided herein may use one or more of the second active ingredients or agents together with Compound 1, Compound 2, or Compound 3. The second active agents may be high molecular weight compounds (eg, proteins), small molecules (eg, , synthetic inorganic, organometallic, or organic molecules) or cell therapies (eg, CAR cells).

Examples of second active agents that may be used in the methods and compositions described herein include one or more of the list including melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin, bendamustine, a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixazomib, oprosomib or marizomib), histone deacetylase inhibitor (eg, panobinostat, ACY241), BET inhibitor (eg, GSK525762A, OTX015, BMS-986158, TEN-010, CPI0610, INCB54329, BAY1238097, FT-1101, C90010, ABBV-075, BI 894999 Inhibitor BCL2 (eg, venetoclax or navitoclax), MCL-1 inhibitor (eg, AZD5991, AMG176, MIK665, S64315, or S63845), corticosteroid (eg, prednisone), dexamethasone; an antibody (eg, a CS1 antibody such as elotuzumab; a CD38 antibody such as daratumumab, isatuximab; or a BCMA antibody or antibody conjugate such as GSK2857916 or BI 836909), a checkpoint inhibitor (as described herein), or CAR cells (as described in this document).

In one embodiment, the second active agent used with Compound 1, Compound 2, or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof in the methods and compositions described herein is dexamethasone.

In some embodiments, dexamethasone is administered at a dosage of 4 mg on days 1 and 8 of a 21-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 4 mg

- 51 045896 on days 1, 4, 8 and 11 of the 21-day cycle. In some embodiments, dexamethasone is administered at a dosage of 4 mg on days 1, 8, and 15 of a 28-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 4 mg on days 1, 4, 8, 11, 15, and 18 of a 28-day cycle. In some embodiments, dexamethasone is administered at a dosage of 4 mg on days 1, 8, 15, and 22 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 4 mg on days 1, 10, 15, and 22 of Cycle 1. In some embodiments, dexamethasone is administered at a dosage of 4 mg on days 1, 3, 15, and 17 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 4 mg on days 1, 3, 14, and 17 of Cycle 1.

In some other embodiments, dexamethasone is administered at a dosage of 8 mg on days 1 and 8 of a 21-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 8 mg on days 1, 4, 8, and 11 of a 21-day cycle. In some embodiments, dexamethasone is administered at a dosage of 8 mg on days 1, 8, and 15 of a 28-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 8 mg on days 1, 4, 8, 11, 15, and 18 of a 28-day cycle. In some embodiments, dexamethasone is administered at a dosage of 8 mg on days 1, 8, 15, and 22 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 8 mg on days 1, 10, 15, and 22 of Cycle 1. In some embodiments, dexamethasone is administered at a dosage of 8 mg on days 1, 3, 15, and 17 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 8 mg on days 1, 3, 14, and 17 of Cycle 1.

In some embodiments, dexamethasone is administered at a dosage of 10 mg on days 1 and 8 of a 21-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 10 mg on days 1, 4, 8, and 11 of a 21-day cycle. In some embodiments, dexamethasone is administered at a dosage of 10 mg on days 1, 8, and 15 of a 28-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 10 mg on days 1, 4, 8, 11, 15, and 18 of a 28-day cycle. In some embodiments, dexamethasone is administered at a dosage of 10 mg on days 1, 8, 15, and 22 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 10 mg on days 1, 10, 15, and 22 of Cycle 1. In some embodiments, dexamethasone is administered at a dosage of 10 mg on days 1, 3, 15, and 17 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 10 mg on days 1, 3, 14, and 17 of Cycle 1.

In some embodiments, dexamethasone is administered at a dosage of 20 mg on days 1 and 8 of a 21-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 20 mg on days 1, 4, 8, and 11 of a 21-day cycle. In some embodiments, dexamethasone is administered at a dosage of 20 mg on days 1, 8, and 15 of a 28-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 20 mg on days 1, 4, 8, 11, 15, and 18 of a 28-day cycle. In some embodiments, dexamethasone is administered at a dosage of 20 mg on days 1, 8, 15, and 22 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 20 mg on days 1, 10, 15, and 22 of Cycle 1. In some embodiments, dexamethasone is administered at a dosage of 20 mg on days 1, 3, 15, and 17 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 20 mg on days 1, 3, 14, and 17 of Cycle 1.

In some embodiments, dexamethasone is administered at a dosage of 40 mg on days 1 and 8 of a 21-day cycle. In some other embodiments, dexamethasone is administered at a dosage of 40 mg on days 1, 4, 8, and 11 of a 21-day cycle. In some embodiments, dexamethasone is administered at a dosage of 40 mg on days 1, 8, and 15 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 40 mg on days 1, 10, 15, and 22 of Cycle 1. In certain other embodiments, dexamethasone is administered at a dosage of 40 mg on days 1, 4, 8, 11, 15, and 18 of a 28-day period. cycle. In other such embodiments, dexamethasone is administered at a dosage of 40 mg on days 1, 8, 15, and 22 of a 28-day cycle. In other such embodiments, dexamethasone is administered at a dosage of 40 mg on days 1, 3, 15, and 17 of a 28-day cycle. In one such embodiment, dexamethasone is administered at a dosage of 40 mg on days 1, 3, 14, and 17 of Cycle 1.

In another embodiment, the second active agent used together with Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof in the methods and compositions described herein is bortezomib. In yet another embodiment, the second active agent used together with Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof in the methods and compositions described herein is daratumumab. In some such embodiments, the methods further comprise administering dexamethasone. In some embodiments, these methods include administering Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, together with a proteasome inhibitor as described herein, with a CD38 inhibitor as described herein document, and a corticosteroid as described herein.

In some embodiments, Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof is administered to

- 52 045896 combinations with checkpoint inhibitors. In one embodiment, a single checkpoint inhibitor is used in combination with Compound 1, or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof with respect to the methods presented herein. In another embodiment, two checkpoint inhibitors are used in combination with Compound 1, or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof with respect to the methods presented herein. In yet another embodiment, three or more checkpoint inhibitors are used in combination with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof with respect to the methods presented herein.

As used herein, the term immune checkpoint inhibitor or checkpoint inhibitor refers to molecules that completely or partially reduce, inhibit, interfere with, or modulate one or more checkpoint proteins. Without being limited by theory, checkpoint proteins regulate T cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86 and PD-1 with its ligands PD-LI and PD-P2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). These proteins are responsible for costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins are responsible for regulating and maintaining self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

In one embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in US Pat. No. 5,811,097; 5811097; 5855887; 6051227; 6207157; 6682736; 6984720 and 7605238, all of which are incorporated herein by reference in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675206). In another embodiment, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is marketed under the trade name Yervoy™.

In one embodiment, the checkpoint inhibitor is a PDT/PD-LI inhibitor. Examples of PD-1/PD-L1 inhibitors include, but are not limited to, those described in US Pat. No. 7,488,802; 7943743; 8008449; 8168757; 8217149 and publications of patent application under the PCT No. WO 2003042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400 and WO 2011 161699, all of which are incorporated herein in their entirety.

In one embodiment, the checkpoint inhibitor is a PD1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106), or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In one embodiment, the anti-FD-1 antibody is nivolumab. Nivolumab is an IgG4 monoclonal antibody against human FD1 and is marketed under the trade name Opdivo™. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized IgG4 monoclonal antibody and is marketed under the trade name Keytruda™. In yet another embodiment, the anti-FD-1 antibody is CT-011, a humanized antibody. CT-011, when administered by itself, did not demonstrate a response in the treatment of relapsed acute myeloid leukemia (AML). In yet another embodiment, the anti-PD-1 antibody is an AMP-224 fusion protein. In another embodiment, the anti-PD-1 antibody is BGBA317. BGB-A317 is a monoclonal antibody that is specifically engineered to bind to Fc gamma receptor I and has a unique PD1 binding signature with high affinity and excellent target specificity.

In one embodiment, the checkpoint inhibitor is a PDL1 inhibitor. In one embodiment, the PD-LI inhibitor is an anti-PD-LI antibody. In one embodiment, the anti-PD-LI antibody is MEDI4736 (durvalumab). In another embodiment, the anti-PD-LI antibody is BMS-936559 (also known as MDX1105-01). In yet another embodiment, the PD-LI inhibitor is atezolizumab (also known as MPDL3280A and Tecentriq®).

In one embodiment, the checkpoint inhibitor is a PDL2 inhibitor. In one embodiment, the PD-R2 inhibitor is an anti-PD-R2 antibody. In one embodiment, the anti-PD-R2 antibody is rHIgM12B7A.

In one embodiment, the checkpoint inhibitor is a gene inhibitor

- 53 045896 lymphocyte activation 3 (LAG-3). In one embodiment, the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brignone et al., J. Immunol., 2007, 179, 4202-4211). In another embodiment, the LAG-3 inhibitor is BMS-986016.

In one embodiment, the checkpoint inhibitor is a B7 inhibitor. In one embodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7H4 inhibitor. In one embodiment, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834).

In one embodiment, the checkpoint inhibitors are a TIM3 (T cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp Med., 2010, 207, 2187-94).

In one embodiment, the checkpoint inhibitor is an OX40 agonist (CD134). In one embodiment, the checkpoint inhibitor is an anti-OX40 antibody. In one embodiment, the anti-OX40 antibody is anti-OX-40. In another embodiment, the anti-OX40 antibody is MEDI6469.

In one embodiment, the checkpoint inhibitor is a GITR agonist. In one embodiment, the checkpoint inhibitor is an anti-GITR antibody. In one embodiment, the anti-GITR antibody is TRX518.

In one embodiment, the checkpoint inhibitor is a CD137 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD137 antibody. In one embodiment, the anti-CD137 antibody is urelumab. In another embodiment, the anti-CD137 antibody is PF-05082566.

In one embodiment, the checkpoint inhibitor is a CD40 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD40 antibody. In one embodiment, the anti-CD40 antibody is CF-870893.

In one embodiment, the checkpoint inhibitor is recombinant human interleukin-15 (rhIL-15).

In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In one embodiment, the IDO inhibitor is INCB024360. In another embodiment, the IDO inhibitor is indoximod.

In some embodiments, the combination therapies provided herein include two or more checkpoint inhibitors described herein (including checkpoint inhibitors of the same or a different class). Moreover, the combination therapies described herein may be used in combination with one or more second active agents described herein as necessary for the treatment of the diseases described herein and as contemplated in the art.

In some embodiments, Compound 1, Compound 2, or Compound 3 may be used in combination with one or more immune cells expressing one or more chimeric antigen receptors (CARs) on their surface (eg, a modified immune element). Typically, CARs contain an extracellular domain of a first protein (eg, antigen binding protein), a transmembrane domain, and an intracellular signaling domain. In some embodiments, once the extracellular domain binds to a target protein, such as a tumor associated antigen (TAA) or tumor-specific antigen (TSA), a signal is generated through the intracellular signaling domain that activates the immune cell, for example, to target and kill the cell expressing the target protein.

Extracellular domains:

The extracellular domains of the CAR bind to the antigen of interest. In some embodiments, the extracellular domain of the CAR includes a receptor or portion of a receptor that binds to the antigen. In some embodiments, the extracellular domain comprises, or is, an antibody or an antigen-binding portion thereof. In specific embodiments, the extracellular domain comprises, or is a single chain Fv (scFv) domain. The single chain Fv domain may comprise, for example, _VL linked to VH via a flexible linker, where VL and VH are antibodies that bind said antigen.

In some embodiments, the antigen recognized by the extracellular domain of a polypeptide described herein is a tumor associated antigen (TAA) or a tumor specific antigen (TSA). In various specific embodiments, the tumor associated antigen or tumor-specific antigen is, without limitation, Her2, prostate stem cell antigen (PSCA), alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA- 125), CA19-9, calretinin, MUC-1, B-cell maturation antigen (BCMA), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-24-associated antigen (MAGE), CD19, CD22, CD27, CD30, CD34, Cd45, CD70, CD99, CD117, EGFRvIII (epidermal growth factor variant III), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell alterna receptor protein

- 54 045896 tive reading frame gamma), Trp-p8, STEAPI (six transmembrane epithelial prostate antigen 1), chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), acute cystic disease fluid protein (GCDFP-15), antigen HMB-45, melan-A protein (melanoma antigen recognized by T lymphocytes, MART-I), muo-DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase type M2 isoenzyme (M2-FC tumor), abnormal ras protein or abnormal p53 protein. In some other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is ave3 integrin (CD61), galactin, or Ral-B.

In some embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is a cancer-testis antigen (CT), e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88 , NY-ES0-1, NY-SAR-35, OY-TES-1, SPANXBI, SPA17, SSX, SYCPI or TPTE.

In some other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is a carbohydrate or ganglioside, for example, fuc-GMI, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.

In some other embodiments, the TAA or TSA recognized as the extracellular domain of the CAR is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29 YBCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigens, ETV6 fusion protein -AML1, HLA-A2, HLA-All, hsp70-2, KIAA0205, Mart2, Mum-1, 2 and 3, neo-PAP, class I myosin, OB-9, pml-RARa fusion protein, PTPRK, K-ras , N-ras, trisophosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pme117), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom/ Mel-40, PRAME, p53, HRa, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) E6 and E7 antigens, TSP-180, MAGE-4 , MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, C0-029, FGF-5, G250, Ga733 (EpCAM) , HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-C0-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP or TPS.

In various specific embodiments, the tumor-associated antigen or tumor-specific antigen are tumor antigens associated with AML, as described in S. Anguille et al. Leukemia (2012), 26, 2186-2196.

Other tumor-associated and tumor-specific antigens are known in the art.

Receptors, antibodies and scFvs that bind to TSA and TAA, suitable for the construction of chimeric antigen receptors, as well as nucleotide sequences that encode them, are known in the art.

In some specific embodiments, the antigen recognized by the extracellular domain of the chimeric antigen receptor is an antigen that is not typically considered a TSA or TAA, but that is nonetheless associated with tumor cells or tumor-induced damage. In some embodiments, for example, the antigen is, for example, a growth factor, cytokine, or interleukin, such as a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines or interleukins may include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF) or interleukin -8 (IL-8). Tumors can also create a hypoxic environment local to the tumor. Thus, in other specific embodiments, the antigen is a hypoxia-associated factor, such as HIF-1a, HIF-1e, HIF-2a, HIF2β, HIF-3a, or HIF-3e. Tumors can also cause localized damage to normal tissue by causing the release of molecules known as pathogen-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in some other specific embodiments, the antigen is a DAMP, e.g., heat shock proteins, high mobility chromatin-bound protein box 1 (HMGB 1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA) or may be deoxyribonucleic acid, adenosine triphosphate, uric acid or heparin sulfate.

In some embodiments, the extracellular domain of the CAR is linked to the transmembrane domain of the polypeptide by a linker, spacer, or loop polypeptide sequence, such as a sequence from CD28 or a sequence from CTLA4. The transmembrane domain may be derived from or derived from the transmembrane domain of any transmembrane protein and may include all or part of such transmembrane domain. In specific embodiments, the transmembrane domain may be derived from, for example, CD8,

- 55 045896

CD16, cytokine receptor and interleukin receptor or growth factor receptor or the like.

In some embodiments, a CAR intracellular domain is or comprises an intracellular domain or protein motif that is expressed on the surface of T cells and initiates activation and/or proliferation of said T cells. Such a domain or motif is capable of transmitting the primary antigen-binding signal, which is necessary for the activation of T lymphocytes in response to antigen binding to the extracellular part of the CAR. Typically this domain or motif contains or is an ITAM (immunoreceptor tyrosine activation motif). ITAM-containing polypeptides suitable for CAR include, for example, the CD3 zeta chain (CD3Z) or ITAM-containing portions thereof. In a specific embodiment, the intracellular domain is a CD3Z intracellular signaling domain. In other specific embodiments, the intracellular domain is a lymphocyte receptor chain, a TKP/CD3 complex protein, an Fe receptor subunit, or an IL-2 receptor subunit. In some embodiments, the CAR further comprises one or more co-stimulatory domains or motifs, for example, as part of an intracellular domain of the polypeptide. One or more costimulatory domains or motifs may be or may comprise one or more of a CD27 polypeptide costimulatory sequence, a CD28 polypeptide costimulatory sequence, an OX40 polypeptide costimulatory sequence (CD134), a 4-1BB polypeptide costimulatory sequence (CD137), or a costimulatory inducible T polypeptide sequence -cell (ICOS) or other co-stimulatory domain or motif, or any combination thereof.

CARs may also contain a T cell survival motif. A T cell survival motif can be any polypeptide sequence or motif that facilitates the survival of T lymphocytes following antigen stimulation. In some embodiments, the T cell survival motif is or is derived from CD3, CD28, IL-7 receptor (IL-7R) intracellular signaling domain, IL-12 receptor intracellular signaling domain, IL-15 receptor intracellular signaling domain, IL-15 receptor intracellular signaling domain -21 or the intracellular signaling domain of the transforming growth factor β receptor (TGFe).

Modified immune cells expressing CAR can be, for example, T lymphocytes (T cells, e.g. CD4+ T cells or CD8+ T cells), cytotoxic lymphocytes (CTL) or natural killer (NK) cells. T lymphocytes used in the compositions and methods presented herein can be native T lymphocytes or MHC-restricted T lymphocytes. In some embodiments, the T lymphocytes are tumor infiltrating lymphocytes (TILs). In some embodiments, T lymphocytes were isolated from a tumor biopsy or were expanded from T lymphocytes isolated from a tumor biopsy. In some other embodiments, T cells have been isolated from or expanded from T lymphocytes isolated from peripheral blood, umbilical cord blood, or lymph. Immune cells to be used to generate modified immune cells expressing CARs can be isolated using conventional conventional methods, for example, blood collection followed by apheresis and, optionally, antibody-mediated cell isolation or sorting.

The modified immune cells are preferably autologous to the individual to whom the modified immune cells are to be administered. In some other embodiments, the modified immune cells are preferably allogeneic to the individual to whom the modified immune cells are to be administered. When allogeneic T lymphocytes or NK cells are used to produce modified T lymphocytes, T lymphocytes or NK cells are preferably selected, which reduces the likelihood of graft-versus-host disease (GVHD) occurring in the individual. For example, in some embodiments, virus-specific T lymphocytes are selected to produce modified T lymphocytes; such lymphocytes are expected to have a significantly reduced native ability to bind to, and thus be activated by, any recipient antigens. In some embodiments, recipient-mediated rejection of allogeneic T lymphocytes can be reduced by coadministration of one or more immunosuppressive agents to the host, for example, cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.

T lymphocytes, for example, unmodified T lymphocytes or T lymphocytes expressing CD3 and CD28, or containing a polypeptide containing a CD3Z signaling domain and a CD28 co-stimulatory domain, can be expanded using antibodies to CD3 and CD28, for example, antibodies attached to granules; see, for example, US patent No. 5948893; 6534055; 6352694; 6692964; 6887466 and 6905681.

Modified immune cells, such as modified T lymphocytes, may optionally contain a suicide gene or safety switch that, if desired, allows substantially all of the modified immune cells to be killed. For example, the modified T lymphocytes, in some embodiments, may contain the HSV thymidine kinase gene

- 56 045896 (HSV-TK), which causes the death of modified T-lymphocytes upon contact with ganciclovir. In another embodiment, the modified T lymphocytes contain an inducible caspase, for example, inducible caspase 9 (icaspase9), for example, a fusion protein between caspase 9 and human FK506-binding protein, which allows dimerization using a specific pharmaceutical small molecule. See Straathof et al., Blood 1 05(11):4247-4254 (2005). In certain embodiments, Compound 1, Compound 2, or Compound 3 provided herein is administered to patients with various types or stages of multiple myeloma in combination with chimeric antigen receptor (CAR) modified T cells. In some embodiments, the CAR T cells are combined with a target B-cell maturation antigen (BCMA), and in more specific embodiments, the CAR T cells are bb2121 or bb21217. In some embodiments, the CAR-modified T cell is JCARH125.

In some embodiments of the various methods provided herein, a control sample is obtained from the subject prior to administration of the treatment compound to the subject, and a reference sample is obtained from the same source and sample. In other embodiments of the various methods provided herein, the control sample is obtained from a healthy subject who does not have cancer, and the reference sample is obtained from the same source and sample.

In some embodiments of the various methods provided herein, the cancer is a hematologic cancer. In some embodiments, the hematologic cancer is metastatic. In some embodiments of the various methods provided herein, the cancer is multiple myeloma.

In some embodiments, the methods provided herein include the treatment, prevention, or control of various types of cancer. In some embodiments, the methods provided herein cover the treatment or prevention of various types of cancer. In some embodiments, the methods presented herein cover the treatment of various types of cancer, such as the cancer presented herein. In one embodiment, the methods provided herein include methods for treating, preventing, or controlling multiple myeloma by administering a therapeutically effective amount of Compound 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof. In one embodiment, the methods provided herein include methods for treating, preventing, or controlling multiple myeloma by administering a therapeutically effective amount of Compound 2 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof. In one embodiment, the methods provided herein include methods for treating, preventing, or controlling multiple myeloma by administering a therapeutically effective amount of Compound 3 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

In specific embodiments, the disclosure is a method of treating, preventing, or controlling cancer in patients with impaired renal function. In specific embodiments, the disclosure provides methods for providing appropriate dosage adjustments for patients with impaired renal function due to, but not limited to, disease, aging, or other patient factors.

In specific embodiments, provided herein are methods of treating, preventing, or controlling MM, including relapsed/refractory multiple myeloma in patients with impaired renal function or a symptom thereof, which comprises administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof, to a patient having relapsed/refractory MM with renal impairment, alone or in combination with a second active agent. In some embodiments, the methods include the step of administering to a subject a compound provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof in combination with a second active agent in amounts effective to treat, prevent, or control relapsed/refractory MM in patients with impaired renal function. In some embodiments, the compound is Compound 1. In some embodiments, the compound is Compound 2. In some embodiments, the compound is Compound 3.

In some embodiments, the level of the biomarker is reduced upon treatment with the compound. In some embodiments, the biomarker is selected from the group consisting of CRBN, IKZF1, IKZF3, CTC, soluble BCMA, survivin, serum free light chain, ZFP91, c-MYC, IRF4, pRB1, and the level of the biomarker is reduced from control levels to treatment with a medicinal compound.

In other embodiments, the level of the biomarker increases upon treatment with the compound. In some embodiments, the biomarker is selected from the group consisting of caspase 1, caspase-3, caspase 7, PARP, ZFNy, TNFa, IL2, p21, p27, BIM, and TCR clonality, and the level of the biomarker is increased compared to the control level before administration of the therapeutic compound.

In some embodiments of the methods provided herein, the biomarker is a protein that is directly or indirectly affected by CRBN, for example, through protein-protein interactions (e.g., certain CRBN substrates or their downstream effectors) or through various cellular reaction cascades ( e.g. signal transmission pathways). In specific embodiments, the biomarker is a CRBN-associated protein (CAP). In some embodiments, the biomarker is the mRNA of a protein that is directly or indirectly affected by CRBN. In other embodiments, the biomarker is a cDNA of a protein that is directly or indirectly affected by CRBN.

Thus, in some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be receptive to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for identifying a subject having a cancer that is likely to be susceptible to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject, wherein the biomarker is an ATS; and (b) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, this document provides a method for identifying

- 58 045896 a subject having a cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; wherein the therapeutic compound is Compound 1 or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker;

wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker;

wherein the therapeutic compound is Compound 3 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to a sample obtained from the subject;

(b) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 1

- 59 045896

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, Compound 1, Compound 2, or Compound 3 is provided herein for use in a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of a biomarker in the sample, wherein the biomarker is an ATS; and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

- 60 045896 (b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample;

wherein the therapeutic compound is Compound 2 o

X or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample;

wherein the therapeutic compound is Compound 3 o

^N '\ /=°

Vnh

X T

NC^F or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample;

wherein the therapeutic compound is Compound 2

- 61 045896

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject, wherein the biomarker is an ATS;

(d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample differs from the level of a biomarker obtained from a control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is a CAP;

(d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

- 62 045896 (b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is an ATS;

(d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 3 o

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of treating cancer in a subject with a therapeutic compound that was administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject, wherein the biomarker is an ATS;

(b) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, the biomarker is a CAP selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC, and IRF4. In some embodiments, the biomarker is a member of the Ikaros family, such as IKZF1 or IKZF3. In a specific embodiment, the biomarker is IKZF1. In another specific embodiment, the biomarker is IKZF3. In a specific embodiment, the biomarker is ZFP91. In some embodiments, the biomarker is a binding partner of IKZF1 and IKZF3, a downstream effector of IKZF1 and IKZF3, or a cellular cascade factor influencing IKZF1 and IKZF3. For example, in some embodiments, the biomarker is a binding partner of IKZF1 or IKZF3, a downstream effector of IKZF1 or IKZF3, or a cellular cascade factor that is influenced by IKZF1 or IKZF3. In a specific embodiment, the biomarker is a downstream effector of IKZF1, such as IRF4. In a specific embodiment, the biomarker is a downstream effector of IKZF3, such as IRF4. In a specific embodiment, the biomarker is a downstream effector of IKZF1, such as c-MYC. In a specific embodiment, the biomarker is a downstream effector of IKZF3, such as c-MYC.

As shown in the examples, the level of biomarker such as CRBN, IKZF1, IKZF3, CTC, soluble BCMA, survivin, free light chain in serum, ZFP91, c-MYC, IRF4, pRB1, is reduced compared to control in response to administration of Compound 1 , Compound 2 or Compound 3. Accordingly, in some embodiments, the biomarker is selected from the group consisting of CRBN, IKZF1, IKZF3, CTC, soluble BCMA, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 and pRB1, and the level of the biomarker decreases in response to administration of Compound 1, Compound 2, or Compound 3. Thus, in some embodiments of the various methods provided herein, the biomarker is CRBN, IKZF1, IKZF3, CTC, soluble BCMA, TIL, survivin, free lung chain in serum, ZFP91, c-MYC, IRF4, pRB1, or the protein (or factor) they influence, and the biomarker level is below the control level.

Thus, in some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be receptive to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, SBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, SBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 1 o

J

NC'-F or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable

- 63 045896 salt.

In another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 2 o

U-J --_- ^{0 0}

J t _NC -'W- _F or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 3 o

[Г N'·/ )=О

Jnc^f or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for identifying a subject having a cancer that is likely to be susceptible to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in a sample obtained from the subject; and (b) diagnosing the subject as likely susceptible to the therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4 or pRB1 is lower than the level of CRBN, IKZF1 , IKZF3, CTC, sBCMA, TIL, survivin, serum free light chain, ZFP91, c-MYC, IRF4, or pRB1 in control; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 1

- 64 045896 or its enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 2 o

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 3 o

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to a sample obtained from the subject;

(b) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4 or pRB1 is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in a control sample and (d) administering to the subject a therapeutically effective amount of the treatment compound;

wherein the therapeutic compound is Compound 1

- 65 045896

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4 or pRB1 is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in a control sample and (d) administering to the subject a therapeutically effective amount of the treatment compound;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4 or pRB1 is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in a control sample and (d) administering to the subject a therapeutically effective amount of the treatment compound;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, Compound 1, Compound 2, or Compound 3 is provided herein for use in a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4 or pRB1 is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in a control sample and (d) administering to the subject a therapeutically effective amount of the treatment compound.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB 1 in the sample and (d) diagnosing a subject as likely susceptible to a therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4, or pRB1 is lower than the level of CRBN, IKZF1, IKZF3, CTC , sBCMA, TIL, survivin, serum free light chain, ZFP91, c-MYC, IRF4, or pRB1 in control;

- 66 045896 wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing subject as likely susceptible to a therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB 1 in the sample and (d) diagnosing a subject as likely susceptible to a therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4, or pRB1 is lower than the level of CRBN, IKZF1, IKZF3, CTC , sBCMA, TIL, survivin, serum free light chain, ZFP91, c-MYC, IRF4, or pRB1 in control;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject, wherein the biomarker is CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample; and (b) diagnosing a subject as likely susceptible to a therapeutic compound if the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, cMYC, IRF4 or pRB1 is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

- 67 045896 (b) introducing a therapeutic compound into the sample;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 1 o

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 2 o

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample;

wherein the therapeutic compound is Compound 3 o

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to a sample obtained from the subject;

(b) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of CrBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, cMYC, IRF4 or pRB1 in the sample is lower than the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

- 68 045896

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and

(d) comparing the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample with the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 obtained from a control sample, wherein a decrease in level compared to the control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject; wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4 or pRB1 in the sample and

(d) comparing the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample with the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 obtained from a control sample, wherein a decrease in level compared to the control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in the sample and (d) comparing the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, serum free light chain, ZFP91, c-MYC, IRF4 or pRB1 in a sample with CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, serum free light chain, ZFP91, c-MYC , IRF4 or pRB1 obtained from a control sample, wherein a decrease in level compared to the control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a method for monitoring the effectiveness of treating cancer in a subject with a therapeutic compound that was administered to the subject, comprising:

(a) determining the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1 in a sample obtained from the subject; and (b) comparing the serum level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL, survivin, free light chain, ZFP91, c-MYC, IRF4, or pRB1 in the sample with the level of CRBN, IKZF1, IKZF3, CTC, sBCMA, TIL , survivin, free light chain in serum, ZFP91, c-MYC, IRF4 or pRB1, derived from con

- 69 045896 troll sample, wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In specific embodiments of the methods described herein, the control sample is a sample before administration of the therapeutic compound.

In a specific embodiment, the biomarker is CRBN and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is CRBN and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, the treatment compound is Compound 1. In an embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is IKZF1 and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is IKZF1 and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, In an embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is IKZF3 and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is IKZF3 and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, In an embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is a CTC and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is a CTC and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, the treatment compound is Compound 1. In an embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is a TIL and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is a TIL and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, the treatment compound is Compound 1. In an embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is survivin and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is survivin and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, the treatment compound is Compound 1. In an embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is a free light chain in serum and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is a free light chain in serum and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another specific embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is ZFP91 and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is ZFP91 and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, the treatment compound is Compound 1. In an embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is c-MYC and the cancer is multiple myeloma (MM). In a specific embodiment, the biomarker is c-MYC and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another specific embodiment, the treatment compound is Compound 2. In another embodiment In one particular embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is IRF4 and the cancer is multiple myeloma (MM). In a specific embodiment, the biomarker is IRF4 and the treatment compound is Compound 1, Compound 2, or Compound 3. B

- 70 045896 in a specific embodiment, the treatment compound is Compound 1. In another specific embodiment, the treatment compound is Compound 2. In yet another specific embodiment, the treatment compound is Compound 3.

In a specific embodiment, the biomarker is pRB 1 and the cancer is multiple myeloma (MM). In a particular embodiment, the biomarker is pRB1 and the treatment compound is Compound 1, Compound 2, or Compound 3. In a particular embodiment, the treatment compound is Compound 1. In another particular embodiment, the treatment compound is Compound 2. In yet another particular embodiment, the treatment compound is Compound 1. In an embodiment, the treatment compound is Compound 3.

In some embodiments, the biomarker is involved in apoptosis. In some embodiments, a biomarker provided herein is involved in the cell cycle. In other embodiments, a biomarker provided herein is involved in T cell activation.

In some embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample containing cancer cells from the subject;

(b) detecting the level of CRBN in the sample and (c) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control CRBN level;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In other embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample containing cancer cells from the subject;

(b) detecting the level of CRBN in the sample and (c) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control CRBN level;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

(a) obtaining a sample containing cancer cells from the subject;

(b) detecting the level of CRBN in the sample and (c) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control CRBN level;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising:

- 71 045896 (a) detection of the level of CRBN in a sample obtained from the subject; and (b) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control level of CRBN; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method for predicting the responsiveness of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample containing cancer cells from the subject;

(b) detecting the level of CRBN in the sample and (c) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control CRBN level;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample containing cancer cells from the subject;

(b) detecting the level of CRBN in the sample and (c) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control CRBN level;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) obtaining a sample containing cancer cells from the subject;

(b) detecting the level of CRBN in the sample and (c) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control CRBN level;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising:

(a) detecting the level of CRBN in a sample obtained from the subject; and (b) diagnosing the subject as likely susceptible to the treatment compound if the level of CRBN in the sample is detectable and below the control level of CRBN; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

Thus, in some embodiments, provided herein is a method for detecting levels of CRBN in a cancer cell and diagnosing a subject as likely susceptible to a therapeutic compound if the level of CRBN in a sample is detectable and below a control CRBN level. In some embodiments, the MM is relapsed, refractory, or resistant to conventional therapy. In one embodiment, the MM is lenalidomide-resistant MM. In another embodiment, the MM is pomalidomide-resistant MM. As described in the examples in section 6, Compound 2 also has immunomodulatory properties in PBMC and CD4+ and CD8+ T cells. Therefore, in one embodiment of the methods provided herein, CRBN is not detected in the cancer cell, but CRBN is detected in the immune cell, and the patient is susceptible to Compound 1. In another embodiment

- 72 045896 ante implementation of the methods proposed herein, CRBN is not detected in the cancer cell, but CRBN is detected in the immune cell, and the patient is susceptible to Compound 2. In yet another embodiment of the methods proposed herein, CRBN is not detected in the cancer cell cell, but CRBN is found in the immune cell, and the patient is susceptible to Compound 1.

In another aspect, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from a subject at different points in time;

(c) determining the level of IKZF1, IKZF3, or both in one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from a subject at different points in time;

(c) determining the level of IKZF1, IKZF3, or both in one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising:

(a) administering a dose of a therapeutic compound to the subject;

(b) obtaining one or more samples from a subject at different points in time;

(c) determining the level of IKZF1, IKZF3, or both in one or more samples and thereby determining whether the dose is appropriate or requires adjustment;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound administered to the subject, comprising:

(a) determining the level of IKZF1, IKZF3, or both in one or more samples obtained from the subject, and thereby determining whether the dose is appropriate or requires adjustment; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some other specific embodiments, the level of the biomarker increases upon treatment with the compound. In some embodiments, the biomarker is selected from the group consisting of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM, and TCR clonality, and the level of the biomarker is increased relative to the level in the control sample to administration of the therapeutic compound. In some embodiments, the biomarker is involved in apoptosis, and the level of the biomarker is increased compared to the level in the control sample before administration of the treatment compound. In other embodiments, the biomarker is involved in T cell activation and the level of the biomarker is increased compared to the level in the control sample before administration of the therapeutic compound.

- 73 045896

Thus, in some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be receptive to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound ^1,0 or an enantiomer thereof, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt

In another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another embodiment, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 3 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method for identifying a subject having a cancer that is likely to be susceptible to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in a sample obtained from the subject; and (b) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

- 74 045896 wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 1 or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 3 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising:

(a) administering the therapeutic compound to a sample obtained from the subject;

(c) determination of the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or

- 75 045896 TCR clonality in the sample and (d) diagnosing a subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase -3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in a control sample and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase -3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in a control sample and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

(b) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase -3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample; and (d) administering to the subject a therapeutically effective amount of a therapeutic compound;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is Compound 1, Compound 2, or Compound 3, or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof for use in a method of treating cancer, comprising:

(a) obtaining a sample from a subject having cancer;

- 76 045896 (b) determination of the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample;

(c) diagnosing a subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase -3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample and (d) administering to the subject a therapeutically effective amount of the treatment compound.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP , IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP , IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of a biomarker in the sample, wherein the biomarker is caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to a therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP , IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

- 77 045896

In some embodiments, provided herein is a method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound that has been administered to the subject, comprising:

(a) determining the level of a biomarker in a sample obtained from the subject, wherein the biomarker is caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM, or TCR clonality in the sample; and (b) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 1 or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) obtaining a sample from the subject;

(b) introducing the therapeutic compound into the sample;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 3

- 78 045896

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising:

(a) administering the therapeutic compound to a sample obtained from the subject;

(b) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of caspase 1 , caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample is higher than the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the control sample;

wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) comparing the level of caspase 1, caspase-3, caspase 7, PARP , IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in a sample with caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality levels obtained from a control sample wherein an increase in level compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) comparing the level of caspase 1, caspase-3, caspase 7, PARP , IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in a sample with caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality levels obtained from a control sample wherein an increase in level compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of a therapeutic compound treating cancer in a subject, comprising:

(a) administering the therapeutic compound to the subject;

(b) obtaining a sample from the subject;

(c) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample and (d) comparing the level of caspase 1, caspase-3, caspase 7, PARP , IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in a sample with the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21,

- 79 045896 p27, BIM or clonality of TCR obtained from a control sample, wherein an increase in level compared to the control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject;

wherein the therapeutic compound is Compound 3 o

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, provided herein is a method for monitoring the effectiveness of treating cancer in a subject with a therapeutic compound that was administered to the subject, comprising:

(a) determining the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in a sample obtained from the subject; and (b) comparing the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality in the sample with the level of caspase 1, caspase-3, caspase 7, PARP, IFNy, TNFa, IL2, p21, p27, BIM or TCR clonality obtained from a control sample, wherein an increase in level compared to the control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject; wherein the therapeutic compound is Compound 1, Compound 2 or Compound 3, or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In specific embodiments of the methods described herein, the control sample is a sample before administration of the therapeutic compound.

In some embodiments of the various methods provided herein, the level of biomarkers is measured by determining the level of the biomarker protein.

In other embodiments of the various methods provided herein, the level of biomarkers is measured by determining the level of biomarker mRNA.

In other embodiments of the various methods provided herein, the level of biomarkers is measured by determining the level of the biomarker cDNA.

In some embodiments of the various methods provided herein, the therapeutic compound is a compound described in section 5.7 below.

In some embodiments, the therapeutic compound is Compound 1 and the cancer is MM. In some embodiments, the therapeutic compound is Compound 2 and the cancer is MM. In other embodiments, the therapeutic compound is Compound 3 and the cancer is MM.

In some embodiments, patient or subject responsiveness, or treatment efficacy, is determined by overall survival (OS), complete remission rate (CRR), objective response rate (ORR), time to progression, disease-free survival (RFS), progression-free survival (PFS) , event-free survival, duration of remission, duration of response and/or time to remission/response of a patient having cancer. In one embodiment, the cancer is a hematological cancer. In certain embodiments, ORR includes all responses in complete remission (CR) (ie, morphologic CR, morphologic CR, cytogenetic CR, molecular CR, and morphologic CR with incomplete blood recovery) and partial remission.

In other embodiments, various methods provided herein are intended to improve overall survival (OS), complete remission rate (CRR), objective response rate (ORR), time to progression, disease-free survival (RFS), progression-free survival ( PFS), event-free survival, duration of remission, duration of response and/or time to remission/response of a patient having cancer, for example, hematological cancer.

In other embodiments, Compound 1, Compound 2, or Compound 3 is provided herein for use in any of the methods provided herein.

5.3 Biomarker detection and quantification methods

In certain embodiments, provided herein are methods for detecting and quantifying the level of a biomarker protein, such as CRBN, a protein that is directly or indirectly affected by CRBN, or a protein that is indicative of a pharmacodynamic (PD) response (e.g., soluble BCMA) to a Compound 1, Compound 2 or Compound 3 from a biological sample, comprising contacting proteins in the sample with a first antibody that immunospecifically binds to a biomarker protein. In some embodiments, the methods provided herein further comprise: (i) contacting a biomarker protein bound to a first antibody with a second antibody having a detectable label, wherein the second antibody immunospecifically binds the biomarker protein and the second antibody immunospecifically binds the biomarker protein. binds to an epitope on the biomarker protein that is different from the epitope of the first antibody; (ii) presence detection

- 80 045896 second antibody associated with a biomarker protein; and (iii) determining the amount of biomarker protein based on the amount of detectable label on the second antibody. In other embodiments, the methods provided herein further include: (i) contacting a biomarker protein bound to a first antibody with a detectably tagged second antibody, wherein the second antibody immunospecifically binds to the first antibody; (ii) detecting the presence of a second antibody associated with the first antibody; and (iii) determining the amount of biomarker protein based on the amount of detectable label on the second antibody.

In some embodiments of the various methods presented herein, the method includes using double-stain immunohistochemistry to determine the level of a biomarker, such as CRBN, or a protein that is directly or indirectly affected by CRBN. In a double staining immunohistochemical assay, a biomarker provided herein and another cancer biomarker are simultaneously detected using a first labeled antibody targeting the biomarker provided herein and a second labeled antibody targeting the cancer biomarker. Such an assay could improve the specificity, accuracy, and sensitivity for detection and measurement of the biomarker presented herein. In some embodiments, the cancer biomarker is a MM biomarker.

In some embodiments of the various methods provided herein, the method includes DNA sequencing. For example, in some embodiments, provided herein are methods for identifying a subject having multiple myeloma that is likely to be responsive to a therapeutic compound, as well as methods for predicting the responsiveness of a subject having multiple myeloma to a therapeutic compound. Patients resistant to initial treatment with a cereblon modulator compound may have a mutation in CRBN. It is important to note that, as shown in the examples, cells that are resistant to cereblon modulators, such as lenalidomide or pomalidomide, may still be sensitive to Compound 1, Compound 2, or Compound 3. Therefore, in some embodiments, the method includes sequencing CRBN DNA on presence of mutations to identify a subject having multiple myeloma who is likely to be susceptible to a therapeutic compound, wherein the treatment compound is Compound 1. In some embodiments, the method includes sequencing CRBN DNA for mutations to identify a subject having multiple myeloma who is likely to be susceptible to a treatment compound, wherein the treatment compound is Compound 2. In some embodiments, the method includes sequencing CRBN DNA for mutations to identify a subject having multiple myeloma who is likely to be susceptible to the treatment compound, wherein the treatment compound is Compound 3 In other embodiments, the method includes sequencing CRBN DNA for mutations to predict the response of a subject having multiple myeloma that is likely to be susceptible to a treatment compound, wherein the treatment compound is Compound 1. In other embodiments, the method includes sequencing CRBN DNA for the presence of mutations to predict the response of a subject having multiple myeloma who is likely to be susceptible to a therapeutic compound, wherein the therapeutic compound is Compound 2. In other embodiments, the method includes sequencing CRBN DNA for mutations to predict the response of a subject having multiple myeloma who is likely to will be susceptible to the medicinal compound, wherein the medicinal compound is Compound 3.

In some embodiments, the methods provided herein are designed to detect T cell activation. In some embodiments, TCR DNA may be sequenced following treatment with Compound 1, Compound 2, or Compound 3. Activation of T cells may result in clonal expansion of a population of T cells with a specific rearrangement of their TCR genes. Rearrangements can be determined by DNA sequencing. Therefore, in some embodiments of the various methods presented herein, the biomarker of the method is TCR clonality, and TCR clonality is measured by sequencing TCR DNA.

Thus, in some embodiments, the method provided herein includes: (i) contacting proteins in the sample with a first antibody that immunospecifically binds to a biomarker provided herein, the first antibody being associated with a first detectable label; (ii) contacting proteins in the sample with a second antibody that immunospecifically binds to a cancer biomarker, wherein the second antibody is associated with a second detectable label; (iii) detecting the presence of a first antibody and a second antibody bound to the proteins; and (iv) determining the level of a biomarker provided herein based on the amount of detectable label on the first antibody and determining the level of the cancer biomarker based on the amount of detectable label on the second antibody. In some embodiments, the cancer biomarker is a MM biomarker.

In certain embodiments, provided herein are methods for detecting and quantifying the protein level of a biomarker, such as Aiolos or Ikaros, or any other

- 81 045896 of the biomarker presented herein from a biological sample, comprising: (i) contacting the biomarker protein with a first antibody; (ii) contacting a biomarker protein bound to the first antibody with a second antibody having a detectable label, wherein the second antibody immunospecifically binds to the first antibody; (iii) detecting the presence of a second antibody complexed with a biomarker protein; and (iv) determining the amount of biomarker protein based on the amount of detectable label on the second antibody using flow cytometry (eg FACS). In addition, it is understood that detection and quantification of the level of a biomarker protein, such as Aiolos or Ikaros, can be performed in cancer cells and non-cancerous cells (eg, CD3+ T cells). For example, the expression of Aiolos and/or Ikaros can be measured by flow cytometry in CD3+ T cells before, during, and/or after treatment with Compound 1, Compound 2, or Compound 3. In some embodiments, the cancer biomarker is a MM biomarker. In some embodiments, the biomarker is a CTC.

In some embodiments, provided herein are methods for assessing the expression of biomarkers, such as CRBN, Aiolos, Ikaros, ZFP91, c-Myc, IRF4, p-caspase-3, as well as TILs, in a biological sample, comprising: (i) collecting the samples bone marrow aspirate before, during and/or after treatment with Compound 1, Compound 2 or Compound 3; (ii) formation of clots from each sample; (iii) fixation of clots, for example, in formalin; (iv) embedding the clot in paraffin or cryoembedding medium (eg, OCT); (v) sectioning the embedded samples for immunostaining; (vi) performing immunohistochemistry (IHC) on sections; (vii) visualization of stained samples under a microscope and (viii) assessment of biomarker expression.

In some embodiments, provided herein are methods for detecting and quantifying tumor infiltrating lymphocytes (TILs) as a biomarker from a biological sample, comprising: (i) staining the histological sample with hematoxylin and eosin (H&E); (ii) visualization of TILs using a light microscope; (iii) detection of TILs based on morphology and staining; (iv) determination of TIL levels based on the number of stained lymphocytes within (i.e., infiltrating) the tumor.

In some embodiments, provided herein are methods for detecting and quantifying the level of RNA (e.g., mRNA, total RNA, or small RNA) of a biomarker, such as CRBN or a biomarker provided herein, from a biological sample, comprising: (a) obtaining the RNA from sample; (b) contacting the RNA with a primer that specifically binds to the RNA sequence to form a first DNA molecule having a sequence complementary to said RNA (ie, cDNA); (c) amplifying cDNA corresponding to the gene segment encoding the biomarker; and (d) determining the level of biomarker RNA based on the amount of amplified DNA. In some embodiments, provided herein are methods for sequencing RNA (RNA-seq) of a biomarker from a biological sample, comprising: (a) isolating RNA from the sample; (b) depletion of ribosomal RNA (rRNA) and enrichment of RNA 3'-polyadenylated (poly(A)) tails, or none of the above; (c) reverse transcription of RNA into cDNA; and (d) cDNA sequencing. In some embodiments, the biomarker(s) are assessed in combination with other biomarker(s) provided herein, such as Aiolos, Ikaros, CRBN, ZFP91, c-MYC, IRF4, beta-caspase 1, beta-caspase-3, p-caspase 7, cleaved PARP, survivin, BIM, sFLC, p21, p27, pRB1, soluble BCMA, CTC, TIL, IL-2, ZFNy, TNFa and TCR clonality.

In some embodiments, provided herein are methods for detecting and quantifying one or more biomarkers that are involved in apoptosis. It is well known to those skilled in the art that there are various methods for detecting apoptosis. One method involves detecting DNA fragmentation, which is a hallmark of apoptosis. Terminal deoxynucleotidyl transferase dUTP single-strand end labeling (TUNEL) is an established method for detecting DNA fragments. Therefore, in some embodiments of the various methods presented herein, the biomarker is involved in apoptosis, and the level of the biomarker is measured using terminal deoxynucleotidyl transferase dUTP single-strand end labeling (TUNEL).

One of the early events of apoptosis involves the movement of membrane phosphatidylserine (PS) from the inside of the plasma membrane to the surface. Annexin V, a Ca2+-dependent phospholipid-binding protein, has high affinity for PS, and fluorochrome-labeled annexin V can be used to detect susceptible PS by flow cytometry.

The use of fluorescent intercalating agents that bind to DNA may further facilitate the distinction between early apoptotic cells and late apoptotic cells. Cells that are early apoptotic cells will exclude fluorescent intercalating agents such as 7-aminoactinomycin D (7-AAD) and propidium iodide (PI), whereas late apoptotic cells will stain positive due to the passage of these dyes into the nucleus where they bind to DNA. 7-AAD and PI have a high DNA binding constant and are efficiently eliminated by intact cells. Thus, in some embodiments it is possible to

- 82 045896 In the various methods presented herein, a biomarker is involved in apoptosis, and the level of the biomarker is measured using annexin-V and 7-AAD. In other embodiments of the various methods presented herein, the biomarker is involved in apoptosis, and the level of the biomarker is measured using Annexin-V and propidium iodide (PI).

In some embodiments of the various methods presented herein, two or more steps are performed sequentially. In other embodiments of the methods presented herein, two or more steps are performed in parallel (eg, at the same time).

Examples of assays presented herein for methods of detecting and quantifying biomarker protein levels such as Aiolos, Ikaros, CRBN, c-MYC, IRF4, cleaved caspase-3, soluble BCMA (sBCMA), soluble FLC (sFLC), activated T cell-associated cytokines, or a combination thereof, are immunoassays such as Western blotting, enzyme-linked immunosorbent assay (ELISA) (eg, sandwich ELISA), immunohistochemistry (IHC), and fluorescence-activated cell sorting (FACS). For example, in some embodiments, serum samples may be collected before, during, and/or after treatment with Compound 1, Compound 2, or Compound 3, and sBCMA may be measured by ELISA. Exemplary assays presented herein for methods of detecting and quantifying RNA levels of a biomarker such as Aiolos, Ikaros, CRBN, c-MYC, IRF4, activated T cell-associated cytokines, or combinations thereof are polymerase chain reaction with reverse transcription-PCR (RT-PCR), such as quantitative real-time PCR (qRT-PCR), and RNA-sequencing.

5.4. Subjects, samples and cell types

In some embodiments, the various methods presented herein use samples (eg, biological samples) from subjects or individuals (eg, patients). The subject may be a patient, such as a patient with cancer (eg, multiple myeloma). The subject may be a mammal, such as a human. The subject may be male or female, as well as an adult, child, or infant. Samples can be analyzed during the active phase of the cancer (eg, MM) or when the cancer (eg, MM) is inactive. In some embodiments, more than one sample may be obtained from a subject.

In some embodiments, the sample used in the methods presented herein contains body fluids of the subject. Non-limiting examples of body fluids include blood (e.g., whole blood), blood plasma, amniotic fluid, aqueous humor, bile, bone marrow, earwax, Cowper's fluid, pre-ejaculatory fluid, lymph, chyme, female ejaculate, tissue fluid, lymph, menstruation, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubricant, vomit, water, feces, internal body fluids (including cerebrospinal fluid surrounding the brain and spinal cord), synovial fluid, intracellular fluid (fluid inside cells), and vitreous (fluid in the eyeball). In some embodiments, the sample is a blood sample. A blood sample can be obtained using conventional methods as described in, for example, Innis et al., eds., PCR Protocols (Academic Press, 1990). Leukocytes can be isolated from blood samples using conventional methods or commercially available kits such as the RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). White blood cell subpopulations, such as mononuclear cells, B cells, T cells, monocytes, granulocytes, or lymphocytes, can be further isolated using conventional methods such as magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) or fluorescence-activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

In one embodiment, the blood sample is from about 0.1 to about 10.0 ml, from about 0.2 to about 7 ml, from about 0.3 to about 5 ml, from about 0.4 to about 3.5 ml or from about 0.5 to about 3 ml. In another embodiment, the blood sample is about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1. 5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 6.0, about 7.0, about 8, 0, about 9.0 or about 10.0 ml.

In some embodiments, a blood sample is collected into a TruCulture tube containing an anti-CD3 antibody for an ex vivo T cell activation assay.

In some embodiments, the bone marrow sample is from about 0.1 to about 10.0 ml, from about 0.2 to about 7 ml, from about 0.3 to about 5 ml, from about 0.4 to about 3.5 ml or from about 0.5 to about 3 ml. In another embodiment, the blood sample is about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1. 5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 6.0, about 7.0, about 8, 0, about 9.0 or about 10.0 ml.

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In some embodiments, the sample used in the present methods includes a biopsy (eg, a tumor biopsy). The biopsy can be from any organ or tissue, such as skin, liver, lungs, heart, colon, kidney, bone marrow, teeth, lymph node, hair, spleen, brain, breast, or other organs. Any biopsy technique known to those skilled in the art can be used to obtain a sample from a subject, for example, open biopsy, closed biopsy, core biopsy, excisional biopsy, incisional biopsy, or fine needle aspiration biopsy.

In one embodiment, a sample used in the methods presented herein is obtained from a subject before the subject receives treatment for a disease or disorder. In some embodiments, a sample is obtained from a subject after treatment with a therapy other than Compound 1, Compound 2, or Compound 3, and before the subject receives treatment with Compound 1, Compound 2, or Compound 3. In a particular embodiment, the sample is obtained from a subject after relapse of the disease or after the patient becomes refractory to therapy other than Compound 1, Compound 2, or Compound 3. In another embodiment, the sample is obtained from the subject while the subject is receiving treatment for the disease or disorder. In another embodiment, a sample is obtained from a subject after the subject is receiving treatment for a disease or disorder. In various embodiments, treatment includes administering to a subject a compound (eg, Compound 1, Compound 2, or Compound 3).

In some embodiments, the sample used in the methods presented herein contains a variety of cells, such as cancer (eg, MM) cells. Such cells may include any type of cell, for example, stem cells, blood cells (eg, peripheral blood mononuclear cells (PBMCs)), lymphocytes, B cells, T cells, monocytes, granulocytes, immune cells, or cancer cells.

Specific cell populations can be generated using a combination of commercially available antibodies (eg, antibodies from Quest Diagnostic (San Juan Capistrano, CA) or Dako (Denmark)).

In some embodiments, the cells in the methods presented herein are PBMC. In some embodiments, the sample used in the methods presented herein is from disease-causing tissue, such as from a human having cancer (eg, MM).

In some embodiments, cell lines are used as disease models to evaluate the effects of compounds, study mechanisms of action, or establish reference levels of biomarkers, etc. In some embodiments, the cells used in the methods presented herein are derived from a cancer (eg, MM) cell line. In one embodiment, the MM cell line is the DF15 cell line. In another embodiment, the MM cell line is the lenalidomide and pomalidomide resistant DF15 cell line (DF15R). In another embodiment, the MM cell line is NCIH929. In another embodiment, the MM cell line is lenalidomide-resistant NCI-H929 (NCI-H929-1051). In another embodiment, the MM cell line is pomalidomide-resistant NCI-H929 (NCI-H929-P01). In another embodiment, the MM cell line is OPM2. In another embodiment, the MM cell line is the OPM2 cell line resistant to 100 nM pomalidomide (OPM2-P01). In another embodiment, the MM cell line is the OPM2 cell line resistant to 1 μM pomalidomide (OPM2-P1). In another embodiment, the MM cell line is the OPM2 cell line resistant to 10 μM pomalidomide (OPM2-P10).

In some embodiments, the methods presented herein are useful for detecting gene rearrangements in cells of an individual. In some embodiments, the methods provided herein are useful for detecting TCR gene rearrangement following treatment with Compound 1, Compound 2, or Compound 3. In some embodiments, the number of cells used in the methods provided herein may vary from one cell to approximately 10 cells. In some embodiments, the number of cells used in the methods provided herein is about 1 x 10 ⁴ , about 5 x 10 ⁴ , about 1 x 10 ⁵ , about 5 x 10 ^{5 , about 1 x 10 6 ,} about 5 x 10 ⁶ , about 1 x 10 ⁷ , about 5 x 10 ⁷ , approximately 1 x 10 ⁸ , approximately 5x10 ⁸ or approximately 1x10 ⁹

The number and type of cells collected from a subject can be monitored, for example, by measuring changes in cell surface markers using standard cell detection methods such as flow cytometry, cell sorting, immunocytochemistry (eg, staining with tissue-specific antibodies or cell marker-specific antibodies) , fluorescence-activated cell sorting (FACS), magnetically activated cell sorting (MACS), evaluation of hematoxylin and eosin (H&E)-stained tumor sections, examination of cell morphology using light or confocal microscopy and/or by measuring changes in gene expression with using methods well known in the art, such as PCR and pro

- 84 045896 gene expression filtering. These methods can also be used to identify cells positive for one or more specific markers.

In some embodiments, the methods presented herein use subsets of cells. Methods for sorting and isolating specific populations of cells are well known in the art and can be based on cell size, morphology, H&E staining, intracellular markers or extracellular markers. Such methods include flow cytometry, flow sorting, FACS, bead-based separations such as magnetic cell sorting, size separations (e.g., sieve, multiple obstacles, or filter), H&E staining, microfluidic device sorting, antibody-based separations, sedimentation , affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, etc., but are not limited to. Fluorescence-activated cell sorting (FACS) is a well-known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, Methods Enzymol. 1987, 151:150-165). Laser excitation of fluorescent fragments in individual particles results in a small electrical charge, allowing positive and negative particles to be electromagnetically separated from a mixture. In one embodiment, antibodies or ligands specific for cell surface markers are labeled with various fluorescent tags. The cells are processed using a cell sorter, which separates the cells based on their ability to bind to the antibodies used. FACS-sorted particles can be directly placed into individual wells of 96-well or 384-well plates to facilitate separation and cloning.

In one embodiment, DNA, RNA (eg, mRNA), or protein is purified from a sample, and the presence or absence of a biomarker is measured by analyzing DNA or RNA sequence, gene expression, or protein expression. In some embodiments, the presence or absence of a biomarker is measured using next generation sequencing (NGS), RNA sequencing (RNA-seq), fluorescence in situ hybridization (FISH), quantitative real-time NLR (qRT-PCR), microarray, flow cytometry , immunohistochemistry or immunofluorescence. In other embodiments, the presence or absence of a biomarker is measured using ELISA or other similar methods known in the art.

5.5 Methods for determining the level of mRNA in a sample

Several methods are known in the art to analyze, detect, or quantify mRNA levels. Examples of methods include, but are not limited to, Northern blots, RNA sequencing, ribonuclease protection assays, PCR-based methods, and the like. The mRNA sequence of a biomarker (eg, CRBN mRNA or a protein directly or indirectly affected by CRBN, or a fragment thereof) can be used to generate a probe that is at least partially complementary to the mRNA sequence. The probe can then be used to detect the mRNA in the sample using any suitable assay such as PCR-based methods, Northern blot, dipstick, etc.

In other embodiments, a nucleic acid assay may be performed to test the activity of a compound in a biological sample. The assay typically comprises a solid support and at least one nucleic acid in contact with the support, wherein the nucleic acid corresponds to at least a portion of the mRNA that has changed in expression during the patient's complex treatment, such as a biomarker mRNA (eg, CRBN or a protein, which directly or indirectly affects CRBN). The assay may also have a means of detecting altered mRNA expression in a sample.

The analysis method can vary depending on the type of mRNA information required. Examples of methods include, but are not limited to, Northern blots and PCR-based methods (eg, qRT-PCR). Methods such as qRT-PCR can also accurately determine the amount of mRNA in a sample.

Any suitable assay platform can be used to determine the presence of mRNA in a sample. For example, the assay may be in the form of a test strip, membrane, chip, disk, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The assay system may have a solid support to which a nucleic acid corresponding to the mRNA is attached. The solid support may comprise, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide. The assay components can be prepared and packaged together as an mRNA detection kit.

If desired, the nucleic acid can be labeled to produce a population of labeled mRNAs. Typically, the sample can be labeled using methods well known in the art (eg, using DNA ligase, terminal transferase or RNA backbone labeling, etc.). See, for example, Ausubel et al., Short Protocols in Molecular Biology (Wiley & Sons, 3rd ed. 1995); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 3rd ed. 2001). In some embodiments, the sample is labeled with a fluorescent label. Examples of fluorescent dyes

- 85 045896 include xanthene dyes, fluorescent dyes (for example, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6 carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy4' ,5'-dichloro-2',7'-dimethoxyfluorescein (JOE)), rhodamine dyes (e.g. rhodamine 110 (R110), KKI.I-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine ( ROX), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine 6G (R6G6 or G6)), cyanine dyes (e.g. Cy3, Cy5 and Cy7), Alexa dyes (e.g. Alexa-fluor-555), coumarin, diethylaminocoumarin, umbelliferone, benzimide dyes (eg, Hoechst 33258), phenanthridine dyes (eg, Texas Red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, fluorescein chlorotriazinyl , eosin dyes, tetramethylrhodamine, lissamine, naptofluorescein and the like, but are not limited to them.

In some embodiments, the mRNA sequences comprise at least one biomarker mRNA provided herein. In some embodiments, the biomarker is selected from the group consisting of mRNA CRBN, IKZF1, IKZF3, ZFP91, c-MYC, IRF4, p21, p27, pRbl, caspase-1, caspase-3, caspase-7, PARP, survivin, BIM, IL-2, TNFa, IFNy or a fragment thereof.

In one embodiment, the biomarker is selected from the group consisting of CRBN, IKZF1, IKZF3, ZFP91, c-MYC, IRF4, p21, p27, pRbl, caspase-1, caspase-3, caspase-7, PARP, survivin, BIM mRNA , IL-2, TNFa, IFNy or a fragment thereof. In one embodiment, the mRNA is CRBN mRNA. In another embodiment, the mRNA is IKZF1 mRNA. In yet another embodiment, the mRNA is IKZF3 mRNA. In another embodiment, the mRNA is c-MYC mRNA. In yet another embodiment, the mRNA is IRF4 mRNA. In some embodiments, the mRNA is ZFP91. In some embodiments, the mRNA is p21. In some embodiments, the mRNA is p27. In some embodiments, the mRNA is pRb1. In some embodiments, the mRNA is caspase-1. In some embodiments, the mRNA is caspase-3. In some embodiments, the mRNA is caspase-7. In some embodiments, the mRNA is PARP. In some embodiments, the mRNA is survivin. In some embodiments, the mRNA is BIM. In some embodiments, the mRNA is IL-2. In some embodiments, the mRNA is TNFa. In some embodiments, the mRNA is IFNy. The nucleic acids may be present at specific targeted positions on the solid support, each of which corresponds to at least a portion of the mRNA sequences that are differentially expressed upon treatment with the compound in a cell or in a patient.

In some embodiments, the biomarker is a plurality of mRNA sequences that are differentially expressed when a cell or patient is treated with a compound compared to control treatment. In some embodiments, the biomarker is a plurality of mRNA sequences that are differentially expressed in two different populations of cells upon treatment with a compound. In some embodiments, the biomarker is a plurality of mRNA sequences that are differentially expressed in CD138+ cells and CD138− cells in response to treatment with Compound 1, Compound 2, or Compound 3.

A typical method for mRNA analysis may include the following steps: 1) obtaining surface-bound probes of interest; 2) hybridization of the mRNA population with surface-bound probes under conditions sufficient to ensure specific binding; (3) washing after hybridization to remove nucleic acids not specifically bound to the surface-bound probes; and (4) detection of hybridized mRNAs. The reagents used in each of these steps and the conditions under which they are used may vary depending on the specific application.

Hybridization can be carried out under suitable hybridization conditions, which can be varied in stringency if desired. Typical conditions are sufficient to obtain probe/target complexes on a solid surface between complementary binding elements, that is, between surface-bound probes of interest and complementary mRNAs in the sample. In some embodiments, stringent hybridization conditions may be used.

Hybridization is usually carried out under stringent hybridization conditions. Standard hybridization techniques (eg, under conditions sufficient to ensure specific binding of the target mRNAs in the sample to the probes) are described in Kallioniemi et al., Science 1992, 258:818-821 and International Patent Application Publication No. WO 93/18186. Several manuals on general methods are available, for example Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993). For a description of methods suitable for in situ hybridization, see Gall et al., Meth. Enzymol. 1981, 21:470-480; Angerer et al., Genetic Engineering: Principles and Methods, Vol 7, pgs 43-65 (Plenum Press, New York, Setlow and Hollaender, eds. 1985). The choice of suitable conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, stringency of washing conditions, etc., will depend on the experimental design

- 86 045896 ment, including the source of the sample, the identity of the capture agents, the expected degree of complementarity, etc., and can be determined by routine experiments for those skilled in the art.

Those skilled in the art will readily appreciate that alternative but comparable hybridization and wash conditions can be used to provide conditions of similar stringency.

After the mRNA hybridization procedure, surface-bound polynucleotides are typically washed to remove unbound nucleic acids. Laundering can be performed using any convenient laundering protocol where the laundering conditions are typically stringent, as described above. Hybridization of the target mRNA to the probes is then detected using standard methods.

Other methods, such as PCR-based methods, can also be used to detect the expression of CRBN or a protein that is directly or indirectly affected by CRBN. Examples of PCR methods can be found in US Pat. No. 6,927,024, which is incorporated herein by reference in its entirety. Examples of real-time PCR methods can be found in US Pat. No. 7,122,799, which is incorporated herein by reference in its entirety. The fluorescent in situ PCR method is described in US Pat. No. 7,186,507, which is incorporated herein by reference in its entirety.

In some embodiments, quantitative reverse transcription PCR (RT-PCR) can be used to both detect and quantify RNA targets (Bustin et al., Clin. Sci. 2005, 109:365-379). Quantitative results obtained using RT-PCR are usually more informative than qualitative data. Thus, in some embodiments, RT-PCR based assays may be useful for measuring mRNA levels during cell-based assays. The RT-PCR method is also applicable for monitoring patient therapy. Examples of RT-PCR based methods can be found, for example, in US Pat. No. 7,101,663, which is incorporated herein by reference in its entirety.

Unlike conventional reverse transcriptase PCR and agarose gel analysis, RTPCR provides quantitative results. An additional advantage of RT-PCR is its relative simplicity and ease of use. RT-PCR instruments such as the Applied Biosystems 7500 are commercially available, as are reagents such as TaqMan® Sequence Detection Chemistry. For example, TaqMan® gene expression assays can be used according to the manufacturer's instructions. These kits are pre-designed gene expression assays for the rapid and reliable detection and quantification of human, mouse and rat mRNA transcripts. A sample RT-PCR program, for example, is 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, then 60°C for 1 min.

To determine the cycle number at which the fluorescence signal associated with a particular amplicon accumulation crosses a threshold value (called CT), the data can be analyzed, for example, using 7500 Real-Time PCR System Sequence Detection software versus using a comparative method for calculating the relative CT quantification. Using this method, the result is expressed as the fold change in expression levels. In some embodiments, the threshold level may be selected to be automatically determined by software. In some embodiments, the threshold level is set above the baseline level, but low enough to be within the exponential growth region of the amplification curve.

In some embodiments, mRNA expression is determined by RNA sequencing (RNA-seq). Methods for RNA-seq are well known to those skilled in the art and are described in Waern et al., Methods Mol Biol., 2011, 759:125-132; Wilhelm et al., Nature Protocols, 2010, 5(2):255-66; and Hoeijmakers et al., Methods Mol Biol., 2013, 923:221-39. Briefly, a typical RNA-seq method may involve the step of (1) isolating RNA; (2) ribosomal RNA depletion; (3) cDNA synthesis and (4) cDNA sequencing by Next-Gen sequencing.

5.6 Methods for determining polypeptide or protein levels in a sample

To measure the level of a biomarker, such as CRBN, or a protein that is directly or indirectly affected by CRBN, such as Aiolos and Ikaros, several protein detection and quantification methods can be used. Any suitable protein quantitation method can be used. In some embodiments, antibody-based methods are used. Examples of methods that can be used include, but are not limited to, immunoblotting (Western blotting), ELISA, immunohistochemistry, flow cytometry, fluorescence activated cell sorting (FACS), bead array cytometry, mass spectroscopy, and the like. Several types of ELISA are commonly used, including direct ELISA, indirect ELISA, and sandwich ELISA.

In some embodiments, the biomarker is selected from the group consisting of proteins CRBN, IKZF1, IKZF3, ZFP91, c-MYC, IRF4, p21, p27, pRb1, caspase-1, caspase-3, caspase-7, PARP, survivin, BIM, IL-2, TNFa, ZFNy, soluble BCMA, free lambda light chain and free kappa light chain. In some embodiments, the biomarker is a protein that is directly targeted

- 87 045896 or indirectly affects CRBN. In some embodiments, the biomarker is a CRBN protein. In a specific embodiment, the biomarker is CRBN and is detected by IHC. In another specific embodiment, the biomarker is CRBN, Aiolos, Ikaros, ZFP91, c-Myc, IRF4, p-caspase-3 and/or TIL, and is determined by IHC. In some embodiments, the biomarker is selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC, IRF4. In some embodiments, the biomarker is selected from the group consisting of Aiolos and Ikaros and is detected using FACS. In some embodiments, the biomarker is selected from the group consisting of Aiolos and Ikaros and is detected by ELISA. In some embodiments, the biomarker is selected from the group consisting of caspase-1, caspase-3, caspase-7, PARP, survivin, BIM, free lambda light chain, and free kappa light chain. In some embodiments, the biomarker is selected from the group consisting of a free lambda light chain and a free kappa light chain. In a specific embodiment, the biomarker is Aiolos. In a specific embodiment, the biomarker is Ikaros. In a specific embodiment, the biomarker is c-MYC. In a specific embodiment, the biomarker is IRF4. In a specific embodiment, the biomarker is cleaved caspase-3. In another specific embodiment, the biomarker is ZFP91. In yet another specific embodiment, the biomarker is p21. In some embodiments, the biomarker is p27. In some embodiments, the biomarker is p27. In some embodiments, the biomarker is pRb1. In some embodiments, the biomarker is caspase-1. In some embodiments, the biomarker is caspase-3. In some embodiments, the biomarker is caspase-7. In some embodiments, the biomarker is PARP. In some embodiments, the biomarker is survivin. In some embodiments, the biomarker is a BIM. In some embodiments, the biomarker is IL-2. In some embodiments, the biomarker is p27. In some embodiments, the biomarker is TNFa. In some embodiments, the biomarker is IFNy. In some embodiments, the biomarker is a soluble BCMA. In some embodiments, the biomarker is a free lambda light chain and a free kappa light chain. In a specific embodiment, the biomarker is a free lambda light chain. In a specific embodiment, the biomarker is a free kappa light chain.

5.7 Connections

In some embodiments of the various methods provided herein, the compound is 4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4yl)oxy)methyl) benzyl)piperazin-1-yl)-3-fluorobenzonitrile (Compound 1) or an enantiomer thereof, or a mixture of enantiomers, a tautomer, an isotopologue or a pharmaceutically acceptable salt.

In some embodiments of the various methods presented herein, the compound is ^)-4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4yl)oxy )methyl)benzyl)piperazin-1-yl)-3-fluorobenzonitrile (Compound 2) or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments of the various methods presented herein, the compound is ^)-4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4yl)oxy )methyl)benzyl)piperazin-1-yl)-3-fluorobenzonitrile (Compound ³ ) or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

The various compounds provided herein may contain chiral centers and may exist as mixtures of enantiomers (eg, racemic mixtures) or mixtures of diastereomers. The methods provided herein cover the use of stereomerically pure

- 88 045896 forms of such compounds, as well as mixtures of these forms. For example, the methods presented herein may use mixtures containing equal or unequal amounts of enantiomers of a particular compound. These isomers can be synthesized asymmetrically or separated using standard techniques such as chiral columns or chiral resolving agents. See Jacques et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen et al., Tetrahedron 1977, 33:2725-2736; Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, Tables of Resolving Agents and Optical Resolutions, p. 268 (Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

Also provided herein are isotopically enriched analogues of the compounds proposed herein. Isotope enrichment (eg, deuteration) of pharmaceuticals to improve pharmacokinetic properties (PK), pharmacodynamic properties (PD), and toxicity profiles has been previously demonstrated for several classes of drugs. See, for example, Lijinsky et al., Food Cosmet. Toxicol., 20: 393 (1982); Lijinsky et al., J. Nat. Cancer Inst., 69: 1127 (1982); Mangold et al., Mutation Res. 308:33 (1994); Gordon et al., Drug Metab. Dispos., 15: 589 (1987); Zello et al., Metabolism, 43: 487 (1994); Gately et al., J. Nucl. Med., 27: 388 (1986); Wade D, Chem. Biol. Interact. 117: 191 (1999).

Without being limited to any particular theory, isotope enrichment of a drug can be used, for example, to: (1) reduce or eliminate unwanted metabolites, (2) increase the half-life of the parent drug, (3) reduce the number of doses required to achieve the desired effect (4) reduce the dose size required to achieve the desired effect, (5) increase the formation of active metabolites, if they are formed, and/or (6) reduce the production of harmful metabolites in specific tissues and/or create a more effective drug and/or a safer drug for combination therapy, whether the combination therapy is planned or not.

Replacing an atom with one of its isotopes often results in a change in the reaction rate of a chemical reaction. This phenomenon is known as the kinetic isotope effect (KIE). For example, if a C-H bond is broken at the rate-determining step of a chemical reaction (i.e., the step with the highest transition state energy), substituting deuterium for that hydrogen atom will reduce the rate of the reaction and the process will slow down. This phenomenon is known as the deuterium kinetic isotope effect (DKIE) (See, for example, Foster et al., Adv. Drug Res., vol. 14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol., vol. 77, pp. 79-88 (1999)).

The DKIE value can be expressed as the ratio between the rates of a given reaction in which the C-H bond is broken, and the same reaction, provided that deuterium is replaced by hydrogen. DKIE can range from about 1 (no isotope effect) to very large numbers, such as 50 or more, meaning the reaction can be fifty times slower or more, provided the deuterium is replaced with hydrogen. Without being bound by theory, high DKIE values may be due in part to a phenomenon known as tunneling, which is a consequence of the uncertainty principle. Tunneling is attributed to the low mass of the hydrogen atom and occurs because transition states involving a proton can sometimes occur in the absence of the required activation energy. Since deuterium has more mass than hydrogen, it is statistically much less likely to be affected by this phenomenon.

Tritium (T) is a radioactive isotope of hydrogen used in research, fusion reactors, neutron generators, and radiopharmaceuticals. Tritium is a hydrogen atom that has 2 neutrons in its nucleus and has an atomic weight close to 3. It occurs naturally in the environment in very low concentrations, most often found in the form T2O. Tritium decays slowly (half-life=12.3 years) and emits low-energy beta particles that cannot penetrate the outer layer of human skin. Internal exposure is the main hazard associated with this isotope, but it must be taken in large quantities to incur significant health risks. Compared to deuterium, smaller amounts of tritium must be consumed before reaching dangerous levels. Substitution of tritium (T) for hydrogen results in an even stronger bond than in the case of deuterium and produces numerically larger isotope effects.

Likewise, substitution of isotopes of other elements, including, but not limited to, C or C in the case of carbon, ³³ S, ³⁴ S or ³⁶ S in the case of sulfur, ¹⁵ N in the case of nitrogen, and ¹⁷ O or ¹⁸ O in the case of oxygen, will provide similar kinetics isotope effects.

The animal's body expresses a variety of enzymes for the purpose of eliminating foreign substances, such as therapeutic agents, from its circulation. Examples of such enzymes include cytochrome P450 (CYP) enzymes, esterases, proteases, reductases, dehydrogenases and monoamine oxidases to react with and convert these foreign substances into more polar intermediates or metabolites for renal excretion. Some of the most common metabolic reactions of pharmaceutical compounds involve the oxidation of a carbon-hydrogen (C-H) bond into either a carbon-oxygen (C-O) or a carbon-carbon (C-C) pi bond. The resulting metabolites may be stable or unstable under physiological conditions, and may have significantly different pharmacokinetic, pharmacodynamic, and short- and long-term toxicity profiles relative to the parent compounds. For many drugs, such oxidation is rapid. As a result, these drugs often require multiple or high daily doses.

Isotopic enrichment at certain positions of a compound provided herein may have a detectable KIE that affects the pharmacokinetic, pharmacological and/or toxicological profiles of the compound provided herein compared to a similar compound having a naturally occurring isotopic composition. In one embodiment, deuterium enrichment occurs at the C-H bond cleavage position during metabolism.

Standard physiological, pharmacological and biochemical procedures are available to test compounds to identify those that have the desired antiproliferative activity.

Such assays include, for example, biochemical assays such as binding assays, radioactive inclusion assays, and various cell-based assays.

The compounds provided herein can be prepared by methods known to one of ordinary skill in the art and by following procedures similar to those described in the Examples section of this document and customary modifications thereof.

It is understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be construed as limiting the scope of the invention. Various changes and modifications to the described embodiments will be apparent to those skilled in the art. Such changes and modifications, including, without limitation, those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, formulations and/or methods of use provided herein, may be made without departing from the spirit and scope of the document. US patents and publications referenced herein are incorporated herein by reference.

5.8 Pharmaceutical compositions

The pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more compounds provided herein, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

These compounds may be formulated into suitable dosage forms such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release preparations or elixirs, for oral administration, or in sterile solutions or suspensions for ophthalmic or parenteral administration, as well as transdermal patch and dry powder inhalers. Typically, the compounds described above are formulated into pharmaceutical compositions using methods and techniques well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, 7th ed. 1999).

In these compositions, effective concentrations of one or more compounds or pharmaceutically acceptable salts thereof are admixed with a suitable pharmaceutical carrier or excipient. In specific embodiments, the concentrations of the compounds in the compositions are effective to deliver an amount that, when administered, treats, prevents, or alleviates one or more symptoms and/or progression of multiple myeloma.

Typically, the compositions are formulated for a single dose administration. To prepare the composition, a bulk portion of the compound is dissolved, suspended, dispersed, or otherwise mixed in the selected excipient at such an effective concentration that the pathological condition is alleviated or improved. Pharmaceutical carriers or excipients suitable for administering the compounds provided herein include any such carriers known to those skilled in the art suitable for the particular route of administration.

In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in a composition or may be combined with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes such as tumor-targeted liposomes, may also be useful as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to those skilled in the art. For example, liposomal dosage forms can be prepared as known in the art. Briefly, liposomes such as multilayer vesicles (MLVs) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) on the inside of a flask. A solution of the compound provided herein in phosphate buffered saline (PBS) lacking divalent cations is added and the flask is stirred until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation and then resuspended in PBS.

The active compound is included in the pharmaceutically acceptable excipient in an amount sufficient

- 90 045896 precise to provide a therapeutically beneficial effect in the absence of undesirable side effects for the patient undergoing treatment. The therapeutically effective concentration can be determined empirically by testing the compounds in the in vitro and in vivo systems described herein and then extrapolated from this for human dosing.

The concentration of the active compound in the pharmaceutical composition will depend on the rates of absorption, tissue distribution, inactivation, metabolism and excretion of the active compound, the physicochemical characteristics of the compound, the dosage schedule and amount of the substance administered, as well as other factors known to those skilled in the art. For example, the amount that is delivered is sufficient to improve one or more symptoms of cancer, including solid tumors and hematologic tumors.

Solutions or suspensions for parenteral, intradermal, subcutaneous or topical use may include any of the following: a sterile diluent such as water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol, dimethyl acetamide or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates and phosphates; and tonicity adjusters such as sodium chloride or dextrose. Parenteral preparations may be contained in ampoules, pen syringes, disposable syringes, or single-use or refillable vials of glass, plastic, or other suitable material.

In cases where compounds exhibit insufficient solubility, methods for solubilizing compounds can be used. Such methods are known to those skilled in the art and include, but are not limited to: the use of co-solvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as TWEEN®, or dissolution in an aqueous solution of sodium bicarbonate.

When the compound(s) are mixed or added, the resulting mixture may be a solution, suspension, emulsion, or the like. The dosage form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the selected carrier or excipient. An effective concentration is sufficient to reduce the symptoms of a disease, disorder or pathology, and can be determined empirically.

Pharmaceutical compositions are intended for administration to humans and animals in unit dose dosage forms such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, as well as oil-water emulsions containing suitable the amount of compounds or pharmaceutically acceptable salts thereof. Pharmaceutically therapeutically active compounds and their salts are formulated and administered in unit dosage forms or multiple dose dosage forms. Unit dosage forms as used herein refer to physically discrete units suitable for use in humans and animals and are individually packaged as is known in the art. Each unit dose contains a predetermined amount of a therapeutically active compound sufficient to produce the desired therapeutic effect, in combination with the necessary pharmaceutical carrier, excipient or diluent. Examples of unit dosage forms include ampoules and syringes, and individually packaged tablets or capsules. Unit dosage forms may be administered in portions or multiples thereof. A multiple-dose dosage form is a plurality of identical single-dose dosage forms packaged in a single container to be administered in separate single-dose dosage forms. Examples of multi-dose dosage forms include vials, tablet or capsule bottles, or pint or gallon bottles. Therefore, multiple dose dosage forms are multiples of unit doses that are not divided in the package.

Dosage forms or compositions can be prepared containing the active ingredient in the range of 0.005 to 100%, the remainder being a non-toxic carrier. For oral administration, a pharmaceutically acceptable non-toxic composition is prepared by adding any of the commonly used excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, cross-carmellose sodium, glucose, sucrose, magnesium carbonate or sodium saccharin. Such compositions include solutions, suspensions, tablets, capsules, powders and sustained release dosage forms such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparing these compositions are known to those skilled in the art.

The active compounds or pharmaceutically acceptable salts can be formulated with carriers that protect the compound from rapid elimination from the body, such as time-release dosage forms or coated formulations.

- 91 045896

The compositions may contain other active compounds to provide the desired combinations of properties. The compounds provided herein, or their pharmaceutically acceptable salts as described herein, may also advantageously be administered for therapeutic or prophylactic purposes together with another pharmacological agent that is known in the art to be useful for treating one or more diseases or medical conditions mentioned above in this document, such as diseases associated with oxidative stress. It should be understood that such combination therapy represents an additional aspect of the compositions and methods of treatment presented herein.

5.9 Sets

In one aspect, provided herein is a kit for identifying a subject having a cancer that is likely to be susceptible to a treatment compound, comprising a means for determining the level of a biomarker in a sample that has been treated with a treatment compound, wherein the treatment compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In one aspect, provided herein is a kit for identifying a subject having a cancer that is likely to be susceptible to a treatment compound, comprising a means for determining the level of a biomarker in a sample that has been treated with a treatment compound, wherein the treatment compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In one aspect, provided herein is a kit for identifying a subject having a cancer that is likely to be susceptible to a treatment compound, comprising a means for determining the level of a biomarker in a sample that has been treated with a treatment compound, wherein the treatment compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a kit for the treatment of cancer comprising an agent for determining the level of a biomarker in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a kit for the treatment of cancer comprising an agent for determining the level of a biomarker in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a kit for the treatment of cancer comprising an agent for determining the level of a biomarker in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound is Compound 3

- 92 045896

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising a means for determining the level of a biomarker in a sample that has been treated with the therapeutic compound, wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising a means for determining the level of a biomarker in a sample that has been treated with the therapeutic compound, wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising a means for determining the level of a biomarker in a sample that has been treated with the therapeutic compound, wherein the therapeutic compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising a means for determining the level of a biomarker in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising a means for determining the level of a biomarker in a sample that has been treated with the therapeutic compound, wherein the therapeutic compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising a means for determining the level of a biomarker in a sample that has been treated with a therapeutic compound, wherein the therapeutic compound is Compound 3

- 93 045896

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for identifying a subject having multiple myeloma who is likely to be susceptible to a treatment compound, comprising a means for determining the level of a biomarker in a sample that has been treated with the treatment compound, wherein the treatment compound is Compound 1

or an enantiomer, mixture of enantiomers, tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for identifying a subject having multiple myeloma who is likely to be susceptible to a treatment compound, comprising a means for determining the level of a biomarker in a sample that has been treated with the treatment compound, wherein the treatment compound is Compound 2

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit for identifying a subject having multiple myeloma who is likely to be susceptible to a treatment compound, comprising a means for determining the level of a biomarker in a sample that has been treated with the treatment compound, wherein the treatment compound is Compound 3

or a tautomer, isotopologue or pharmaceutically acceptable salt thereof.

In some embodiments, the biomarker detected by the various kits provided herein is CRBN. In some embodiments, the biomarker detected by the various kits provided herein is CRBN-associated protein (CAP). In some embodiments, the biomarker includes one ATS. In some embodiments, the biomarker includes two ATS. In still other embodiments, the biomarker includes three ATS. In yet other embodiments, the biomarker includes four ATS. In some embodiments, the biomarker includes five or more ATS.

In some embodiments, the biomarker detected by the various kits provided herein is an ATS selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC, IRF4. In some embodiments, the biomarker is selected from the group consisting of IKZF1 and IKZF3. In some embodiments, the biomarker is IKZF1. In some embodiments, the biomarker is IKZF3. In other embodiments, the biomarker is ZFP91. In yet another embodiment, the biomarker is c-MYC. In other embodiments, the biomarker is IRF4.

In some embodiments, the biomarker detected by the various kits provided herein is involved in apoptosis. In some embodiments, the biomarker detected by the various kits provided herein is involved in apoptosis and is selected from the group consisting of CTC, caspase-1, caspase-3, caspase-7, PARP, BIM, survivin, free lambda light chain serum and free kappa light chain in serum. In some embodiments, the biomarker detected by the various kits provided herein is involved in apoptosis and is selected from the group of free serum lambda light chain and free serum kappa light chain. In some embodiments, the biomarker detected by the various kits provided herein is CTC. In some embodiments, the biomarker detected by the various kits provided herein is caspase-3. In some embodiments, the biomarker detected by the various kits provided herein is caspase-7. In some embodiments, the biomarker detected by the various kits is

- 94 045896 presented in this document is survivin. In some embodiments, the biomarker detected by the various kits provided herein is BIM. In some embodiments, the biomarker detected by the various kits provided herein is caspase-1. In some embodiments, the biomarker detected by the various kits provided herein is PARP. In specific embodiments, the biomarker detected by the various kits provided herein is free lambda light chain in serum. In specific embodiments, the biomarker detected by the various kits provided herein is free kappa light chain in serum.

In some embodiments, the biomarker detected by the various kits provided herein is involved in the cell cycle. In some embodiments, the biomarker detected by the various kits provided herein is involved in the cell cycle and is selected from the group consisting of p21, p27, and pRb1. In specific embodiments, the biomarker detected by the various kits provided herein is p21. In specific embodiments, the biomarker detected by the various kits provided herein is p27. In specific embodiments, the biomarker detected by the various kits provided herein is pRb1.

In still other embodiments, the biomarker detected by the various kits provided herein is associated with T cell activation. In some embodiments, the biomarker detected by the various kits provided herein is associated with T cell activation and is selected from the group consisting of the T cell activation associated cytokines IL-2, TNFa, and IFNy.

In some embodiments of the various kits provided herein, the sample is obtained from a tumor biopsy, a node biopsy, a liquid biopsy (eg, blood), or a bone marrow biopsy.

In some embodiments of the various kits provided herein, the cancer is a blood cancer. In some embodiments, the blood cancer is selected from the group consisting of multiple myeloma. In some embodiments, the multiple myeloma is relapsed, refractory, or resistant to conventional therapy.

In some embodiments, provided herein is a kit for determining the mRNA level of one or more biomarkers. In some embodiments, the kit includes one or more probes that specifically bind to the mRNA of one or more biomarkers. In some embodiments, the kit further includes a wash solution. In some embodiments, the kit further includes reagents for performing a hybridization assay, means for isolating or purifying mRNA, means for detection, and positive and negative controls. In some embodiments, the kit further includes reagents for generating cDNA from mRNA. In some embodiments, the kit further includes reagents for sequencing cDNA derived from mRNA. In some embodiments, the kit further includes instructions for using the kit. The kit can be adapted for home, clinical or research use.

In some embodiments, provided herein is a kit for determining the protein level of one or more biomarkers. In some embodiments, the kits contain a test strip coated with an antibody that recognizes the biomarker protein, wash solutions, assay reagents, protein isolation or purification means, detection means, and positive and negative controls. In some embodiments, the kit further includes instructions for using the kit. The kit can be adapted for home, clinical or research use.

Such a kit may use, for example, test strips, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a glass slide, a multiwell plate, or an optical fiber. The solid support of the kit may be, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide. The biological sample may be, for example, a cell culture, cell line, tissue, organ, organelle, biological fluid, blood sample, urine sample, or skin sample.

In another embodiment, the kit contains a solid support, nucleic acids attached to the support, wherein the nucleic acids are complementary to at least 20, 50, 100, 200, 350 or more bases of the mRNA, and means for detecting expression of the mRNA in a biological sample.

In a specific embodiment, the pharmaceutical or assay kit contains the compound or pharmaceutical composition thereof in a container and further contains components for RNA extraction in one or more containers. In another specific embodiment, the pharmaceutical or assay kit contains a compound or pharmaceutical in a container.

- 95 045896 composition and additionally contains in one or more containers components for RTPCR, real-time qPCR, deep sequencing or microarray analysis.

In some embodiments, the kits provided herein utilize tools to detect biomarker expression using quantitative real-time PCR (qRT-PCR), microarray analysis, flow cytometry, FACS, FISH, DNA sequencing, RNA sequencing, hematoxylin staining and eosin (H&E), immunohistochemistry or immunofluorescence. In other embodiments, biomarker expression is measured using ELISA-based methodologies or other similar methods known in the art.

In another specific embodiment, the pharmaceutical or assay kit contains a compound or pharmaceutical composition thereof in a container and further contains protein isolation components in one or more containers. In another specific embodiment, the pharmaceutical or assay kit contains a compound or pharmaceutical composition in a container and further contains flow cytometry or ELISA components in one or more containers.

In another aspect, provided herein are biomarker measurement kits that provide the materials needed to measure the content of one or more biomarker gene products or a subset of biomarkers (e.g., one, two, three, four, five or more biomarkers) provided herein . Such kits may include the materials and reagents needed to measure DNA, RNA, protein, or cell populations. In some embodiments, such kits include microarrays, where the microarray consists of oligonucleotides and/or DNA and/or RNA fragments that hybridize to one or more biomarker gene products or a subset of biomarkers provided herein, or any combination thereof. In some embodiments, such kits may include PCR primers for either the RNA product, cDNA copies of the RNA product of the biomarkers or a subset of the biomarkers, or both. In some embodiments, such kits may include PCR primers as well as qPCR probes. In some embodiments, such kits may include multiple primers and multiple probes, some of the probes having different fluorophores, allowing the simultaneous measurement of multiple biomarker gene products or a subset of the biomarkers provided herein. In some embodiments, such kits may further include materials and reagents for generating cDNA from RNA. In some embodiments, such kits may include antibodies specific to the protein products of biomarkers or a subset of biomarkers provided herein. Such kits may further include materials and reagents for isolating RNA and/or proteins from a biological sample. In addition, such kits may include materials and reagents for synthesizing cDNA from RNA isolated from a biological sample. In some embodiments, such kits may include a computer program product embedded in a computer-readable medium for predicting whether a patient is clinically sensitive to a compound. In some embodiments, the kits may include a computer program product embedded in a computer-readable medium along with instructions.

In some embodiments, such kits measure the expression of one or more nucleic acid products of biomarkers or a subset of biomarkers provided herein. In accordance with this embodiment, the kits may contain materials and reagents that are needed to measure the expression of specific nucleic acid products of biomarkers or a subset of biomarkers presented herein. For example, a microarray or RT-PCR kit may be customized for a specific condition and contain only those reagents and materials needed to measure levels of specific RNA transcription products of biomarkers or a subset of biomarkers presented herein to predict whether hematological cancer in a patient clinically sensitive to the compound. Alternatively, in some embodiments, the kits may include the materials and reagents needed to measure the expression of specific nucleic acid products of genes other than the biomarkers provided herein. For example, in some embodiments, the kits contain the materials and reagents needed to measure expression levels 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 , 50 or more biomarker genes provided herein, in addition to the reagents and materials necessary to measure the expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, according to at least 40, at least 45, at least 50 or more genes other than the biomarkers presented herein. In other embodiments, the kits contain the reagents and materials necessary to measure the expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6,

- 96 045896 at least 7, at least 8, at least at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more biomarkers presented herein, and 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or more genes that are not biomarkers, presented in this document. In specific embodiments, the kits contain the reagents and materials necessary to measure the expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more biomarkers presented herein, and 1-10, 1100, 1-150, 1-200, 1-300, 1-400, 1-500, 1-1000, 25-100, 25- 200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 genes that are not biomarkers presented in this document.

Nucleic acid microarray kits typically contain probes attached to the surface of a solid support. In one such embodiment, the probes can be either oligonucleotides or longer probes, including probes ranging from 150 nucleotides to 800 nucleotides in length. The probes may be labeled with a detectable label. In a specific embodiment, the probes are specific for one or more biomarker gene products provided herein. Microarray kits may contain instructions for performing the assay and methods for interpreting and analyzing the data obtained from the assay. In a specific embodiment, the kits contain instructions for predicting whether a patient's hematologic cancer is clinically sensitive to a compound. The kits may also contain hybridization reagents and/or reagents necessary to detect the signal generated when the probe hybridizes to a target nucleic acid sequence. Typically, the materials and reagents for microarray kits are contained in one or more containers. Each component of the kit is usually contained in its own suitable container.

In some embodiments, the nucleic acid microarray kit contains the materials and reagents necessary to measure expression levels of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more biomarker genes provided herein, or combinations thereof, in addition to the reagents and materials necessary to measure the expression levels of at least 1, at least 2, at least 3, at least 4 , at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, according at least 35, at least 40, at least 45, at least 50 or more genes other than the biomarker genes presented herein. In other embodiments, the nucleic acid microarray kit contains the reagents and materials necessary to measure the expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more biomarker genes provided herein, or any combination thereof 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or more genes that are not biomarkers, provided in this document. In another embodiment, the nucleic acid microarray kit contains the reagents and materials necessary to measure the expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more biomarkers presented herein, or any combination thereof, and 1-10, 1-100, 1-150, 1-200, 1-300, 1 -400, 1-500, 1-1000, 25-100, 25-200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300, 100-400 , 100-500, 100-1000, or 500-1000 genes that are not biomarkers presented in this document.

qPCR kits typically contain preselected primers specific for specific nucleic acid sequences. qPCR kits may also include enzymes suitable for amplification of nucleic acids (eg, polymerases such as Taq polymerase), deoxynucleotides and buffers required for the amplification reaction. qPCR kits may also include probes specific for nucleic acid sequences associated with or indicative of a condition. The probes may or may not be labeled with a fluorophore. The probes may or may not be labeled with a quencher molecule. In some embodiments, qPCR kits also contain components suitable for reverse transcription of RNA, including enzymes (eg

- 97 045896 measures, reverse transcriptases such as AMV, MMLV, etc.) and primers for reverse transcription, as well as deoxynucleotides and buffers necessary for the reverse transcription reaction. Each component of a qPCR kit is usually contained in its own suitable container. Thus, these kits typically contain separate containers suitable for each individual reagent, enzyme, primer and probe. In addition, qPCR kits may contain instructions for performing the reaction and methods for interpreting and analyzing the data obtained from the reaction. In a specific embodiment, the kits contain instructions for predicting whether a patient with or suspected of having multiple myeloma is clinically sensitive to a compound.

Antibody-based kits may include, for example: (1) a first antibody (which may or may not be attached to a solid support) that binds to the peptide, polypeptide or protein of interest; and, optionally, (2) a second, different antibody that binds to either the first antibody or a peptide, polypeptide, or protein and is conjugated to a detectable label (eg, a fluorescent label, radioactive isotope, or enzyme). In a specific embodiment, the peptide, polypeptide or protein of interest is associated with or indicative of a condition (eg, disease). Antibody kits may also include immunoprecipitation beads. Each component of antibody kits is typically contained in its own suitable container. Thus, these kits typically contain separate containers suitable for each antibody and reagent. In addition, antibody kits may contain instructions for performing the assay and methods for interpreting and analyzing data obtained from the assay. In a specific embodiment, the kits contain instructions for predicting whether a patient's hematologic cancer is clinically sensitive to a compound.

In one embodiment, the kit provided herein includes a compound provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof. The kits may further include additional active agents, including, but not limited to, those disclosed herein.

The kits provided herein may further include devices that are used to administer the active ingredients. Examples of such devices include, but are not limited to, syringes, droppers, patches, and inhalers.

The kits may further include cells or blood for transplantation, as well as pharmaceutically acceptable carriers that can be used to administer one or more active ingredients. For example, if the active ingredient is provided in a solid form that needs to be reconstituted for parenteral administration, the kit may include a sealed container with a suitable carrier in which the active ingredient can be dissolved to form a sterile, particulate-free solution that is suitable for parenteral administration. Examples of pharmaceutically acceptable carriers include Water for Injection USP; aqueous vehicles (such as, but not limited to, saline injection, Ringer's injection, dextrose injection, dextrose saline injection, and lactated Ringer's injection); water-miscible carriers (such as, but not limited to, ethyl alcohol, polyethylene glycol and polypropylene glycol); and non-aqueous vehicles (such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate).

In some embodiments of the methods and kits provided herein, solid supports are used for protein purification, sample labeling, or solid phase assays. Examples of solid phases suitable for carrying out the methods disclosed herein include beads, particles, colloids, discrete surfaces, tubes, multiwell plates, microtiter plates, slides, membranes, gels and electrodes. When the solid phase is particulate material (eg, granules), in one embodiment, it is distributed into the wells of multiwell plates to allow parallel processing of solid substrates.

It should be noted that any combination of the above embodiments, for example, with respect to one or more reagents such as, but not limited to, nucleic acid primers, solid support, etc., is also contemplated with respect to any of the various methods and/or kits presented in this document.

Certain embodiments of the present invention are illustrated by the following non-limiting examples.

6. Examples

The following examples are performed using standard techniques that are well known and routine for those skilled in the art, unless otherwise described in detail. Examples are for illustration purposes only.

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Example 1. Synthesis of 4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)benzyl)piperazin-1-yl)- 3-fluorobenzonitrile (Compound 1) o

2-Amino-5-methoxy-5-oxopentanoic acid.

To a suspension of 2-aminopentanedic acid (250 g, 1.70 mol) in dry methanol (2.5 L) under nitrogen was added trimethylsilyl chloride (277 g, 2.55 mol) over 30 minutes. The resulting clear solution was stirred at room temperature (20°C) for 30 minutes. ¹ H NMR showed that the starting material was completely consumed. The reaction mixture was used in the next step without further processing.

1H NMR: 400 MHz CD3OD δ: 4.17-4.15 (m, 1H), 3.71 (s, 3H), 2.70-2.60 (m, 2H), 2.33-2.25 (m, 2H).

2-((tert-Butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid.

Triethylamine (275 g, 2.72 mol) and di-tert-butyl dicarbonate (447.35 g, 2.05 mol) were added to the resulting solution. The reaction mixture was stirred at 25°C for 2 hours. The solution was evaporated to dryness, then water (2.5 L) was added to dissolve the residue. The resulting aqueous phase was washed with ethyl acetate (200 ml), then acidified to pH=3 with HCl (1 N) and extracted with ethyl acetate (1 L x 3). The organic layers were washed with brine (800 ml), dried over sodium sulfate, filtered and evaporated to give 2-(tert-butoxycarbonylamino)-5-methoxy-5-oxopentanoic acid (250 g, 56% yield, two steps) as white solid matter.

1H NMR: 400 MHz CD ₃ OD δ: 4.18-4.11 (m, 1H), 3.69 (s, 3H), 2.48-2.43 (m, 2H), 2.21-2.15 (m, 1H), 1.951.91 (m, 1H), 1.46 (s, 9H).

Methyl 5-amino-4-(tert-butoxycarbonylamino)-5-oxopentanoate.

To a solution of 2-(tert-butoxycarbonylamino)-5-methoxy-5-oxopentanoic acid (200 g, 765 mmol) in 1,4-dioxane (1.5 L) was added di-tert-butyl dicarbonate (267 g, 1. 22 mmol) and pyridine (121 g, 1.53 mol). After the reaction mixture was stirred at 25°C for 30 minutes, ammonium carbonate (182 g, 2.30 mol) was added to the mixture and stirred for an additional 16 hours at 25°C. The organic solvent was removed on a rotary evaporator, the residue was acidified with HCl (6 M) to pH=3, and then extracted with ethyl acetate (800 ml x 3). The combined organic phase was washed with brine (800 ml), dried over sodium sulfate and filtered. Volatiles were removed under reduced pressure to give methyl 5-amino-4-(tert-butoxycarbonylamino)-5-oxopentanoate (180 g, 90% yield) as a white solid.

1H NMR: 400 MHz CDCl ₃ δ: 6.51 (s, 1H), 5.94 (s, 1H), 5.43 (s, 1H), 4.21 (s, 1H), 3.63 (s, 3H), 2.59-2.40 (m, 2H), 2.15-2.11 (m, 1H), 1.94-1.90 (m, 1H), 1.42 (s, 9H).

Methyl 4,5-diamino-5-oxopentanoate hydrochloride.

A mixture of methyl 5-amino-4-(tert-butoxycarbonylamino)-5-oxopentanoate (180 g, 692 mmol) and HCl/ethyl acetate (300 ml, 4 M) was stirred at 25°C for 12 hours. The precipitated solid was collected by vacuum filtration and washed with ethyl acetate (500 ml) to obtain methyl 4,5-diamino-5-oxopentanoate hydrochloride (130 g, 95% yield) as a white solid.

1H NMR: 400 MHz CD3OD δ: 4.00-3.96 (m, 1H), 3.70 (s, 3H), 2.59-2.52 (m, 2H), 2.22-2.13 (m, 2H).

Methyl 3-hydroxy-2-methylbenzoate.

Four loads (200 g each) were carried out in parallel. To a solution of 3-hydroxy-2-methylbenzoic acid (200 g, 1.31 mol) in methanol (4.0 L) was added concentrated sulfuric acid (47.7 g, 486 mmol). The reaction mixture was stirred at 60°C for 17 hours. The reaction mixture was evaporated to 800 ml. The resulting mixture was cooled to 20°C and slowly poured into water (400 ml) over 30 min. Water (1200 ml) was added at 20°C over 3 hours and the resulting mixture was stirred at 20°C for 1 hour. The precipitated solid was collected by vacuum filtration (four batches combined) and washed three times with water/methanol (1000 ml, 9: 1) to pH > 3. The solid was dried in vacuo at 45°C to obtain methyl 3-hydroxy-2-methyl benzoate (700 g, 80.4% yield) as a gray solid.

Methyl 3-[tert-butyl(dimethyl)silyl]oxy-2-methylbenzoate.

Two loads (240 g each) were carried out in parallel. To a solution of methyl 3-hydroxy-2-methylbenzoate (240 g, 1.44 mol) in N.N-dimethylformamide (1.40 L) was added imidazole (246 g, 3.61 mol) and tert-butyldimethylsilyl chloride (238 g, 1.58 mol ) at 5°C. After addition, the mixture was heated to 20°C and stirred for 6 hours. Isopropyl acetate (1700 ml) was added and then water (2000 ml) was added slowly while the temperature was maintained below 30°C. The resulting mixture was stirred and the organic phase was separated. The combined organic phase (combined two loads) was washed with water (1700 ml x 3) and evaporated to -1500 ml (KF < 0.05%). The product was stored as a solution in isopropyl acetate, which was used for the next step without further purification.

- 99 045896

Methyl 2-(bromomethyl)-3-[tert-butyl(dimethyl)silyl]oxybenzoate.

Two loads (~375 g each) were carried out in parallel. Nbromosuccinimide (274 g, 1.54 mol) and azobisisobutyronitrile (4.40 g, 26. 8 mmol). The reaction mixture was heated to 70°C for at least 1 hour and stirred at 70°C for 4 hours. The reaction mixture was cooled to 20°C and kept at 20°C for at least 1 hour. Two loads of solid (succinimide) was removed by filtration and washed with isopropyl acetate (700 ml). The filtrate was washed with a saturated solution of sodium sulfite (700 g) in water (6000 ml), then with water (1500 ml). The organic layer was distilled under vacuum at 45°C to dryness to obtain methyl 2-(bromomethyl)-3-[tert-butyl(dimethyl)silyl]oxybenzoate (920 g, 95.5% yield) as a dark orange oil.

¹ H NMR: 400 MHz DMSO-d ₆ δ: 7.45 (d, J=6.8 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 7.13 (t, J=7.2 Hz, 1H), 4.95 ( s, 2H), 1.02 (s, 9H), 0.29 (s, 6H).

Methyl 5-amino-4-[4-[tert-butyl(dimethyl)silyl]oxy-1-oxoisoindolin-2-yl]-5-oxopentanoate.

To a stirred solution of methyl 4,5-diamino-5-oxopentanoate hydrochloride (74.5 g, 379 mmol) in acetonitrile (2.50 L) was added methyl 2-(bromomethyl)-3-[tert-butyl(dimethyl)silyl] hydroxybenzoate (125 g, 348 mmol). Diisopropylethylamine (89.9 g, 696 mmol) was added to the suspension via a dropping funnel over 10 min and then the mixture was stirred at 60°C for 16 h. The reaction mixture was diluted with ethyl acetate (1.0 L), washed successively with HCl (1H, 1.0 L), sodium bicarbonate (us, 1.0 L) and saline (1.0 L). The organic layer was evaporated to give crude methyl 5-amino-4-[4-[tert-butyl(dimethyl)silyl]oxy-1-oxoisoindolin-2-yl]-5-oxopentanoate (108 g, crude product) as a pale yellow solid. LC-MS: m/z 407.3 [M+1]+.

Methyl 5-amino-4-(4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate

To a stirred cold solution of methyl 5-amino-4-[4-[tert-butyl(dimethyl)silyl]oxy-1oxoisoindolin-2-yl]-5-oxopentanoate (108 g, 266 mmol) in N-dimethylformamide (350 ml) Potassium carbonate (14.7 g, 106 mmol) in water (40 ml) was added in portions over 5 min. The resulting reaction mixture was stirred at 15°C for 15 hours. The reaction mixture was cooled in an ice bath and HCl (12 M, 15 ml) was slowly added at 0-5°C. Acetonitrile (200 ml) was added to the mixture and a solid precipitate formed. The suspension was stirred at room temperature for 10 minutes and filtered. The filter cake was washed with ethyl acetate (200 ml x 5) to obtain the product (55 g). The filtrate was evaporated under high vacuum to obtain crude product (100 g) which was dissolved in dichloromethane (1.0 L) and left to stand at 15°C for 16 hours. A white solid formed which was filtered to obtain 5 g of product. The solids were combined to give methyl 5-amino-4-(4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate (60 g, 77% yield) as a white solid.

^1H NMR: 400 MHz DMSO-d6 δ: 7.58 (s, 1H), 7.31 (t, J=8.0 Hz, 1H), 7.19-7.14 (m, 2H), 7.01 (d, J=7.6 Hz, 1H) , 4.75-4.71 (m, 1H), 4.50 (d, J=17.6 Hz, 1H), 4.32 (d, J=17.6 Hz, 1H), 3.51 (s, 3H), 2.29-2.18 (m, 3H), 2.091.99 (m, 1H).

Methyl 5-amino-4-[4-[[4-(bromomethyl)phenyl]methoxy]-1-oxoisoindolin-2-yl]-5-oxopentanoate

Two reactions (25 g, 85.5 mmol) were carried out in parallel. A mixture of 1,4-bis(bromomethyl)benzene (67.7 g, 257 mmol), potassium carbonate (11.8 g, 85.5 mmol) and methyl 5-amino-4-(4-hydroxy-1-oxo- Isoindolin-2yl)-5-oxopentanoate (25 g, 85.5 mmol) in acetonitrile (1 L) was stirred at 60°C for 16 hours. The two batches were combined and the mixture was cooled to 15°C and filtered. The filtrate was concentrated and purified by silica gel column chromatography (eluted with 50% petroleum ether in ethyl acetate to 100% ethyl acetate) to give methyl 5-amino-4-[4-[[4-(bromomethyl)phenyl]methoxy]-1-oxoisoindolin2-yl ]-5-oxopentanoate (52 g, 63% yield) as a white solid.

1H NMR: 400 MHz DMSO-d6 δ: 7.59 (s, 1H), 7.50-7.44 (m, 5H), 7.32-7.28 (m, 2H), 7.19 (s, 1H), 5.26 (s, 2H), 4.79 -4.71 (m, 3H), 4.55 (d, J=17.6 Hz, 1H), 4.43 (d, J=17.6 Hz, 1H), 3.52 (s, 3H), 2.30-2.19 (m, 3H), 2.102. 08 (m, 1H).

3-[4-[[4-(Bromomethyl)phenyl]methoxy]-1-oxoisoindolin-2-yl]piperidin-2,6-dione

Two reactions (28.5 g, 60.0 mmol) were carried out in parallel. Methyl 5-amino-4-[4-[[4-(bromomethyl)phenyl]methoxy]-1-oxoisoindolin-2-yl]-5-oxopentanoate (28.5 g, 60.0 mmol) was dissolved in tetrahydrofuran (720 ml) and the solution was cooled with a dry ice/acetone bath to -70°C. While stirring, potassium tert-butoxide (7.4 g, 66.0 mmol) was added to the clear solution in one portion. The reaction mixture turned pale yellow and stirring was continued for another 2 hours at 70°C. Cooled HCl solution (1H, 260 ml) was quickly added to the reaction mixture, maintaining the temperature at -70°C. The mixture immediately turned milky white and the dry ice/acetone bath was removed. The mixture was evaporated to remove most of the tetrahydrofuran. After evaporation of the reaction mixture, a white solid precipitated. The white suspension was diluted with water (500 ml) and then filtered. The filter cake was washed with water (500 ml) and dried in a vacuum oven at 40°C for 12 hours, then washed with ethyl acetate (500 ml). The charges were combined to give 3-[4-[[4-(bromomethyl)phenyl]methoxy]-1

- 100 045896 oxoisoindolin-2-yl]piperidin-2,6-dione (49.85 g, 93%) as a pale yellow solid.

1H NMR: 400 MHz DMSO-d ₆ δ: 10.95 (s, 1H), 7.51-7.41 (m, 5H), 7.35-7.28 (m, 2H), 5.23 (s, 2H), 5.125.07 (m, 1H ), 4.70 (s, 2H), 4.41 (d, J=17.6 Hz, 1H), 4.25 (d, J=17.6 Hz, 1H), 2.90-2.84 (m, 1H), 2.58-2.53 (m, 1H) , 2.44-2.41 (m, 1H), 1.98-1.95 (m, 1H).

4-(4-(4-(((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)benzyl)piperazin-1 yl)-3-fluorobenzonitrile.

3-(4-((4-(Bromomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidin-2,6-dione (5.0 g, 11.28 mmol) was added to the flask with 3-fluoro -4-(piperazin-1-yl)benzonitrile (2.315 g, 11.28 mmol), diisopropylethylamine (5.91 ml, 33.8 mmol) and acetonitrile (100 ml). The reaction mixture was stirred at 40°C for 18 hours. Volatile organic compounds were removed under reduced pressure and purified by standard methods to obtain 4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)- 1-oxoisoindolin-4yl)oxy)methyl)benzyl)piperazin-1-yl)-3-fluorobenzonitrile.

1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 7.68 (dd, J=1.96, 13.45 Hz, 1H), 7.56 (dd, J=1.77, 8.38 Hz, 1H), 7.43-7.52 ( m, 3H), 7.30-7.38 (m, 4H), 7.11 (t, J=8.80 Hz, 1H), 5.24 (s, 2H), 5.11 (dd, J=5.14, 13.33 Hz, 1H), 4.37-4.46 (m, 1H), 4.22-4.30 (m, 1H), 3.54 (s, 2H), 3.12-3.23 (m, 4H), 2.84-2.98 (m, 1H), 2.52-2.62 (m, 5H), 2.36 -2.48 (m, 1H), 1.92-2.04 (m, 1H).- MS (ESI) m/z 568.2 [M+1]+. Anal. calculation for C ₃₂ H ₃₀ FN ₅ O ₄ : C, 67.71; N, 5.33; N, 12.34. Observed: C, 67.50; N, 5.44; N 12.34.

Example 2. Synthesis of ^)-4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)benzyl)piperazine-1- yl)-3-fluorobenzonitrile (Compound 2) o

tert-Butyl (4S)-5-amino-4-(benzyloxycarbonylamino)-5-oxo-pentanoate. To a solution of (2S)-2(benzyloxycarbonylamino)-5-tert-butoxy-5-oxopentanoic acid (150 g, 445 mmol) in 1,4-dioxane (1.50 L) was added di-tert-butyl dicarbonate (155 g , 711 mmol), pyridine (70.3 g, 889 mmol) and ammonium bicarbonate (105 g, 1.33 mol). The reaction mixture was stirred at 18°C for 16 hours and then evaporated. The residue was dissolved in ethyl acetate (5.0 L) and water (5.0 L), the organic layer was separated and washed with HCl (3.0 mL, 1 N), saturated sodium hydrogen carbonate solution (3.0 L), brine (3 0 L), dried over anhydrous sodium sulfate, filtered and evaporated to give crude tert-butyl (4S)-5-amino-4-(benzyloxycarbonylamino)-5-oxopentanoate (450 g, crude) as a white solid, which used in the next step without further purification.

1H NMR 400 MHz DMSO-d ₆ δ: 7.35-7.30 (m, 5H), 7.02 (s, 1H), 5.01 (d, J=3.2 Hz, 1H), 3.93-3.90 (m, 1H), 2.20 (t , J=8.0 Hz, 2H), 1.88-1.84 (m, 1H), 1.72-1.69 (m, 1H), 1.35 (s, 9H).

tert-Butyl (4S)-4,5-diamino-5-oxopentanoate.

To a solution of tert-butyl (4S)-5-amino-4-(benzyloxycarbonylamino)-5-oxopentanoate (112 g, 333 mmol) in methanol (1.0 L) was added 10% palladium on carbon (15 g) under a nitrogen atmosphere . The suspension was degassed under vacuum and purged with hydrogen several times. The mixture was stirred under an atmosphere of hydrogen (40 psi) at 30°C for 16 hours. The reaction mixture was filtered and the filtrate was evaporated to give crude tert-butyl (4S)-4,5-diamino-5-oxopentanoate as colorless oils

1H NMR 400 MHz DMSO-d ₆ δ: 7.30 (s, 1H), 6.95 (s, 1H), 3.10-3.07 (m, 1H), 2.27-2.23 (m, 2H), 1.69-1.78 (m, 1H) , 1.59-1.55 (m, 1H), 1.38 (s, 9H).

Methyl 3-hydroxy-2-methyl benzoate.

Four loads (200 g each) were carried out in parallel. To a solution of 3-hydroxy-2-methylbenzoic acid (200 g, 1.31 mol) in methanol (4.0 L) was added concentrated sulfuric acid (47.7 g, 486 mmol). The reaction mixture was stirred at 60°C for 17 hours. The reaction mixture was evaporated to 800 ml. The resulting mixture was cooled to 20°C and slowly poured into water (400 ml) over 30 min. Water (1200 ml) was added at 20°C over 3 hours and the resulting mixture was stirred at 20°C for 1 hour. The precipitated solid was collected by vacuum filtration (four batches combined) and washed three times with water/methanol (1000 ml, 9: 1) to pH > 3. The solid was dried in vacuo at 45°C to obtain methyl 3-hydroxy-2-methyl benzoate (700 g, 80.4% yield) as a gray solid.

1H NMR: 400 MHz DMSO-d6 δ: 9.70 (s, 1H), 7.18 (t, J=6.8 Hz, 1H), 7.09 (t, J=7.6 Hz, 1H), 7.00 (t, J=6.8 Hz, 1H), 3.81 (s, 3H), 2.29 (s, 3H).

Methyl 3-[tert-butyl(dimethyl)silyl]oxy-2-methylbenzoate.

Two loads (240 g each) were carried out in parallel. To a solution of methyl 3-hydroxy-2-methylbenzoate (240 g, 1.44 mol) in N,N-dimethylformamide (1.40 L) was added imidazole (246 g, 3.61 mol) and tert-butyldimethylsilyl chloride (238 g, 1. 58 mol) at 5°C. After addition, the mixture was heated to 20°C and stirred for 6 hours. Isopropyl acetate (1700 ml) was added and then water (2000 ml) was added slowly while the temperature was maintained below 30°C. The resulting mixture was stirred, followed by separation of the organic phase. Combined organic phases (combined two per

- 101 045896 loads) were washed with water (1700 ml x 3) and evaporated to ~1500 ml (KF < 0.05%). The product was stored as a solution in isopropyl acetate, which was used for the next step without further purification.

Methyl 2-(bromomethyl)-3-[tert-butyl(dimethyl)silyl]oxy-benzoate. Two loads (~375 g each) were carried out in parallel. To a solution of methyl 3-[tert-butyl(dimethyl)silyl]oxy-2-methylbenzoate (~375 g, 1.34 mol) in isopropyl acetate was added N-bromosuccinimide (274 g, 1.54 mol) and azobisisobutyronitrile (4.40 g, 26.8 mmol). The reaction mixture was heated to 70°C for at least 1 hour and stirred at 70°C for 4 hours. The reaction mixture was cooled to 20°C and kept at 20°C for at least 1 hour. Two loads of solid (succinimide) was removed by filtration and washed with isopropyl acetate (700 ml). The filtrate was washed with a saturated solution of sodium sulfite (700 g) in water (6000 ml), then with water (1500 ml). The organic layer was distilled under vacuum at 45°C to dryness to obtain methyl 2-(bromomethyl)-3-[tert-butyl(dimethyl)silyl]oxybenzoate (920 g, 95.5% yield) as a dark orange oil.

¹ H NMR: 400 MHz DMSO-d ₆ δ: 7.45 (d, J=6.8 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 7.13 (t, J=7.2 Hz, 1H), 4.95 ( s, 2H), 1.02 (s, 9H), 0.29 (s, 6H).

tert-Butyl (4S)-5-amino-4-[4-[tert-butyl(dimethyl)silyl]oxy-1-oxoisoindolin-2-yl]-5-oxopentanoate. To a solution of tert-butyl (4S)-4,5-diamino-5-oxopentanoate (130 g, 643 mmol) in acetonitrile (4.0 L) was added methyl 2-(bromomethyl)-3-[tert-butyl(dimethyl) silyl]oxybenzoate (210 g, 584 mmol) and diisopropylethylamine (113 g, 877 mmol). The reaction mixture was stirred at 50°C for 16 hours. The reaction mixture was evaporated to remove most of the acetonitrile, the residue was dissolved in methyl tert-butyl ether (2.0 L) and water (1.5 L), the organic layer was washed with saturated potassium dihydrogen phosphate solution (1.0 L x 2), brine (1.0 L), dried over anhydrous sodium sulfate, filtered and evaporated to give crude tert-butyl (4S)-5-amino-4-[4-[tert-butyl(dimethyl )silyl]oxy-1-oxoisoindolin-2-yl]-5-oxopentanoate (524 g), which was used in the next step without further purification.

tert-Butyl (48)-5-amino-4-(4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate. To a solution of tert-butyl (48)-5-amino-4-[4-[tert-butyl(dimethyl)silyl]oxy-1-oxoisoindolin-2-yl]-5-oxopentanoate (275 g, 613 mmol) in methanol (2 .0 L) tetrabutylammonium fluoride trihydrate (38.7 g, 123 mmol) was added. The mixture was stirred at 18°C for 16 hours. The reaction mixture was evaporated to remove most of the methanol, the residue was dissolved in dichloromethane/water (3 L/2 L), the organic layer was washed with brine (1.0 L), dried over anhydrous sulfate sodium, filtered and evaporated to give the crude product which was purified on a silica gel column to give the product (260 g). The product was added to acetonitrile (750 ml) and the mixture was stirred at 60°C for 2 hours, cooled to 18°C and stirred for a further 2 hours. The solid was filtered and the precipitate was dried to give tert-butyl (4S)-5- amino 4-(4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate (248 g, 60.5% yield) as a gray solid.

¹ H NMR 400 MHz DMSO-d6 δ: 10.00 (s, 1H), 7.54 (s, 1H), 7.29 (t, J=7.6 Hz, 1H), 7.14 (d, J=4.8 Hz, 2H), 4.72- 4.68 (m, 1H), 4.49-4.28 (m, 2H), 2.17-1.97 (m, 4H), 1.31 (s, 9H).

4-(4-(4-(chloromethyl)benzyl)piperazin-1-yl)-3-fluorobenzonitrile.

1,4-bis(Chloromethyl)benzene (51.2 g, 292 mmol) was placed in a flask with acetonitrile (195 ml) and N,Ndimethylformamide (195 ml). The reaction mixture was stirred at ambient temperature until the solids were completely dissolved. Diisopropylamine (51.1 mL, 292 mmol) was then added along with 3-fluoro-4-(piperazin-1-yl)benzonitrile (20 g, 97 mmol). The reaction mixture was heated at 60°C for 1 hour. Acetonitrile was removed under reduced pressure. The remaining mixture was washed with ethyl acetate (1.0 L), water (700 ml) and saline (300 ml). The organic layer was separated and the aqueous layer was extracted twice with ethyl acetate. Volatile organic compounds were removed under reduced pressure. The solid was dissolved in a minimal amount of dichloromethane and purified on a silica gel column (0-100% ethyl acetate in hexane for 3 L). Fractions containing the desired product were combined and volatile organic compounds were removed under reduced pressure. The residue was dissolved in a minimal amount of dichloromethane and purified a second time on a silica gel column (isocratic 10% ethyl acetate in hexane for 800 ml, then 20-80% ethyl acetate in hexane for 4 L). Fractions containing the desired product were combined and VOCs were removed under reduced pressure to give 4-(4-(4-(chloromethyl)benzyl)piperazin-1-yl)-3-fluorobenzonitrile (22.7 g, 66.0 mmol , yield 67.7%) as a grayish solid.

^1H NMR (400 MHz, CDCl ₃ ) δ ppm 7.33-7.39 (m, 5H) 7.29 (d, J=1.96 Hz, 1H) 7.25 (d, J=1.96 Hz, 1H) 6.91 (t, J=8.56 Hz , 1H) 4.60 (s, 2H) 3.58 (s, 2H) 3.19-3.27 (m, 4H) 2.58-2.66 (m, 4H). MS (ESI) m/z 344.2 [M+1]+.

(b)-tert-Butyl 5-amino-4-(4-((4-((4-(4-cyano-2-fluorophenyl)piperazin-1-yl)methyl)benzyl)oxy)-1oxoisoindoline-2- yl)-5-oxopentanoate.

(b)-tert-Butyl 5-amino-4-(4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate (22.05 g, 65.9 mmol) was added to the flask with 4-(4- (4-(chloromethyl)benzyl)piperazin-1-yl)-3-fluorobenzonitrile (22.67 g, 65.9 mmol), potassium carbonate (18.23 g, 132 mmol) and N-dimethylformamide (330 ml). Reactionary

- 102 045896 the mixture was heated at 45°C for 16 hours. The reaction mixture was diluted with ethyl acetate (50 ml), filtered. The filtrate was washed with ethyl acetate (900 ml), water (600 ml) and saline (200 ml). The organic layer was separated and partitioned with water (600 ml). The organic layer was separated and all organic layers were combined, dried over sodium sulfate and volatiles were removed under reduced pressure. The residue was treated with 20% ethyl acetate in hexane and the volatiles were removed under reduced pressure to give ^)-tert-butyl 5-amino-4-(4-((4-((4-(4-cyano-2-fluorophenyl)piperazine -1yl)methyl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (44.02 g, 68.6 mmol, 104% yield) as a grayish solid. The yield was slightly higher than quantitative, since some amount of N^dimethylformamide remained.

¹ H NMR (400 MHz, CDCls) δ ppm 7.43-7.49 (m, 2 H) 7.40 (s, 4H) 7.36 (dd, J=8.38, 1.28 Hz, 1H) 7.29 (d, J=1.96 Hz, 1H) 7.26 (d, J=1.83 Hz, 1H) 7.11 (dd, J=7.64, 1.16 Hz, 1H) 6.92 (t, J=8.50 Hz, 1H) 6.23 (br s, 1H) 5.24-5.32 (m, 1H) 5.15 (s, 2H) 4.86-4.94 (m, 1H) 4.38-4.55 (m, 2H) 3.61 (s, 2H) 3.18-3.32 (m, 4H) 2.58-2.70 (m, 4H) 2.09-2.47 (m, 4H) 1.43 (s, 8 H). MS (ESI) m/z 642.4 [M+1]+.

^)-4-(4-(4-(((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)benzyl)piperazin1 -yl)-3-fluorobenzonitrile .

^)-tert-Butyl 5-amino-4-(4-((4-((4-(4-cyano-2-fluorophenyl)piperazin-1 -yl)methyl)benzyl)oxy)-1 oxoisoindoline-2- yl)-5-oxopentanoate (12.1 g, 18.86 mmol) was added to the vial with acetonitrile (189 ml) and benzenesulfonic acid (3.96 g, 24.51 mmol). The reaction mixture was placed under vacuum and purged with nitrogen. This procedure was repeated once more and the resulting mixture was then heated to 85°C overnight under a nitrogen atmosphere. The reaction mixture was poured warm directly into 2 separatory funnels containing dichloromethane (1000 ml) and ethyl acetate (300 ml). To this mixture were added saturated sodium hydrogen carbonate solution (900 ml), water (100 ml) and saline solution (450 ml). The organic layer was separated and the aqueous layer was extracted with dichloromethane (800 ml) and ethyl acetate (200 ml). The combined organic layers were dried over anhydrous magnesium sulfate and evaporated. Purify using standard methods to obtain the title compound.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 10.96 (s, 1H) 7.68 (dd, J=13.45, 1.83 Hz, 1H) 7.56 (dd, J=8.44, 1.83 Hz, 1H) 7.43-7.52 ( m, 3 H) 7.29-7.39 (m, 4H) 7.11 (t, J=8.80 Hz, 1H) 5.24 (s, 2H) 5.11 (dd, J=13.20, 5.14 Hz, 1H) 4.22-4.46 (m, 2H ) 3.54 (s, 2H) 3.12-3.22 (m, 4H) 2.85-2.97 (m, 1H) 2.53-2.62 (m, 2H) 2.38-2.48 (m, 2H) 1.93-2.03 (m, 1H). MS (ESI) m/z 568.2 [M+1]+.

Example 3 Compound 1, Compound 2 and Compound 3 have strong antiproliferative activity in multiple myeloma cells

The relative effectiveness of Compound 2 and Compound 3 in accelerating the degradation of Aiolos CRL4 ^crbn E3 protein by ubiquitin ligase was studied in DF15 myeloma cells at various time points (45 min, 90 min and 3 h). DF15 cells expressing Enhanced ProLabel (ePL) Aiolos were incubated with Compound 2 or Compound 3 for 45 min, 90 min, or 3 h. Cell extracts were then prepared and Aiolos ePL protein was quantified using the ePL luminescent assay. The values shown in table. 1 correspond to the Aiolos content versus concentration data presented in FIG. 1. The results showed that the time-dependent and concentration-dependent loss of Aiolos was caused by both enantiomers (Fig. 1).

Table 1. Compounds 2 and 3 contributed to the destruction of Aiolos

Compound Destruction of Aiolos 1C ₅₀ (µM) 45 minutes 90 minutes 3 hours Connection 2 >0.1 (27% inhibition at 0.001 µM) 0.00036 0.000031 Connection 3 >0.1 (13% inhibition at 0.001 µM) 0.0059 0.00045

Taken together, the experiments showed that Compounds 2 and Compound 3 degrade Aiolos in a time- and concentration-dependent manner. The data also demonstrate that Aiolos degradation is an effective biomarker for monitoring the effectiveness of these compounds.

Example 4 Compound 2-Induced Apoptosis and Suppression of Factors Essential for Tumor Survival and Synergistic with Dexamethasone

To determine whether Compound 2 acts synergistically with dexamethasone, the effect of adding dexamethasone on the loss of known CRL4 ubiquitin ligase substrates ^CRBN E3, Aiolos, Ikaros, and ZFP91, was examined using pomalidomide-sensitive (OPM2) or resistant (OPM2-P1) myeloma cells. Dexamethasone alone or in combination with Compound 2 resulted in the destruction of Aiolos and Ikaros in OPM2 and OPM2-P1 cells after 72 hours of treatment (Fig. 2). It is important to note that a large loss of downstream effector proteins (c-Myc and IRF4) was observed when 10 or 100 nM dexamethasone was added to Compound 2 (Fig. 2). In addition, increased induction of apoptotic markers (Bcl-2-interacting mediator [BIM], cleaved caspase-3, cleaved caspase-7 and

- 103 045896 cleaved poly[adenosine diphosphate ribose] polymerase [PARP]) was observed after 72 h, as measured by Western blotting (Fig. 3).

Similarly, five-day incubation of OPM2 myeloma cells with Compound 2 alone significantly reduced the expression levels of Aiolos and Ikaros, as well as ZFP91 (Fig. 4). The disruption of Ikaros and Aiolos was followed by a decrease in the levels of two additional and very important transcription factors, c-Myc and IRF4 (Fig. 4). Loss or reduction of these factors was associated with increased levels of the cyclin-dependent kinase inhibitor p21 and induction of apoptosis as measured by cleaved caspase-3 (Fig. 4). The results indicate that Compound 2 exerts its antitumor activity by abrogating the expression of key multiple myeloma survival factors, including IRF4 and cMyc.

Taken together, these results indicate that Aiolos, Ikaros, ZFP91, c-Myc, and IRF4, as well as apoptotic markers such as Bcl-2-interacting mediator (BIM), cleaved caspase-3, cleaved caspase-7, and cleaved poly(adenosine diphosphate) ribose) polymerase (PARP) and p21 may serve as biomarkers to monitor the effectiveness of Compound 2 treatment.

Example 5: Destruction of Cereblon and Ikaros is required for the antiproliferative activity of Compound 2

To determine whether cereblon could serve as a biomarker to predict response to Compound 2, the DF15R myeloma cell line was treated with Compound 2. The DF15R cell line is a multiple myeloma cell line that has developed resistance to lenalidomide and pomalidomide by continuous administration of pomalidomide (up to 100 μM) and which has no detectable levels of CRBN protein. Treatment of this cell line with Compound 2 did not show any disruption of Ikaros or Aiolos (Fig. 5A). Reintroduction of CRBN protein into DF15R cells reactivated Compound 2-induced degradation of Ikaros and Aiolos (Fig. 5A). These results indicate that CRBN is important in mediating the response to Compound 2. Thus, CRBN levels may serve as a biomarker to assess potential response to Compound 2.

Compound 2 was shown to inhibit Ikaros, Aiolos, and ZFP91 (Figure 4) and CRBN was found to be required for the degradation of Ikaros and Aiolos (Figure 5A). To determine whether disruption of Ikaros, Aiolos, or ZFP91 is responsible for the effects on c-Myc, IRF4, and p21 or on apoptosis induced by Compound 2, stabilized mutants of all three proteins were generated and overexpressed one at a time in OPM2 cells (Fig. 5B).

Successful overexpression of stabilized Ikaros mutants, Ikaros-Q146H or Ikaros-G151A, prevented Ikaros degradation and abrogated the Compound 2-induced effects on c-Myc, IRF4, p21, and cleaved caspase-3 (Fig. 5B). However, a stabilizing mutation in ZFP91 (Q400H) failed to abolish the effect of Compound 2, although the mutation was able to effectively protect the protein from degradation in the presence of Compound 2 (Fig. 5B).

The effect of stabilizing mutations on cell proliferation was also measured. Consistent with the effect on downstream proteins, overexpression of stabilized Ikaros mutants, Ikaros-Q146H or Ikaros-G151A, abolished the antiproliferative effects of Compound 2 (Table 2). Specifically, the concentration at which cell proliferation was inhibited by 50% following treatment with Compound 2 increased more than 200-fold (>100 nM) in cells overexpressing stabilized Ikaros mutants compared to parental cells (0.488 nM) (Table 2 ). There was no apparent downstream effect of stabilizing ZFP91 or reducing sensitivity to the antiproliferative activity of Compound 2 (Fig. 5B and Table 2). The potential contribution of Aiolos disruption to the inhibition of MM cell growth by Compound 2 could not be confirmed in cells overexpressing the Aiolos mutant due to the less efficient stabilization of Aiolos to degradation compared to Ikaros in this particular assay.

Taken together, these results further demonstrate that degradation of Ikaros is required for Compound 2 activity and indicate that Ikaros is an important biomarker of Compound 2 activity.

Table 2. Inhibition of ORM2 cell proliferation

Compound Inhibition of ORM2 cell proliferation K% (nM) Parent ьskie Ikaros Ikaros- Q146H Ikaros- G151A Aiolos Aiolos Q147H ZFP91- Q400H Compound 2 0.08 0.13 > 100 > 100 0.08 0.06 0.08 Pomalidom eid 64 91 > 100 > 100 89 56 73 Lenalide id > 100 > 100 > 100 > 100 > 100 > 100 > 100

- 104 045896

Example 6 Compound 2-Induced Ikaros and Aiolos Disruption Correlated with G1 Cell Cycle Arrest and Apoptosis in Multiple Myeloma Cells

To evaluate the functional response of multiple myeloma cells to Compound 2 treatment, the lenalidomide-resistant myeloma cell line H929-1051 was treated with Compound 2 for 4 hours (Fig. 6A) or 72 hours (Fig. 6B). Incubation with concentrations as low as 1 nM Compound 2 promoted sustained degradation of Ikaros and Aiolos over 4 h (Fig. 6A).

Consistent with this, after 72 h of treatment with Compound 2, there was a marked decrease in pRB1 phosphorylation and an increase in levels of the CDK inhibitor p27 in H929-1051 cells (Fig. 6B). These events were accompanied by a decrease in survivin levels, a dramatic increase in the level of the proapoptotic extra-long isoform of the Bim protein (BimEL), and cleavage of caspase-1, caspase-7, caspase-3, and PARP (Fig. 6B). The results indicate that the antitumor activity of Compound 2 in cells resistant to lenalidomide and/or pomalidomide is due to the destruction of Ikaros and Aiolos, leading to cell cycle arrest and apoptosis. In addition, the almost complete and sustained destruction of Aiolos and Ikaros demonstrated the in vitro efficacy of Compound 2 even against cells resistant to lenalidomide and pomalidomide treatment (Figure 10). These results demonstrate that Compound 2 induces cell cycle arrest and apoptosis, as well as the detection of Ikaros, Aiolos, IRF4, c-Myc, p27, phospho-Rb, BimEL, cleaved caspase-1, -caspase-7, -caspase-3 and -PARPs may serve as biomarkers for Compound 2 treatment.

Example 7 Compound 2-Induced Destruction of Aiolos and Ikaros and Increased Apoptosis in Myeloma Cell Lines

The effects of continuous exposure to Compound 2 on the degradation and reexpression of Aiolos and Ikaros, as well as the induction of apoptosis, were measured in the multiple myeloma cell lines H929-1051 and OPM2-P10, which are resistant to lenalidomide and pomalidomide, respectively. Cell lines were treated with a single continuous exposure to Compound 2 without washout, and Aiolos and Ikaros levels were measured by flow cytometry. After treatment, there was more than 70% destruction of Aiolos and Ikaros within 3-6 hours, depending on the cell line (Fig. 7A and 7B). Substrate depletion persisted for up to 70 h in H929-1051 cells or up to 100 h in OPM2-P10 cells (Fig. 7A and 7B). Increasing the concentration 10-fold to 0.1 μM resulted in virtually no additional destruction (Fig. 7A and 7B).

In H929-1051 cells, degradation of Aiolos lasted from 75 to 100 h, whereas degradation of Ikaros levels continued for a shorter period of time (Fig. 7A and 7B). Degradation of both proteins was maintained for more than 150 h in OPM2-P10 cells (Fig. 7A and 7B).

The effect of transient exposure to Compound 2 on the degradation of Aiolos and Ikaros in H929-1051 cells was also assessed. Brief exposure for 15 min at 0.1 μM Compound 2 caused destruction of Aiolos and Ikaros (Fig. 8). Although the degree of destruction (25 to 50%) was not complete, a prolonged pharmacodynamic (PD) effect was observed that lasted between 25 and 50 hours after exposure (Fig. 8).

To better understand the kinetics of Compound 2 exposure and degradation of Aiolos and Ikaros, longer exposure to Compound 2 (0.01 and 0.1 μM) was examined for 6 h followed by washout. Treatment with Compound 2 resulted in destruction of Aiolos and Ikaros by more than 70% (Fig. 9). Washing out Compound 2 after a single 6-hour exposure resulted in rapid recovery of both proteins from degradation (Figure 9). Recovery of more than 75% of the substrate was observed after 25 h in cells exposed to 0.01 μM Compound 2, and ranging from 75 to 100 h in cultures exposed to 0.1 μM Compound 2 (Fig. 9).

The effect of disruption of Aiolos and Ikaros on the induction of apoptosis was also assessed. The almost complete and prolonged substrate suppression observed with continuous exposure to Compound 2 coincided with the induction of apoptosis in MM cells (Fig. 10). Continuous single exposure to 0.01 and 0.1 μM Compound 2 resulted in the induction of apoptosis in H929-1051 and OPM2-P10 cells (ranging from 50 to 75 hours) (Fig. 10). However, temporary exposure to Compound 2 for 15 min or 6 h prevented the induction of apoptosis (Fig. 10).

Taken together, these observations indicate that both the magnitude and duration of destruction of Aiolos and Ikaros are important mediators of apoptosis induction in the MM cell models tested. Additionally, these data demonstrate the use of Aiolos and Ikaros and that markers of apoptosis, such as cleaved caspase-3, are biomarkers of response to Compound 2, and also suggest that these biomarkers may be useful in determining or adjusting the dose of Compound 1 , Connections 2 or Connections 3.

Example 8 Compound 2 Inhibited the Proliferation of Acquired Resistance Multiple Myeloma Cell Lines with Low CRBN

The activity of Compound 2 was tested in myeloma cells that have acquired resistance to lenalidomide or pomalidomide due to prolonged exposure to either compound and, in the process, have acquired reduced levels of cereblon (Table 2). Cells were treated for 5 days and then assessed using an ATP assay (CellTiter-Glo). The percentage of control was calculated by subtracting the background and normalizing to the DMSO control (100% of control). Regarding

- 105 045896 The significant percentage of cereblon in cell lines with acquired resistance to lenalidomide or pomalidomide was determined using Western blotting and is presented in table. 3 relative to CRBN levels in parental cell lines taken as 100%.

In fig. 11 shows IC50 concentration response curves comparing the activity of Compound 2 and pomalidomide in the parental lines (DF15, NCI-H929 and OPM2), a lenalidomide-resistant cell line (NCI-H929-1051) or five pomalidomide-resistant cell lines (NCI-H929-P01, OPM2-P01, OPM2-P1, OPM2-P10 and DF15R).

Table 3. Cereblon protein expression levels in multiple myeloma cell lines,

Pom=pomalidomide;

*background level, not actual cereblon, which is absent in a given cell line

Comparison of cell lines resistant to pomalidomide or lenalidomide demonstrated that CRBN protein levels were lower than those of the sensitive parental cell lines (Table 3).

Proliferation was assessed using the CellTitre-Glo® assay. The results of the study show that the cell lines were sensitive to Compound 2, even in multiple models of acquired resistance where CRBN levels were low, as determined by quantifying the levels of ATP present in the medium after 5 days (Fig. 11). However, Compound 2 showed little antiproliferative activity against the DF15R cell line, which lacks CRBN expression (FIGS. 11 and 5A).

Taken together, these data indicate that Compound 2 has very potent activity against multiple myeloma in models of acquired resistance with low but detectable levels of CRBN. Thus, detectable CRBN levels may be a biomarker for predicting patient susceptibility to Compound 2.

Example 9 Compound 2 alone and in combination with dexamethasone induced apoptosis in lenalidomide-resistant multiple myeloma

Dexamethasone was evaluated for its ability to induce apoptosis as a single agent or in combination with Compound 2. Induction of apoptosis was measured using Caspase-Glo in lenalidomide-resistant multiple myeloma cells (H929-1051). Dexamethasone was distributed at 20 concentrations using an acoustic dispenser. The plates were sealed and stored at room temperature for the next day. The final concentrations of compounds analyzed were: dexamethasone (0.8 to 0.00002 μM) and Compound 2 (0.001, 0.01, or 0.1 μM). Cells were dispensed into assay plates using a Multidrop dispenser and duplicate assay plates were made. Apoptosis was measured 72 hours after compound treatment using the Caspase-Glo 3/7 and CellTiter-Glo assays. Caspase-Glo 3/7 luminescence was normalized to CellTiter-Glo luminescence to account for differences in cell number. The change in folding in the test sample was calculated as follows: normalized caspase value of the treated sample/average of the normalized value of the DMSO control.

The apoptotic activity of dexamethasone alone or in combination with lenalidomide, pomalidomide or Compound 2 was measured by caspase 3 induction. The results showed that Compound 2 synergized with dexamethasone in reducing cell viability and enhanced the apoptotic ability of dexamethasone in a concentration-dependent manner (Fig. 12). In a dose-response relationship, treatment with dexamethasone alone resulted in a slight increase in apoptosis, consistent with changes in caspase-3, compared with DMSO treatment (Fig. 12). In contrast, the onset of dexamethasone activity was shifted by 1 log in the presence of Compound 2. Treatment with three different concentrations of Compound 2 enhanced the apoptotic activity of dexamethasone as measured by caspase-3 (Fig. 12).

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In addition, even the lowest dose of Compound 2 resulted in a strong induction of apoptosis. As little as 10 nM dexamethasone increased the ability of Compound 2 to initiate cell death, and low to subnanomolar concentrations of Compound 2 enhanced the apoptotic effects of dexamethasone (Figure 12). This demonstrated the ability of dexamethasone plus Compound 2 to induce apoptosis in lenalidomide-resistant multiple myeloma cells and demonstrates that measurement of caspase-3 can serve as a biomarker for Compound 2 treatment.

Example 10: Ex Vivo Effect of Compound 2 on the Maturation of Myeloid Progenitors to Adult Neutrophils

Ex vivo cultures of CD34+ bone marrow (BM) cells from healthy donors (HD) were used to study the effect of Compound 2 on neutropenia. Preliminary data indicate that restoration of mature neutrophil levels to at least 50% of the untreated control level in the assay system used in this study correlates with failure to induce or recover from clinically significant neutropenia. Therefore, we monitored the effect of Compound 2 on mature neutrophils.

Ikaros protein levels were analyzed by flow cytometry after each exposure period and on days 19, 21, and 23, corresponding to days 3, 5, and 7 after the last exposure, respectively. CD34+ cells obtained from healthy donor bone marrow were exposed to different concentrations of Compound 2 and different incubation schedules. The percentage of Ikaros (normalized to the DMSO control) was measured after exposure to Compound 2 at concentrations of 1, 10 and 100 nM for 2, 4 and 6 hours on each of 1, 2 or 3 consecutive days starting on day 14. At the end of the exposure period, Compound 2 was removed and the cells were incubated in the absence of Compound 2 (recovery period). Ikaros was measured by flow cytometry after incubation with Compound 2 (days 14 to 16) and during recovery on days 19, 21, and 23. Data were collected for two donors and are presented in FIG. 13. Ikaros levels decreased during exposure to Compound 2 and recovered after drug withdrawal in a concentration-dependent manner, with no significant differences observed across different exposure regimens (Figure 13). Ikaros levels began to return to normal at least 3 days after washout, which preceded complete restoration of late-stage neutrophil precursor maturation. These data suggest that degradation of Ikaros in late-stage neutrophil progenitors may be an important mediator of neutropenia in Compound 2 recipients. In addition, the data suggest that restoration of Ikaros levels precedes restoration of neutrophil progenitor maturation and that successful treatment of neutropenia in patients with MM treated with Compound 2 is possible using appropriate dosing regimens.

To further examine the role of Ikaros in the maturation of neutrophil precursors, Ikaros protein levels were analyzed by flow cytometry after each exposure and one day after the last exposure to Compound 2. Ikaros was completely degraded after a single exposure to Compound 2 at all concentrations and all exposure patterns (Figures 14 and 15 ). After 3 days of exposure to Compound 2, Ikaros recovery began in a concentration-dependent manner just 24 hours after drug washout and returned to control levels after 10 days (Fig. 14; 16B). After 5 days of exposure to Compound 2, recovery of Ikaros began on day 17, three days after drug washout (Figure 15). Full recovery to control levels was observed 6 days after the last exposure at a concentration of 10 nM, while a recovery of more than 50% was observed 8 days after exposure to Compound 2 at a concentration of 100 nM (Fig. 15).

The data support the hypothesis that Compound 2-induced disruption of Ikaros is a factor driving the cessation of neutrophil precursor maturation and that resumption of Ikaros restoration precedes recovery of mature neutrophils, as shown in FIG. 16V. Taken together, these data suggest that more intensive dosing of Compound 2 (multiple doses per day), compared with once daily dosing, may not have a negative impact on the induction and recovery from neutropenia in patients.

Example 11 Compound 2 Enhances Antitumor Activity of Immune Cells from Healthy Human Donors

PBMCs isolated from healthy donors were cultured in RPMI 1640 medium supplemented with 10% FBS at a density of 1x106 cells/ml.

K562 cells were maintained in log phase and cell density and viability were monitored by trypan blue exclusion using a ViCELL® XR Cell Viability Analyzer (Beckman Coulter, Brea, CA).

Freshly isolated human PBMCs were cultured with recombinant IL-2 at a concentration of 20 U/ml for 72 hours. Peripheral blood mononuclear cells were then centrifuged and resuspended in fresh complete RPMI medium to 2x 106 cells/ml. Cells were then treated with DMSO or Compound 2 at the indicated concentrations and incubated for an additional 72 hours. PBMCs were washed twice in fresh complete RPMI prior to coculture. K562 cells re

- 107 045896 were suspended to a cell density of 1x 10 ⁶ cells/ml and stained with 1 µM CellTrace CFSE according to the manufacturer's instructions. K562-labeled cells were then seeded in 96-well round bottom plates at 1 x 10 ⁵ cells/well. Peripheral blood mononuclear cells were then transferred into the same 96-well plate at a ratio of 1:15 in triplicate and incubated at 37°C for 4 hours. Specific lysis of target cells by PBMC-mediated apoptosis was measured using V-fluorescein annexation. isothiocyanate (FITC) and propidium iodide (PI) according to the manufacturer's instructions, and the samples were subjected to FACS Array scanning. Unlabeled K562 cells, CellTrace CFSE-labeled K562 cells, and Annexin V-FITC-labeled and PI-labeled untreated K562 cells were included in each assay as controls. All myeloma cell lines were maintained in log phase and cell density and viability were monitored by trypan blue exclusion using a Vi-CELL XR Cell Viability Analyzer. Ninety-six-well plates were precoated with anti-CD3 antibody (OKT3, 3 μg/ml) and incubated at 4°C overnight before starting the experiment. Frozen donor PBMCs were thawed at 37°C for 2 min in RPMI medium supplemented with 10% FBS, then cell number and viability were measured on a Vi-CELL® (Beckman Coulter). Peripheral blood mononuclear cells were washed and diluted to 1x 10 ⁶ cells/ml and distributed into compound-treated plates in a total volume of 200 μl. Cells were incubated in the presence of compounds for 2 h before being transferred to anti-CD3 antibody-coated plates and incubated for an additional 72 h at 37°C. After 72 h, PBMCs were centrifuged and the cells were washed twice with RPMI + 10% FBS. Untreated MM cell lines (H929 and H929-1051) were labeled with CellTrace CFSE according to the manufacturer's instructions and resuspended at a total concentration of 0.1 x 10 ⁶ cells/ml in a U-shaped 96-well plate in a total volume of 100 μl. Peripheral blood mononuclear cells were counted and added to MM cells at a target:effector (T:E) ratio of 1:5. After 24 h of coculture, specific target cell lysis by PBMC-induced apoptosis was measured using Annexin V-AF647 and 7-AAD according to the manufacturer's instructions, and samples were analyzed on an Attune NxT cytometer (Thermo Fisher).

96-well plates were precoated with anti-CD3 antibody (OKT3, 3 μg/ml) and incubated at 4°C overnight before starting the experiment. Frozen donor PBMCs were thawed at 37°C for 2 min in RPMI medium supplemented with 10% FBS, then cell number and viability were measured on a Vi-CELL analyzer. Peripheral blood mononuclear cells were washed and diluted to 1 x 10 ⁶ cells/ml and distributed into compound-treated plates in a total volume of 200 μl. Cells were incubated in the presence of Compound 2 for 2 hours before being transferred to plates coated with anti-CD3 antibodies and incubated for an additional 72 hours. At the same time, MM cell lines (NCI-H929, H929-1051, OPM2, OPM2-P10) diluted to a final concentration of 0.1 x 10 ⁶ cells/ml and labeled with CellTrace CFSE according to the manufacturer's instructions. Multiple myeloma cell lines were then distributed into compound-treated plates at a total volume of 200 μl and incubated for 72 h. After 72 h, PBMC and MM cells were counted and transferred to a 96-well U-bottom plate at a final T:E ratio 1:5. After 24 h of coculture, specific target cell lysis by PBMC-mediated apoptosis was measured using Annexin V-AF647 and 7-AAD according to the manufacturer's instructions, and samples were analyzed on an Attune NxT cytometer.

This co-culture model was used to determine the direct effects of Compound 2 on the antitumor activity of PBMCs taken from healthy donors. Compound 2 treatment of IL-2-activated PBMCs induced the killing of untreated K562 cells in a concentration-dependent manner (FIGS. 17A and 17B). PBMC treated with Compound 2 (IC ₅₀ =5.9 pM) effectively achieved 50% direct killing of K562 cells.

The effects of Compound 2 on the activity of PBMC incubated with Compound 2 against MM cells were further analyzed in cell lines displaying a resistance phenotype in order to compare with the response in sensitive cells. In another coculture model, donor PBMCs were pretreated with Compound 2 for 2 h before culturing on anti-CD3 antibody-coated plates for 72 h. Anti-CD3 antibody-activated PBMCs treated with Compound 2 showed increased tumor cell lysis, concentration-dependent, untreated lenalidomide-sensitive (NCI-H929; _IC50 =0.005 μM) and lenalidomide-resistant (H929-1051; _IC50 =0.0002 μM) MM cell lines to a similar extent (FIGS. 18A and 18B). A similar level of tumor cell killing by PBMC-mediated apoptosis was observed in co-cultured lenalidomide-sensitive and lenalidomide-resistant tumor cells, indicating that PBMCs were primed to induce apoptosis in tumor cells regardless of their resistance phenotype.

Because preincubation of immune cells with Compound 2 enhanced targeting and lysis of MM cells, the effect of preincubation of MM cells with Compound 2 on their susceptibility to immune-mediated death was also examined (FIGS. 19A-19D). Four MM cell lines

- 108 045896 and aHTu-CD3 antibody-activated PBMCs alone were preincubated with Compound 2 for 72 hours. When anti-CD3 antibody-activated PBMCs and MM lines were both pretreated with Compound 2 and then cocultured, the effects on PBMC-induced MM cell lysis was enhanced in terms of activity and magnitude of cell death response. Comparing the IC50 values of individual MM cell cultures versus co-cultures of immune and tumor cells, Compound 2 enhanced killing of NCI-H929 cells by ~7000-fold, and it enhanced killing of H929-1051 cells by ~6000-fold (Fig. 19A and 19B).

PBMC treated with Compound 2 significantly induced tumor lysis of untreated K562 and MM cell lines. Additionally, tumor cell death was significantly increased when both PBMC and MM cell lines were pretreated with Compound 2, suggesting that in addition to its potent cellular independent effects, Compound 2 may also enhance the immunogenicity of MM cell lines. Taken together, the results indicate a combination of significant cell-autonomous and immunogenic effects on MM cells, as well as its immunomodulatory properties.

Example 12 Compound 2-Activated Effector Lymphocytes and Cytokines

The immunomodulatory activity of Compound 2 on healthy T-cell receptor (TCR)-activated PBMCs was assessed. In a panel of cells from nine healthy PBMC donors, incubation with Compound 2 in the presence of anti-CD3 antibody stimulation for 72 hours induced IL-2 secretion with a mean half-maximal effective concentration (EC ₅₀ ) of 14 pM (FIG. 20). This increase in cytokine secretion was observed already 24 hours after administration of Compound 2, while the peak production of effector cytokines induced by Compound 2 was higher than the placebo control by 5.5 times for IL-2, 4.5 times for interferon gamma (IFN -γ) and 2-fold for tumor necrosis factor alpha (TNF-α) (Fig. 21A-21C, respectively). These data demonstrate that Compound 2 can activate T cells and stimulate their secretion of cytokines such as TNF-α, IFN-γ and IL-2. Additionally, these data indicate that these cytokines may serve as biomarkers of activated T cells in response to Compound 2.

Example 13 Compound 2-Induced Substrate Destruction in T Cells

The effect of Compound 2 on Ikaros degradation in CD4+ and CD8+ T cells was examined over 72 hours. The ability of Compound 2 to induce the release of effector cytokines correlated with Ikaros degradation in CD4+ T cells, a known transcriptional repressor of IL-2 (FIGS. 22A-22C). . In CD4+ T cells, after 24 hours, treatment with Compound 2 demonstrated sustained disruption of Ikaros (24 hour IC ₅₀ =0.0003 μM) (FIG. 22A). Activity was also observed in the CD8+ T cell population, with a 24 hour IC50 of Compound 2 of 0.0005 μM (data not shown). The ability of Compound 2 to degrade Ikaros was also observed at the 48 and 72 hour time points in both CD4+ (FIGS. 22B and 22C) and CD8+ (data not shown) T cell populations. These data demonstrate that Compound 2 degrades Ikaros in T cells, which correlates with the induction of effector cytokine release. Thus, Ikaros and Aiolos may serve as biomarkers of T cell activation.

Example 14. Effect of Compound 2 in combination with dexamethasone on immunomodulation/induction of interleukin-2

The effect of Compound 2 in combination with various concentrations of dexamethasone on the ability of PBMC to produce IL-2 was examined. Peripheral blood mononuclear cells from 4 healthy donors were preincubated with Compound 2 for 2 hours before stimulation with anti-CD3 antibody-coated beads for a further 72 hours. When used in combination with 10 nM dexamethasone, Compound 2 was still able to induce IL- 2 to levels at least 10 times greater than the placebo control, with a peak response achieved at 0.01 nM Compound 2 (Fig. 23C). However, the combination of Compound 2 with higher concentrations of dexamethasone (100 nM) showed little induction of IL-2 compared to baseline.

Taken together, these data indicate that the immunomodulatory activity of Compound 2 was maintained in the presence of low levels (10 nM) of dexamethasone, with Compound 2 exhibiting impressive synergy in autonomic killing of MM cells.

Example 15: Pharmacodynamics, efficacy and prognostic assessment using biomarkers

Pharmacodynamics (PD), efficacy, and prognostic endpoints can be assessed by analyzing various biomarkers in the patient's blood and bone marrow. Biomarkers to be assessed include degradation of Aiolos and Ikaros; cytokine profile upon ex vivo stimulation; phenotypic analysis of immune cells (for example, T cells); expression levels of CRBN, Aiolos, Ikaros, tumor infiltrating lymphocytes (TIL), c-Myc, IRF4, p-caspase 3; cytogenicity, mutations and clonality of TCRs; soluble BCMA, free light chain (FLC) and circulating tumor cells (CTC).

Bone marrow aspiration (BMA) is performed on the patient before treatment, during treatment, and at the end of the study. Approximately 6 ml of IMA is taken, and 1 ml of a fixed IMA clot is taken in the wedge

- 109 045896 cial center is used for immunohistochemistry (IHC) to evaluate the expression levels of CRBN, Aiolos, Ikaros, ZFP91, c-Myc, IRF4, p-caspase-3, as well as TILs as biomarkers. The remaining VMA is then subjected to CD138+ selection to isolate myeloma cells, and the ^CD138− and CD138+ fractions are collected. A portion of CD138' cells is used for fluorescence in situ hybridization (FISH). The remaining CD138+ cells are aliquoted for RNA and DNA extraction and analysis, additional FISH analysis, and viable frozen CD138+ cells. Bone marrow mononuclear cells (BMMNC) are isolated from the CD138 fraction ^by Ficoll density gradient centrifugation. BMMNC CD138 ^- are aliquoted for RNA and DNA isolation and analysis, for immunoprofiling of viable frozen CD138 ^- cells and frozen cell pellets for TCR clonality.

Whole blood from patients is also analyzed. Peripheral blood mononuclear cells (PBMCs) and/or serum/plasma are isolated from the samples. Whole blood is also used directly for the analysis of some biomarkers.

Whole blood testing can be used to determine T cell activation as a biomarker. Blood is taken during screening and during treatment according to all regimens. Blood is drawn into a TruCulture tube containing anti-CD3 antibodies to stimulate T cells. The tube is incubated at 37°C for 42 ± 4 hours. The medium is separated from the cells and frozen at -70°C. Samples are analyzed for T cell activation using a custom cytokine panel (Myriad RBM, Inc.) that includes cytokines indicative of T cell activation such as interferon gamma (ZFNy), tumor necrosis factor alpha (TNFa), and interleukin-gamma. 2 (IL-2).

Serum is isolated from whole blood and soluble BCMA (sBCMA) is measured by ELISA as a biomarker before and during treatment in all cycles and regimens. Additionally, serum free light chain (sFLC) is measured as a biomarker in the same serum samples as sBCMA. The FreeLite test will be used to measure the ratio between kappa light chain and lambda light chain (k/l ratio).

PBMCs isolated from whole blood are analyzed for protein expression of the biomarkers Aiolos and Ikaros using FACS and/or ELISA. Isolation of PBMCs to measure expression levels of these biomarkers is performed on patients enrolled in all regimens on Day 1, during treatment, and at the end of the study.

In addition, PBMC biomarker analysis involves immunophenotyping after collecting whole blood samples before and during treatment. Immune modulation is assessed using FACS assays for T cell subsets (CD3, CD4, CD8, Treg, Teff, Tmem), B cells and NK cells.

Finally, the number of circulating tumor cells (CTCs) present in the peripheral blood is quantified as a biomarker. CTCs are identified and analyzed using FACS assays used to assess minimal residual disease (MRD). CTCs are measured in the blood in the first days and during treatment for all regimens.

Biomarkers are used to determine the combined activity of Compound 2 with low-dose dexamethasone and to evaluate dosing regimens of Compound 2. In addition, the effectiveness of Compound 2 in combination with dexamethasone compared to other cereblon modulator compounds is assessed according to biomarkers. Collectively, assessment of these biomarkers can provide guidance for patient selection, regimen design, predict response to therapy, and/or determine treatment effectiveness.

Example 16: Using Biomarkers to Change Treatment for Relapsed and Refractory Multiple Myeloma

The biomarkers presented herein can be used to determine whether treatment should be modified, such as extending or shortening the regimen. Assessing Aiolos/Ikaros repression and T cell activation and proliferation may guide treatment regimens.

To determine whether the optimal dose has been achieved, blood is collected during treatment, PBMCs are isolated, and Aiolos/Ikaros repression is measured using FACS. In one scenario, the amount of Aiolos/Ikaros recovery above the assay background is <15%. In addition, IHC shows that the correlation of bone marrow Aiolos/Ikaros repression across multiple dose levels confirms that bone marrow Aiolos repression is also within 15% of the low end of the assay. Additionally, compared to the next lowest dose level, there is little to no further PD response, suggesting that the effects of PD are modest. Measurement of T cell activation and proliferation is determined by assessing the increase in soluble CD25 in serum/plasma as well as the increase in Ki67 in CD8+ and CD4+ cells. The results indicating that T cell activation and proliferation plateau with dose and that there is no loss of _Teff populations indicate that the optimal dose has been achieved. The absence of grade 3 or higher neutropenia also demonstrates that PD is optimized and indicates that the regimen may be extended rather than dose increased. In this scenario, biomarkers indicate that the optimal biologic dose has been achieved and the regimen can be continued without modification.

Alternatively, biomarker data may indicate that the regimen requires modification.

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In another scenario, treatment results using a dosing schedule may indicate that protein expression levels of the Aiolos/Ikaros biomarkers are restored to greater than 15% above background during treatment in PBMCs and/or IMs. In addition, there may be no plateau for the suppression of Aiolos biomarker expression in PBMC and/or BM at increasing doses. Additionally, assessing T cell activation and proliferation as a biomarker may indicate that T cell activation is not maximal. This suggests that the effects of PD were not optimized by the MTD treatment regimen. The addition of unwanted neutropenia and incomplete or absent recovery of ANC during treatment indicates that the rest window provided by the treatment regimen is insufficient to sufficiently restore neutrophil maturation. Thus, the regimen can be shortened to increase the recovery window and resume dose escalation. Taken together, these two scenarios demonstrate that biomarkers can help guide treatment planning for patients with CMB in multiple myeloma.

Example 17: Use of Biomarkers for Patient Selection and as Prognostic Markers for Treatment of Relapsed and Refractory Multiple Myeloma with Cereblon Modulators

Biomarkers can also be used to guide patient selection and to predict response or resistance to treatment of relapsed/refractory multiple myeloma with a cereblon modulator, such as Compound 2, or an enantiomer, a mixture of enantiomers, a tautomer, or a pharmaceutically acceptable salt.

Bone marrow samples are collected before treatment for screening purposes, during treatment, and after completion of the study. Approximately 6 ml of VMA is collected and CD138 fractionation can be performed. In addition, ex vivo drug testing can be performed on primary samples. Ex vivo data can be combined with clinical data collected from CD138 populations. Collectively, the data that is collected from the biomarkers can be used to guide patient selection for treatment with compounds.

Example 18: Phase 1, multicenter, open-label study to evaluate the safety, pharmacokinetics, and preliminary efficacy of Compound 2 in combination with dexamethasone in subjects with relapsed and refractory multiple myeloma

Indications: Relapsed and refractory multiple myeloma (RRMM).

Primary goals:

Assess the pharmacokinetics (PK), safety/tolerability, and determine the maximum tolerated dose (MTO)/Part 2 recommended dose (RP2D) of Compound 2 in combination with dexamethasone according to a minimum of two dosing regimens of Compound 2.

Secondary Goals:

Assess the preliminary efficacy of Compound 2 in combination with dexamethasone.

Study design

This is an open-label, multicenter, international, Phase 1 study to evaluate the safety, PK/PD, and preliminary efficacy of Compound 2 in combination with dexamethasone in subjects with RRMM. Patients with RRMM previously treated with at least 3 previous regimens, including lenalidomide or pomalidomide, a proteasome inhibitor, and an anti-CD38 antibody, will be eligible to participate.

The study will be conducted in two parts: Part 1 will evaluate the PK/PD and safety of escalating doses of Compound 2 with concurrent standard dose dexamethasone and determine the MTD/RP2D for the combination when administered under at least two different dosing schedules. Part 2 will consist of one expansion cohort(s) of Compound 2 in RP2D plus dexamethasone for one or more dosing regimens. In addition to safety, PK and PD assessments, all subjects will undergo monthly response assessment according to the International Multiple Myeloma Study Group (IMWG) uniform response criteria (Rajkumar et al., Blood, 2011, 117(18):4691-5 ; Kumar et al., Lancet Oncol., 2016,17(8):e328-e346) and may continue study treatment until disease progression, intolerable toxicity, or the physician or subject decides to discontinue study treatment.

The study will be conducted in accordance with the International Conference on Harmonization (ICH) Technical Requirements for Registration of Pharmaceuticals for Human Use/Good Clinical Practice (GCP) and applicable regulatory requirements.

Part 1 (Increasing the dose)

Cohorts of subjects with RRMM will receive escalating doses of Compound 2 plus a fixed dose of dexamethasone (40 mg/dose; 20 mg/dose for subjects >75 years) to evaluate its safety, MTD/RP2D and PK/PD profiles. Part 1 will evaluate at least two different dosing regimens, the first of which consists of 10 consecutive days of once-daily dosing (OID), followed by 4 days of no treatment x 2 times per 28-day cycle (so called the 20/28 scheme). The second regimen will consist of dosing twice daily (bid) for 3 consecutive days with

- 111 045896 subsequent 11 days of absence of study treatment x 2 times per cycle (the so-called 6/28 scheme). The initial dose cohorts will receive 0.1 mg/day of Compound 2 QD on a 20/28 schedule and 0.2 mg BID on a 6/28 schedule. Switching between dosage regimens is not allowed. Additional dosing regimens consisting of dosing Compound twice daily (QD) or twice daily (BID) followed by no treatment days x 2 times in a 28-day cycle (e.g., 5 days of dose 1 r/d or twice a day followed by 9 days of no treatment x 2 times per 28-day cycle or 7 days of dosing once or twice a day followed by 7 days of no treatment x 2 times per 28-day cycle and 21 days of once-daily dosing followed by 7 days of no treatment in a 28-day cycle) may be studied after review of safety and PK/PD results in combination with the initial 20/28 and 6/28 regimens and subsequent investigational regimens.

For all dosing regimens, cycle 1, days 1-28 will represent the dose-limiting toxicity (DLT) assessment period for purposes of determining the MTD. Subjects will be assessed for DLT if they receive the prescribed dose of Compound 2 on at least 16 of the 20 days of dosing on the 20/28 schedule and on at least 5 of the 6 days of dosing (10 doses) on the 6/28 schedule (or at least 80% of the prescribed dose on alternative regimens) in cycle 1, or experience DLT. Subjects not eligible for DLT assessment will be replaced.

In each regimen, cohorts of three or more subjects will receive Compound 2 at doses that will be increased by 100% in successive cohorts until two grade 2 treatment-emergent adverse events occur that cannot be clearly and conclusively attributed to extraneous reasons. Thereafter, dose increases will occur, not exceeding 50%, until the first DLT occurs. Once the first DLT is available, a Bayesian dose escalation technique will be used using logistic regression in any dosing regimen with the assigned dose of Compound 2, number of doses per day (QD and BID), and number of consecutive days of dosing for each regimen as covariates .

No dose escalation among subjects is permitted during the DLT evaluation period, however, in Cycle 2 onwards, subjects who tolerate their assigned dose of Compound 2 may be escalated to the highest dose level that has been shown to be adequately tolerated in at least one cohort of subjects. within the prescribed dosing regimen.

Part 2 (Cohort Expansion)

Upon completion of Part 1, a non-comparative extension study of Compound 2 plus dexamethasone will be conducted in 20 subjects with one or more dosing regimens to further evaluate its safety, PD, and efficacy in RP2D and regimen.

Study Population

Subjects aged >18 years with RRMM, refractory to last line of treatment, previously treated with at least 3 previous regimens, including lenalidomide or pomalidomide, a proteasome inhibitor, and an anti-CD38 antibody, having an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0- 2, measurable disease manifestation and adequate bone marrow, kidney and heart function can be recorded. Subjects with a history of allogeneic transplantation, non- or oligosecretory MM, plasma cell leukemia, or primary refractory MM (i.e., no history of at least mild response to a prior treatment regimen) are excluded.

Number of subjects

Approximately 120 subjects with RRMM from North America and Europe will be enrolled. Approximately 80 subjects will be assigned to one of two dosing regimens (QD or BID) to determine MTD/RP2D with concurrent dexamethasone for each regimen in Part 1. Twenty subjects per dosing regimen (total n=40) will be enrolled in Part 2 to receive Compound 2 in MTD/RP2D with dexamethasone.

Estimates of the number of subjects included in Part 1 are based on the following assumptions: 40 subjects for each dosing regimen, including a minimum of 9 subjects treated in MTD/RP2D for each regimen. In estimating the number of subjects included in Part 2, it is assumed that there are 20 subjects per dosing regimen. These estimates may be modified based on the actual number of cohorts, subjects in the cohort included in each dosing regimen (Part 1), and whether additional dosing regimens are evaluated in Part 2.

Inclusion criteria

Subjects must meet the following criteria for enrollment in the study: Subject's age > 18 years at the time of signing the informed consent form (ICF).

The subject must understand and voluntarily sign the ICF prior to any study-related assessments/procedures.

Subject is willing and able to adhere to study attendance and other protocol requirements.

Eastern Cooperative Oncology Group (ECOG) performance status 0, 1, or 2

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Subjects must have a documented diagnosis of MM and measurable disease upon enrollment. Measurable disease manifestation is defined as: (a) M protein count >0.5 g/dL by sPEP; or (b) > urine collection 200 mg/24 h using uPEP; or (c) serum FLC levels >100 mg/L, including light chain, and abnormal kappa/lambda (κ/λ) ratio in subjects without measurable Mprotein in serum or urine; or (d) for subjects with immunoglobulin A (IgA) myeloma whose disease can only be reliably measured by quantitative immunoglobulin measurement, a serum IgA level >0.50 g/dL.

All subjects must: (a) have received at least 3 prior anti-myeloma treatment regimens, including at least 2 consecutive cycles of lenalidomide, pomalidomide, proteasome inhibitor, glucocorticoid, and anti-CD38 antibody (note: induction with or without bone marrow transplant and maintenance with or without therapy is considered one regimen); (b) have documented disease progression at or within 60 days of the last dose of the latest myeloma therapy; (c) in addition to criteria (a and b) above, subjects included in Part 2 must have disease resistant to an immunomodulatory agent (lenalidomide and/or pomalidomide), a glucocorticoid, a proteasome inhibitor, and an anti-CD38 antibody. Refractory disease is defined as disease that does not respond to therapy (failure to achieve minimal response or development of progressive disease) or progresses within 60 days of the last dose.

Subjects must have the following laboratory values: (a) Absolute neutrophil count (ANC) > 1.25 x 10 ⁹ /L without growth factor support for > 7 days (> 14 days for pegfilgrastim). ANC > 1.00 x 10 ⁹ /L allowed for dose escalation cohorts (Part 2); (b) Hemoglobin (Hgb) > 8 g/dL; (c) Platelets (plt) > 75x10 ⁹ /L without transfusion for > 7 days; (d) Corrected serum calcium level < 13.5 mg/dL (< 3.4 mmol/L); (f) 24-hour creatinine clearance (CrCl) > 45 ml/min; (f) AST/SGOT and ALT/SGPT < 3.0 x upper limit of normal (ULN); (g) Serum bilirubin <1.5 x ULN or <3.0 mg/dL for subjects with documented Gilbert's syndrome; (h) Uric acid < 7.5 mg/dL (446 µmol/L); (i) PT/INR <1.5 x ULN and partial thromboplastin time (PTT) <1.5 x ULN (for subjects not receiving therapeutic anticoagulants). Note: Subjects receiving treatment for a thromboembolic event that occurred >3 months prior to enrollment are eligible as long as they are on a stable anticoagulation regimen with warfarin, low molecular weight heparin, or another approved therapeutic anticoagulation regimen.

Women of childbearing potential (FCBP) must: (a) have two negative pregnancy tests confirmed by the investigator before initiating study therapy. They must agree to an ongoing pregnancy test during the study and after discontinuation of Compound 2. This is done even if the subject practices true abstinence* from heterosexual intercourse; (b) either abstain from heterosexual intercourse (which must be verified monthly), or agree to use and be able to comply with two reliable forms of contraception provided to the subject at the time of informed consent, without interruption, 28 days before the start of Compound 2, during study therapy (including during dosing interruptions) and for 28 days after discontinuation of study therapy. Note: A woman of childbearing potential (FCBP) is a woman who: 1) has achieved menstruation at some point and, 2) has not undergone a hysterectomy or bilateral oophorectomy, or 3) has not been naturally postmenopausal (amenorrhea after cancer therapy does not exclude childbearing potential) at for at least 24 consecutive months (i.e., had menstruation at any time in the previous 24 consecutive months).

Male subjects must practice true abstinence (which should be verified on a monthly basis) or agree to use a condom during sexual intercourse with a pregnant woman or woman of childbearing potential while participating in the study (even during dosage breaks) and for at least 3 months after discontinuation of Compound 2 provided to the subject at the time of informed consent, even if the subject has undergone a successful vasectomy. Complete abstinence is acceptable provided it is consistent with the subject's preferred and usual lifestyle. Intermittent abstinence (eg, calendar ovulation, symptothermal method, post-ovulation methods) and withdrawal (withdrawal) are not acceptable methods of contraception.

Men must agree to refrain from donating sperm while taking Compound 2 and for 90 days after stopping it. Women must agree to refrain from donating eggs while taking Compound 2 and for 28 days after stopping it.

All subjects must agree to refrain from donating blood while taking Compound 2 and for 28 days after stopping it.

Exclusion criteria

The presence of any of the following will disqualify the entity from registration:

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The subject has a significant medical condition, laboratory abnormality, or psychiatric illness that may prevent the subject from participating in the study.

The subject has any condition, including the presence of laboratory abnormalities, that would place the subject at an unacceptable risk if he/she were to participate in the study.

The subject suffers from any condition that impairs the ability to interpret test data.

Subject suffers from non- or oligosecretory multiple myeloma.

Subject with refractory primary multiple myeloma (i.e., no history of at least mild response to a previous treatment regimen).

The subject suffers from plasma cell leukemia or active leptomeningeal myelomatosis.

The subject suffers from systemic light chain amyloidosis or polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin lesions syndrome (POEMS).

Subject suffers from immunoglobulin M (IgM) myeloma.

Subject has a history of allogeneic bone marrow transplantation.

Subject is undergoing dialysis.

Subjects with peripheral neuropathy > grade 2.

Subjects with a gastrointestinal disorder that may significantly alter the absorption of Compound 2.

The subject has impaired cardiac function or clinically significant cardiac disease, including any of the following: (a) LVEF <45% as determined by ECHO or MUGA scan at screening; (b) Complete left bundle branch block, bifascicular block, or other clinically significant electrocardiographic (ECG) abnormality at screening; (c) QT prolongation on the screening ECG, determined by repeatedly demonstrating a QTc interval >480 milliseconds (ms) using the Frederick QT correction formula; history or current risk factors for torsade de pointes (eg, heart failure, hypokalemia, or family history of long QT syndrome); and simultaneous administration of drugs that prolong the QT/QTc interval; (d) Congestive heart failure (class III-IV, according to the functional classification of the New York Heart Association); (f) Myocardial infarction < 6 months before the start of Compound 2 administration; (f) Unstable or poorly controlled angina, including Prinzmetal angina.

Concomitant administration of strong CYP3A modulators

Subject has previously received systemic treatment for myeloma with an investigational agent (eg, anti-PD-1, anti-PD-S) < 5 half-lives prior to initiation of Compound 2; the subject has previously been exposed to an approved myeloma treatment (including therapeutic monoclonal antibodies such as anti-CD38 or α-anti-SLAM-7) <5 half-lives or within 4 weeks prior to initiation of Compound 2, whichever is shorter.

Subject underwent major surgery <2 weeks prior to initiation of Compound 2. Note: Subjects must have recovered from any clinically significant effects of recent surgery.

The subject is pregnant or lactating, or intends to become pregnant or donate eggs while participating in the study.

The subject has a known human immunodeficiency virus (HIV) infection.

The subject has a known active or chronic hepatitis B or C virus (HBV/HCV) infection.

The subject has a history of concurrent secondary cancer requiring active long-term systemic treatment.

Subjects had a history of previous malignancies other than MM, unless the subject had been disease free for >3 years OR the subject had one of the following noninvasive malignancies that had been treated with curative intent without known recurrence: (a) Basal or squamous cell skin cancer; (b) Carcinoma in situ of the cervix or breast; (c) Stage 1 bladder cancer; (d) Incidental histologic findings of localized prostate cancer, such as stage 1a or 1b (T1a or T1b) tumor using tumor/node/metastasis (TNM) malignancy classification OR prostate cancer that has been treated with curative intent.

The subject has a history of anaphylaxis to thalidomide, lenalidomide, pomalidomide, or dexamethasone.

The subject has a known or suspected hypersensitivity to the excipients (excipients include dimethylsilica, anhydrous colloidal silica, mannitol, fumaric acid, and stearic acid) contained in Compound 2 or dexamethasone.

The subject underwent any of the following within 14 days of starting Compound 2: (a) plasmapheresis; (b) radiotherapy, other than local therapy, to relieve symptoms associated with

- 114 045896 bone lesions associated with MM.

The subject has received immunosuppressive treatment for 14 days prior to the first dose of Compound 2. Exceptions to this criterion include the following: (a) Intranasal, inhaled, topical or topical corticosteroid injections (eg, intra-articular injections); (b) Systemic corticosteroids in doses not exceeding 10 mg/day of prednisone or its equivalent; (c) Steroids as premedication for hypersensitivity reactions (eg, premedication for computed tomography [CT] scans).

The subject is unable or unwilling to complete the venous thromboembolism (VTE) prophylaxis protocol.

Duration of the study

The average length of study participation per subject is expected to be approximately 6 months. Full registration is expected to take approximately 21 months to complete (18 months for Stage 1 and 3 months for Stage 2). Completion of active treatment and post-treatment follow-up are expected to take an additional 6 to 12 months. The entire study is expected to last approximately 33 months.

The end of the trial is defined as the date of the last visit of the last subject to complete post-treatment follow-up or the date of the last follow-up result from the last subject required for primary, secondary and/or exploratory analyzes as specified in the protocol, whichever is later. .

Treatment regimens being studied

Compound 2 will be administered orally either once daily for subjects enrolled in the 20/28 regimen (or alternative once daily regimens) or twice daily for subjects enrolled in the 6/28 regimen (or alternate twice daily regimens). day). For subjects enrolled in a once-daily dosing regimen, Compound 2 should be administered in the morning with at least 240 ml (milliliters) of water after an overnight fast of at least 6 hours. Subjects should abstain from eating or taking other medications for at least 2 hours after each morning dose. Subjects enrolled in the BID regimen will follow the above instructions as indicated for the BID regimen for the first dose of each dosing day. The second dose should be administered 12 ± 2 hours after the morning dose, at least 4 hours after and 2 hours before meals.

For both dosing regimens, dexamethasone can be administered with Compound 2 on an empty stomach or at least 2 hours after Compound 2 with food (except on PK assessment days, when both should be administered at the same time).

Review of key performance indicators

The primary efficacy variable is the best overall response rate (ORR), defined as the proportion of subjects whose best response > OR, according to the IMWG uniform response criteria (Rajkumar et al., Blood, 2011, 117(18):4691-5; Kumar et al., Lancet Oncol., 2016, 17(8):e328e346). Subjects will receive monthly response assessments. Myeloma response is determined by a site investigator based on laboratory tests (serum protein electrophoresis [sPEP], urinary protein electrophoresis [uPEP], immunofixation electrophoresis [IFE], serum free light chain [sFLC] levels, quantitative immunoglobulin A [IgA], bone marrow analysis to quantify plasma cells, as appropriate) performed at a central reference laboratory and/or locally (eg, corrected serum calcium, computed tomography [CT], positron emission tomography/computed scan [PET] /CT] or magnetic resonance imaging [MRI] to evaluate plasmacytoma and/or CT, PET/CT, MRI or skeletal examination to evaluate bone involvement). Additional efficacy variables include time to response, duration of response, and progression-free survival.

All safety subjects with a valid baseline and at least one post-baseline response assessment will be included in the effectiveness analysis. If treatment is discontinued for reasons other than disease progression, subjects will be asked to continue response assessments according to the established assessment schedule until progression, withdrawal of consent, death, or initiation of new systemic myeloma therapy, whichever occurs first.

Review of key safety assessment indicators

Safety variables for this study include treatment-emergent adverse events (TEAEs) and changes from baseline in physical findings/vital signs, selected laboratory analytes, and 12-lead electrocardiograms (ECGs). Additional safety indicators include exposure to study drug (both Compound 2 and dexamethasone), assessments of concomitant medication use, and a pregnancy test for women of childbearing potential (FPP).

Review of pharmacokinetic evaluations

PK profiles (initial dose and steady state) will be assessed for Compound 2, its

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R-enantiomer (Compound 3) and dexamethasone. Exposure-response assays can be performed as needed to assist in the identification of Compound 2 RP2D.

Review of pharmacokinetic evaluations

Biomarkers will be assessed in blood and bone marrow at baseline and at specific time points during treatment with the study drug. Changes from baseline in Aiolos and Ikaros levels in peripheral blood mononuclear cells (PBMCs) and myeloma cells in bone marrow, immune phenotypes in peripheral blood and bone marrow, and levels of proinflammatory cytokines (eg, interleukin-2 [IL-2], interferon-gamma [IFN-γ]) will be assessed as a function of dose and schedule. Longitudinal quantification of sFLC and soluble B-cell maturation antigen (sBCMA) levels as well as circulating tumor cells will be performed as a means to evaluate early treatment effects. Changes from baseline in expression of cereblon and downstream markers (eg, c-Myc, IRF-4), as well as gene expression in myeloma cells will be assessed as a means to identify potential markers of response and resistance to Compound 2 plus dexamethasone. Finally, MRD detection will be performed in subjects who undergo bone marrow evaluation to confirm complete response (CR).

Point estimates and two-sided 95% confidence intervals for overall response rates (ORR) will be reported. Additional efficacy variables include duration of response, time to response, and progression-free survival (PFS). Effectiveness results will be summarized using frequency tabulations for categorical variables or descriptive statistics for time-to-event variables. Efficacy analyzes will be presented as population-based safety and efficacy (SE) estimates, with population-based EE results considered primary.

Interim results of pharmacodynamic analysis

Aiolos expression was measured by flow cytometry in CD3+ T cells before and during treatment with Compound 2 at two different dosing regimens (administration once daily on days 1-10 and days 15-24 of a 28-day cycle and twice daily administration on days 1-3 and days 15-17 of the 28-day cycle). As shown in FIG. 24A and 24B, the degradation of Aiolos was dose dependent and was restored during breaks in dosing of the compound in both regimens.

Ikaros expression was measured by flow cytometry in CD3+ T cells before and during treatment with Compound 2 at two different dosing regimens (administration once daily on days 1-10 and days 15-24 of a 28-day cycle and twice daily administration on days 1-3 and days 15-17 of the 28-day cycle). As shown in FIG. 25A and 25B, the degradation of Ikaros was dose dependent and was restored during breaks in dosing of the compound in both regimens.

Bone marrow aspirate samples were collected before and during treatment with Compound 2 (cycle 1). Each sample was clotted and formed into formalin-fixed, paraffin-embedded blocks. Immunohistochemical (IHC) analyzes were used to evaluate biomarker expression. IHC scores were determined based on the percentage and intensity of positive staining. As shown in FIG. 26A-26E, Compound 2 induced the destruction of Aiolos, Ikaros and ZFP91 in the tumor compartment. Inhibition of Aiolos and Ikaros leads to decreased expression of c-Myc and IRF4, which leads to apoptosis of tumor cells and can be used to indicate response to treatment with Compound 2.

Serum samples were collected before and during treatment with Compound 2 at specific time points. sBCMA expression was measured by ELISA assay. As shown in FIG. 27, sBCMA signal was reduced by treatment with Compound 2. Samples from one subject showed a >80% reduction in sBCMA compared to baseline, and the subject had a favorable response to treatment (very good partial response; VGPR). The degree of reduction and duration of effect may predict response to treatment with Compound 2.

Serum samples were collected before and during treatment with Compound 2 at specific time points. Serum free light chain (sFLC) can be used to measure efficacy in multiple myeloma. The short half-life of sFLC makes it possible to monitor the dynamic effect of combination treatment on disease severity. As shown in FIG. 28, the sFLC signal decreased upon treatment with Compound 2, indicating the killing of tumor cells. Two subjects showed a >80% reduction in sFLC during the first treatment cycle and a positive response to treatment (PR and VGPR, respectively). The degree of reduction from baseline and duration of effect may predict response to treatment with Compound 2.

The embodiments described above are intended to be examples only and those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents of specific compounds, materials, and techniques. Such equivalents are intended to be included within the scope of this invention and are covered by the appended claims.

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Claims (72)

CLAIM 1. A method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level; wherein the therapeutic compound is Compound 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, and wherein the biomarker comprises one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 2. A method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level; wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof and wherein the biomarker comprises one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 3. A method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising: (a) administering the therapeutic compound to the subject; - 117 045896 (b) obtaining a sample from the subject; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level; wherein the therapeutic compound is Compound 3 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 4. A method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) introducing the therapeutic compound into the sample; (c) determining the level of biomarker in the sample; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; wherein the therapeutic compound is Compound 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 5. A method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) introducing the therapeutic compound into the sample; (c) determining the level of the biomarker in the sample; and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level - 118 045896 the level of biomarker in the sample differs from the control level of the biomarker; wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of: cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 6. A method for identifying a subject having cancer that is likely to be susceptible to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) introducing the therapeutic compound into the sample; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control biomarker level; wherein the therapeutic compound is Compound 3 o or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 7. A method of treating cancer, comprising: (a) obtaining a sample from a subject having cancer; (b) determining the level of biomarker in the sample; (c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering a therapeutically effective amount of a therapeutic compound to a subject who has been diagnosed as likely to be susceptible to the therapeutic compound; wherein the therapeutic compound is Compound 1 - 119 045896 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 8. A method of treating cancer, comprising: (a) obtaining a sample from a subject having cancer; (b) determining the level of biomarker in the sample; (c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering a therapeutically effective amount of a therapeutic compound to a subject who has been diagnosed as likely to be susceptible to the therapeutic compound; wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 9. A method of treating cancer, comprising: (a) obtaining a sample from a subject having cancer; (b) determining the level of biomarker in the sample; (c) diagnosing a subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample differs from the control level of the biomarker; and (d) administering a therapeutically effective amount of a therapeutic compound to a subject who has been diagnosed as likely to be susceptible to the therapeutic compound; wherein the therapeutic compound is Compound 3 - 120 045896 o or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 10. A method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 1 o or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 11. A method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 2 - 121 045896 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 12. A method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 3 o or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 13. A method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) introducing the therapeutic compound into the sample; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 1 - 122 045896 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of: cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 14. A method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) introducing the therapeutic compound into the sample; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 15. A method of predicting the susceptibility of a subject having or suspected of having cancer to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) introducing the therapeutic compound into the sample; (c) determining the level of the biomarker in the sample and (d) diagnosing the subject as likely susceptible to the treatment compound if the level of the biomarker in the sample differs from the level of the biomarker obtained from the control sample; wherein the therapeutic compound is Compound 3 - 123 045896 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of: cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 16. The method according to any one of claims 1 to 15, characterized in that the level of the biomarker in the sample is higher than the control level of the biomarker. 17. The method according to any one of claims 1 to 15, characterized in that the level of the biomarker in the sample is lower than the control level of the biomarker 18. A method of monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of a biomarker in the sample and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject; wherein the therapeutic compound is Compound 1 o or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of: cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 19. A method of monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of biomarker in the sample and - 124 045896 (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject; wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 20. A method of monitoring the effectiveness of a therapeutic compound in treating cancer in a subject, comprising: (a) administering the therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) determining the level of a biomarker in the sample and (d) comparing the level of a biomarker in the sample with the level of a biomarker obtained from a control sample, wherein the change in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating cancer in the subject; wherein the therapeutic compound is Compound 3 or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and where the biomarker contains one or more of the following: (i) cereblon (CRBN); (ii) a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4; (iii) an apoptotic marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3), cleaved caspase-7 (c-caspase-7), cleaved PARP , survivin, BIM BCL-2-like protein 11 (BIM) and serum-free light chain (sFLC); (iv) a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1); (v) a marker of T cell activation selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy) and T cell receptor clonality (TCR); and/or (vi) circulating tumor cells (CTCs), where the cancer is multiple myeloma (MM). 21. The method of claims 18 to 20, wherein an increased level of the biomarker compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject. 22. The method of claims 18 to 20, wherein a reduced level of the biomarker compared to a control level indicates the effectiveness of the therapeutic compound in treating cancer in the subject. 23. The method according to any one of claims 1-6 and 10-17, further comprising administering a therapeutically effective amount of a therapeutic compound to a subject who has been diagnosed as likely to have - 125 045896 sensitive to medicinal compounds. 24. The method according to any one of claims 7-9 and 23, further comprising administering a therapeutically effective amount of the second active agent or maintenance therapy. 25. The method of claim 24, wherein the second active agent is selected from the group consisting of high molecular weight compounds, low molecular weight compounds or cell therapies, and the second active agent is optionally selected from the group consisting of melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin, bendamustine , proteasome inhibitor, histone deacetylase inhibitor, BET inhibitor, BCL2 inhibitor, MCL-1 inhibitor, corticosteroid, dexamethasone, antibody, checkpoint inhibitor and CAR cells. 26. The method of any one of claims 1 to 25, wherein the control sample is obtained from the subject prior to administration of the therapeutic compound to the subject, and wherein the control sample is obtained from the same source as the sample. 27. The method according to any one of claims 1 to 25, characterized in that the control sample is obtained from a healthy subject who does not have cancer, and wherein the control sample is obtained from the same source as the sample. 28. The method according to any one of claims 1 to 25, wherein the control sample is obtained from a subject receiving an anti-cancer compound that is not the specified therapeutic compound, and wherein the control sample is obtained from the same source as the sample. 29. The method according to any one of 1-28, characterized in that said anticancer compound is selected from the group consisting of lenalidomide or pomalidomide. 30. The method according to any one of claims 1-29, characterized in that the MM is recurrent, refractory or resistant to traditional therapy. 31. Method according to any one of claims 1 to 30, characterized in that the biomarker is cereblon (CRBN). 32. The method according to any one of claims 1 to 30, characterized in that the biomarker is a CRBN-associated protein selected from the group consisting of IKZF1, IKZF3, ZFP91, c-MYC and IRF4. 33. The method according to any one of claims 1 to 30, characterized in that the biomarker is an apoptosis marker selected from the group consisting of cleaved caspase-1 (c-caspase-1), cleaved caspase-3 (c-caspase-3 ), cleaved caspase-7 (c-caspase-7), cleaved PARP, survivin, BIM BCL-2-like protein 11 (BIM), and serum-free light chain (sFLC). 34. The method according to any one of claims 1 to 30, characterized in that the biomarker is a cell cycle arrest marker selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and retinoblastoma protein (pRb1 ). 35. The method according to any one of claims 1 to 30, characterized in that the biomarker is a T cell activation marker selected from the group consisting of soluble CD25 (sCD25), interleukin-2 (IL2), tumor necrosis factor alpha (TNFa) ), interferon gamma (IFNy) and T cell receptor (TCR) clonality. 36. Method according to any one of claims 1 to 30, characterized in that the biomarker is circulating tumor cells (CTCs). 37. The method according to claim 32, characterized in that the biomarker is IKZF1. 38. The method according to claim 32, characterized in that the biomarker is IKZF3. 39. The method according to claim 32, characterized in that the biomarker is ZFP91. 40. The method according to claim 32, characterized in that the biomarker is c-MYC. 41. The method according to claim 32, characterized in that the biomarker is IRF4. 42. The method according to claim 33, characterized in that the biomarker is cleaved caspase-3 (p-caspase-3). 43. The method according to claim 33, characterized in that the biomarker is cleaved caspase-1 (p-caspase-1). 44. The method according to claim 33, characterized in that the biomarker is cleaved caspase-7 (p-caspase-7). 45. The method according to claim 33, characterized in that the biomarker is cleaved PARP. 46. The method according to claim 33, characterized in that the biomarker is survivin. 47. The method according to claim 33, characterized in that the biomarker is BCL-2-like protein 11 (BIM). 48. The method according to claim 34, characterized in that the biomarker is p21. 49. The method according to claim 34, characterized in that the biomarker is p27. 50. The method according to claim 34, characterized in that the biomarker is pRb1. 51. The method according to claim 35, characterized in that the biomarker is soluble CD25 (sCD25). 52. The method according to claim 35, characterized in that the biomarker is IL-2. 53. The method according to claim 35, characterized in that the biomarker is TNFa. - 126 045896 54. The method according to claim 35, characterized in that the biomarker is IFNy. 55. The method according to claim 35, characterized in that the biomarker is T-cell receptor (TCR) clonality. 56. The method of claim 55, wherein the biomarker is measured by sequencing TCR DNA. 57. A method of identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be receptive to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level; wherein the therapeutic compound is Compound 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof and wherein the biomarker is CRBN. 58. A method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be responsive to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level; wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof and wherein the biomarker is CRBN. 59. A method for identifying a subject with multiple myeloma that is relapsed, refractory, or resistant to conventional therapy and who is likely to be responsive to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level; wherein the therapeutic compound is Compound 3όc or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and wherein the biomarker is CRBN. 60. A method of predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level; wherein the therapeutic compound is Compound 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof and wherein the biomarker is CRBN. - 127 045896 61. A method of predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level; wherein the therapeutic compound is Compound 2 or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof, and wherein the biomarker is CRBN. 62. A method of predicting the susceptibility of a subject having multiple myeloma to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) determining the level of the biomarker in the sample and (c) diagnosing the subject as likely susceptible to the therapeutic compound if the level of the biomarker in the sample is below the control biomarker level; wherein the therapeutic compound is Compound 3 o or a tautomer, isotopologue or pharmaceutically acceptable salt thereof and wherein the biomarker is CRBN. 63. The method of any one of claims 57 to 62, comprising diagnosing a subject as likely susceptible to a therapeutic compound if the level of a biomarker in the sample is below a control biomarker level. 64. The method of any one of claims 57 to 62, comprising diagnosing a subject as likely susceptible to a therapeutic compound if a biomarker is detected in the sample. 65. The method according to any one of claims 1-54 or 57-64, characterized in that the level of the biomarker is measured by determining the level of the biomarker protein. 66. The method according to any one of claims 1-54 or 57-64, characterized in that the level of the biomarker is measured by determining the level of mRNA of the biomarker. 67. The method according to any one of claims 1-54 or 57-64, characterized in that the level of the biomarker is measured by determining the level of cDNA of the biomarker. 68. The method according to any one of claims 1-54 or 57-64, characterized in that the biomarker is determined by RNA sequencing (RNA-seq). 69. The method of claim 65, comprising contacting proteins in the sample with a first antibody that immunospecifically binds to the biomarker protein. 70. The method according to claim 69, further comprising: (a) contacting a biomarker protein bound to the first antibody with a second antibody having a detectable label, wherein the second antibody immunospecifically binds to the biomarker protein, and wherein the second antibody immunospecifically binds to a different epitope on the biomarker protein than the first antibody; (b) detecting the presence of a second antibody associated with the biomarker protein; and (c) determining the amount of biomarker protein based on the amount of detectable label on the second antibody. 71. The method according to claim 69, further including: (a) contacting a first antibody bound to the biomarker protein with a second antibody having a detectable label, wherein the second antibody immunospecifically binds to the first antibody; (b) detecting the presence of a second antibody bound to the first antibody; and (c) determining the amount of biomarker protein based on the amount of detectable label on the second antibody. 72. A method of determining or adjusting a dose for treating a subject having multiple myeloma with a therapeutic compound, comprising: (a) administering a dose of a therapeutic compound to the subject; - 128 -