WO2022261138A1 - Disrupted ikaros signaling as biomarker for btk inhibition - Google Patents

Disrupted ikaros signaling as biomarker for btk inhibition Download PDF

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Publication number
WO2022261138A1
WO2022261138A1 PCT/US2022/032563 US2022032563W WO2022261138A1 WO 2022261138 A1 WO2022261138 A1 WO 2022261138A1 US 2022032563 W US2022032563 W US 2022032563W WO 2022261138 A1 WO2022261138 A1 WO 2022261138A1
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aspects
ikaros
btk
responder
subject
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PCT/US2022/032563
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French (fr)
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Emmanuel Normant
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Tg Therapeutics, Inc.
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Publication of WO2022261138A1 publication Critical patent/WO2022261138A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates to methods of treating hematologic malignancies, such as, e.g., B-cell non-Hodgkin lymphomas (B-NHLs).
  • B-NHLs B-cell non-Hodgkin lymphomas
  • the present disclosure provides methods for treating various B-NHLs by administering a BTK inhibitor (e.g., TG-1701) to a subject that has been determined to be a responder to a BTK inhibitor (e.g., TG-1701) through, e.g., phosphoproteomic analyses.
  • the present disclosure also provides methods and kits for identifying if a subject is a responder to a BTK inhibitor.
  • B-cell non-Hodgkin lymphomas account for up to 4% of globally diagnosed cancers (Fisher et al., Oncogene 23:6524–6534 (2004)). B-NHLs are divided into low and high grades, typically corresponding to indolent (slow-growing) lymphomas, such as chronic lymphocytic leukemia (CLL), and aggressive lymphomas, such as mantle cell lymphoma (MCL), respectively (Quintanilla-ML, Hematol Oncol.35:37-45 (2017)).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • BTKi B-cell receptor
  • the first- in-class BTKi ibrutinib
  • CLL Cyrd, JC et al., N Engl J Med 369: 32-42 (2013)
  • MCL Wang, ML et al., N Engl J Med 369: 507-516 (2013)
  • W Waldenström's macroglobulinemia
  • ABSC-DLBCL activated B-cell-like diffuse large B-cell lymphoma
  • BTK inhibitors such as ibrutinib
  • ibrutinib the activity of BTK inhibitors, such as ibrutinib
  • the activity of BTK inhibitors has often been limited due to 1) off-target activity that precluded their use in combination with anti- CD20 antibodies; and 2) acquired resistance due to the development of mutations that either effect the irreversible binding of the BTKi to BTK, or activate the PLC ⁇ 2 enzyme, a downstream enzyme in the BTK pathway (Woyach, JA et al., N Engl J Med 370:2286- 2294 (2014)).
  • BTK C481S cysteine-to-serine mutation at the BTK catalytic site
  • next-generation sequencing NGS-based genomic techniques, including whole exome and targeted deep sequencing, have been instrumental in identifying the BTK C481S mutation as a genetic cause of BTKi resistances (Doostparast, TA and Wang. K., Drug Discov Today 23: 1776-1783 (2016); Wacker, SA et al., Nat Chem Biol 8:235-237 (2012)).
  • phosphoproteomic profiling can help understand the role of BTKi in CLL patients; in one report, CLL cells from patients with an unmutated IGVH status showed higher basal phosphorylation than patients with IGVH-mutated status (Beckmann, L et al., “MARCKS affects cell motility and response to BTK inhibitors in CLL,” Blood (2021)).
  • TG-1701 is a novel, orally available, irreversible, and highly specific BTKi that exhibits improved selectivity when compared to ibrutinib (Normant, E. et al., “TG-1701 A Novel, Orally Available, and Covalently-Bound BTK Inhibitor,” EHA Library, 215080, Abstr. No.638 (June 152018)), and shows activity in various in vitro and in vivo models of B-NHL.
  • TG-1701 is currently under study in patients with relapsed/refractory (R/R) B-NHL, alone and in combination with ublituximab, a glycoengineered anti-CD20 antibody, and umbralisib, a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor (also referred to as the “U2” regimen).
  • R/R relapsed/refractory
  • U2 casein kinase-1 ⁇ inhibitor
  • B-NHL B-cell non-Hodgkin lymphoma
  • methods of treating B-cell non-Hodgkin lymphoma comprising administering to a subject that is a TG- 1701 responder a therapeutically effective amount of BTK inhibitor TG-1701, wherein prior to said administration, B-NHL cells of the TG-1701 responder contain at least one phosphopeptide selected from SEQ ID NOS: 1-95.
  • the at least one phosphopeptide is SEQ ID NO: 1.
  • the presence of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis.
  • the B-NHL cells of the TG-1701 responder lack at least one phosphopeptide selected from SEQ ID NOS: 1-95 after administration of TG-1701 to the subject compared to before administration of TG-1701.
  • the at least one phosphopeptide is SEQ ID NO: 1.
  • the lack of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis.
  • the B-NHL cells of the TG-1701 responder comprise an increased quantity of transcripts of an Ikaros-repressed gene signature after the administration of TG-1701 to the subject compared to the quantity of transcripts before the administration of TG-1701.
  • the transcripts of the Ikaros-repressed gene signature comprise one or more of TXNIP, CD36, CA2, YOD1, CFP, DENND3, YES1, NBEAL2, TMC8, PSTPIP2, CD97, DAAM1, NT5E, LYZ, SDK2, TSC22D4, GYPC, FAM129A, TPM3, GNAQ, and/or LUZP1.
  • the B-NHL cells of the TG-1701 responder comprise a decreased quantity of transcripts of an Ikaros-enhanced gene signature after the administration of TG-1701 to the subject compared to the quantity of transcripts before the administration of TG-1701.
  • the transcripts of an Ikaros-enhanced gene signature comprise one or more of TCL1A, CBX5, HNRNPA0, PDHB, BCL2, DYNLL1, SUPT16H, CAMK2D, ALDH6A1, PPP2R5C, ERGIC1, BUB3, SORD, SEPHS1, CTNNBL1, CCT5, and/or APOBEC3G.
  • the quantity of transcripts is determined by at least one amplification-based method.
  • the amplification-based method is Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification.
  • the quantity of transcripts is determined by qPCR.
  • the quantity of transcripts is determined by at least one non- amplification-based method.
  • the non-amplification-based method is a hybridization-based method or a sequencing-based method.
  • the hybridization-based method is a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, or in situ hybridization.
  • the sequencing-based method is a next-generation sequencing (NGS) method.
  • NGS next-generation sequencing
  • the quantity of transcripts is determined by a combination of amplification-based and non-amplification-based methods.
  • the B-NHL is a chronic lymphocytic leukemia, a mantle cell lymphoma, a follicular lymphoma, a diffuse large B-cell lymphoma, a marginal zone B- cell lymphoma, a Burkitt lymphoma, or a lymphoplasmacytic lymphoma.
  • the method further comprises administering to a subject that is a TG-1701 responder a therapeutically effective amount of an anti-CD20 antibody.
  • the method further comprises administering to a subject that is a TG-1701 responder a therapeutically effective amount of a dual PI3K ⁇ and casein kinase- 1 ⁇ inhibitor.
  • the therapeutically effective amount of TG-1701 is between about 100 mg/day and about 400 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 100 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 200 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 300 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 400 mg/day.
  • the subject is a mammal.
  • kits comprising: (i) at least one antibody that binds to at least one phosphopeptide selected from SEQ ID NOs: 1-95; (ii) optionally, reagents to perform a western blot analysis; and/or reagents to perform a phospho-flow analysis; and (iii) instructions for treating B-NHL in a TG-1701 responder according to the methods disclosed herein.
  • the at least one phosphopeptide is SEQ ID NO: 1.
  • the kit further comprises reagents to perform a Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification.
  • the kit further comprises reagents to perform a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, next-generation sequencing (NGS) method, or in situ hybridization.
  • PCR Polymerase Chain Reaction
  • RT-PCR Real-Time Polymerase Chain Reaction
  • qPCR Quantitative Polymerase Chain Reaction
  • the kit further comprises reagents to perform a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, next-generation sequencing (NGS) method, or in situ hybridization.
  • NGS next-generation sequencing
  • FIG.1A shows binding of TG-1701 and ibrutinib 1 ⁇ M against a panel of 441 kinases using the DiscoverX technology. The size of each dark grey circle is proportional to the strength of the binding.
  • FIG.1B TG- 1701 and ibrutinib BTK wt anti-kinase activities were tested using a 33 P-ATP filtration assay.
  • FIG.1C DoHH2 BTK-expressing cells were incubated with ibrutinib or TG- 1701, lysed, and lysates were incubated with a fluorescent ibrutinib probe. Total BTK was assessed by Western blot.
  • FIG.1D increasing concentrations of ibrutinib or TG- 1701 were incubated with DoHH-2 cells. The BCR pathway is then activated with 10 ⁇ g/ml goat F(ab’) 2 anti-IgM for 18h and levels of different downstream enzymes assessed using Western blot.
  • FIG.1E TG-1701 or ibrutinib were dosed orally in the Mino MCL xenograft model.
  • FIG.1F intratumor levels of several BCR-related kinases were assessed by Western blot.
  • FIGS.2A-2F show that phosphoproteomic analysis of six CLL patients treated with TG-1701 can segregate TG-1701 responders and non-responders.
  • FIG.2A BTK occupancy was assessed in all patients in the study at 9 different times points. The occupancy of the 6 CLL patients are shown at 4 hours after treatment with TG-1701.
  • FIG.2B Correlation of TG-1701 Cmax and the daily dose received by the 6 patients.
  • FIG.2C Best tumor reduction in all six patients at cycle 3, day 1 (C3D1). * Patient AIK- 0003 lymphocytosis at C3D1 ranks the response as a stable disease (SD).
  • FIG.2D Phosphoproteomic profiling and principal components (PC) analysis were performed on all 6 CLL patients (left panel), three responders (middle panel), and three non-responders (right panel).
  • PC principal components
  • Pre-treatment samples are designated and the 4 hour post- treatment samples are designated in white (POST). Percentages refer to the total variance explained for each component.
  • FIG.2E Quantified phosphopeptides in responders. Volcano plot of the responder-only samples. The non-responders do not exhibit any TG- 1701-driven changes.
  • FIG.2F The phosphosites that are up- or down- regulated are shown for each single three responder patients. The non-responders did not show any changes in the phosphoproteomic analysis.
  • FIG.3B Comparative multi-dimensional (MDS) analysis of RNA-seq data. The closer the samples, the more similar their RNA-Seq signature.
  • FIG.3C Immunoblot evaluation of p-BTK, Ikaros and Ikaros downstream factors MYC and IRF4, in PBMC lysates from two CLL patients with distinct responses to TG-1701.
  • FIGS.4A-4H show that impairment of Ikaros signaling is associated with B-NHL response to TG-1701 in both clinical and preclinical settings. Change of Ikaros-regulated factors upon TG-1701 treatment in responders (R) and non-responders (NR), according to total proteome data (FIG.4A) and RNA-seq analysis (FIG.4B) of the same samples. For each category, the average of the three patients is displayed.
  • FIG.4C shows YES1 (an Ikaros-repressed gene) and MYC (an Ikaros-enhanced gene) mRNA changes after treatment with TG-1701.
  • FIG.4D depicts immunoblot evaluation of p-BTK, Ikaros, and Ikaros downstream factors, MYC and IRF4, using one representative responder and one non-responder PBMC lysates.
  • P-BTK detection was assessed to confirm on-target activity at 4 hours post-treatment.
  • MCL REC-1 cells were treated for 24 hours with 1 ⁇ M ibrutinib or TG-1701, and variations in Ikaros-regulated factors were quantified using qPCR (FIG.4E) and Western blot (FIG.4F).
  • FIGS.5A-5F show that Ikaros modulation is associated with TG-1701 efficacy in distinct in vitro and in vivo MCL models of ibrutinib resistance.
  • FIG.5A Viability of BTK wt and BTK C481S REC-1 cells exposed to increasing doses of ibrutinib and TG-1701 were evaluated by CellTiter Glo assay.
  • FIG.5B REC-1 and REC-1-BTK C481S cells were exposed for 1h to 1 ⁇ M TG-1701, washed-out for the indicated times, and levels of phospho-BTK and loading control GADPH were assessed using immunoblotting. Values below immunoblot correspond to the densitometric quantification of p-BTK/GADPH ratio.
  • FIG.5C Regulation of Ikaros-regulated factors after 4 hours of treatment with TG- 1701 or ibrutinib (1 ⁇ M) in REC-1 and REC-1-BTK C481S cells according to total proteome data.
  • FIG.5D Ikaros gene signatures were evaluated by qPCR in REC-1 and REC-1-BTK C481S cells exposed for 24 h to 1 ⁇ M ibrutinib or TG-1701. In REC-1- BTK C481S cells, values were referred to untreated REC-1 cells (control).
  • FIGS.6A-6D show in vivo activity of TG-1701 on BTKi-sensitive and BTKi- resistant MCL mouse models.
  • FIG.6A TG-1701 was dosed orally in BKTi-sensitive (REC-1-GFP+LUC+) and BTKi-resistant (UPN-IbruR) MCL xenograft models and tumor volumes were recorded at the endpoint (17 days) by bioluminescence signal recording (REC-1) or external calipers (UPN-IbruR).
  • FIGS.7A-7D FIG.7A: Inhibition of BTK and BTK C481S kinase activity by TG- 1701 and ibrutinib were assessed using a 33 P-ATP filtration assay.
  • FIG.7B Proteomic profiling and principal components analysis were performed on REC-1 and BTK C481S derivative after treatment with ibrutinib or TG-1701 (1 ⁇ M) for 4 hours.
  • FIG.7C Western blot examination of REC-1-BTK C481 whole cell extracts following a 24-hour treatment with ibrutinib or TG-1701.
  • FIG.7D Viability of BTKi-sensitive (UPN-wt) and insensitive (UPN-IbruR) UPN-1 derived cell lines exposed to increasing doses of ibrutinib and TG-1701 was evaluated by a CellTiter Glo assay.
  • FIG.8 depicts a schematic of the BTK occupancy assay developed using a MSD chemoluminescent platform at Bioagilytix. The % occupancy was calculated as shown in the figure.
  • a BTK inhibitor e.g., TG-1701
  • a BTK inhibitor responder e.g., a TG- 1701 responder
  • the BTK inhibitor (e.g., TG-1701) responder is a subject whose B-NHL cells, prior to treatment, comprise at least one phosphopeptide (e.g., Ikaros), not comprised by B-NHL cells of a non-responder, which is dephosphorylated after treatment with the BTK inhibitor (e.g., TG-1701).
  • the B-NHL cells of the BTK inhibitor (e.g., TG- 1701) responder further comprise at least one transcript of a gene of an Ikaros-enhanced gene signature and/or lack at least one transcript of a gene of an Ikaros-enhanced gene signature.
  • the methods further comprise treating B-NHL by administering TG-1701 to a TG-1701 responder, in combination with an anti-CD20 antibody (e.g., ublituximab) and/or umbrasilib.
  • a or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of "either/or.”
  • “and/or,” where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term "effective amount” or “therapeutically effective amount” refers to an amount of a compound, or combination of one or more compounds that, when administered (either sequentially or simultaneously) elicits the desired biological or medicinal response, e.g., either destroys the target cancer cells or slows or arrests the progression of the cancer in a patient.
  • the therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the patient and disease condition being treated, e.g., the weight and age of the patient, the severity of the disease condition, the manner of administration and the like, which may readily be determined by one skilled in the art.
  • the term also applies to a dose that will induce a particular response in target cells, e.g., reduction of cell proliferation and/or cell migration.
  • the "therapeutically effective amount” as used herein refers to the amount of TG-1701, or a pharmaceutically acceptable salt thereof, and/or the amount of an anti-CD20 antibody and/or of a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor that, when administered separately or in combination, have a beneficial effect.
  • the "therapeutically effective amount” as used herein refers to the amount of TG- 1701, or a pharmaceutically acceptable salt thereof, and the amount of an anti-CD20 antibody and a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor that, when administered separately or in combination, have a beneficial effect.
  • the combined effect is additive. In some aspects, the combined effect is synergistic.
  • the amount of TG-1701, or a pharmaceutically acceptable salt thereof, and/or the amount of the CD20 antibody and/or the dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor may be used in a "sub- therapeutic amount", i.e., less than the therapeutically effective amounts of each compound when used alone.
  • the term "about” refers to approximately, in the region of, roughly, or around.
  • cancer refers to or describe the physiological condition in mammals in which a population of cells are characterized by uncontrolled or unregulated cell growth. Examples of cancer include, e.g., carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • B-cell cancer or “B-cell malignancy” refers to an uncontrolled or unregulated growth of B-cells in the blood, bone marrow, or lymph node.
  • B-cell malignancy is a type of hematological malignancy (or hematological cancer) that includes lymphomas, leukemias, and myelomas.
  • the B-cell malignancy may be indolent or aggressive.
  • Non-limiting examples of B-cell malignancies that may be treated with the methods or kits disclosed herein include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) (referred to herein as “B-cell NHL” or “B-NHL”), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), which includes extranodal MZL, nodal MZL, and splenic MZL, hairy cell leukemia (HCL), Burkitt's lymphoma (BL), and Richter's transformation.
  • ALL acute lymphocytic leukemia
  • AML acute my
  • the DLBCL is an activated B-cell DLBCL (ABC-DLBCL), a germinal center B-cell like DLBCL (GBC-DLBCL), a double hit DLBCL (DH-DLBCL), or a triple hit DLBCL (TH-DLBCL).
  • ABS-DLBCL activated B-cell DLBCL
  • GBC-DLBCL germinal center B-cell like DLBCL
  • DH-DLBCL double hit DLBCL
  • TH-DLBCL triple hit DLBCL
  • high risk CLL means CLL characterized by at least one of the following genetic mutations: 17p del; 11q del; p53; unmutated IgVH together with ZAP-70+ and/or CD38+; and trisomy 12, and complex karyotype.
  • the B-NHLs that may be treated with the methods or kits disclosed herein include the following B-NHL subtypes: chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, or a lymphoplasmacytic lymphoma.
  • tumor and tumor cells refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.
  • cancer cell refers to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).
  • tumor cell will be modified by the term “non-tumorigenic” when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.
  • subject refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment (e.g., TG-1701).
  • a particular treatment e.g., TG-1701
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • an “effective amount” of an antibody or an agent as disclosed herein is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” can be determined empirically and in a routine manner by those skilled in the art, in relation to the stated purpose.
  • the term "therapeutically effective amount” refers to the amount of an agent (e.g., monoclonal antibody, small molecule, chemotherapeutic drug, etc%), as disclosed herein, that is effective to "treat” a disease or disorder in a subject or mammal.
  • the therapeutically effective amount of the agent or drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • treating To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.
  • Terms such as “treating,” “treatment,” “to treat,” “having a therapeutic effect,” alleviating,” “to alleviate,” or “slowing the progression of” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder, such as a B-cell malignancy, and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • a subject is successfully "treated" for a B-cell malignancy according to the methods of the present invention if the patient shows one or more of the following: reduction in cachexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass (e.g., by scan), reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrence or progressive disease, tumor response, complete response (CR), partial response (PR), stable disease (SD), progression free survival (PFS), overall survival (OS), each as measured by standards set by the National Cancer Institute and the U.S. Food and Drug Administration for the approval of new drugs. See, Johnson et al., J. Clin.
  • the "therapeutic effect,” as defined above, also encompasses a reduction in toxicity or adverse side effects, and/or an improvement in tolerability.
  • guidelines provided by standard international workshops for particular B-cell malignancies are used to assess tumor response, such as, for CLL, as set forth in Hallek, M. et al., Blood 111:5446-5456 (2008); for NHL, as set forth in Cheson, B.D. et al., J Clin Oncol 25:579-586 (2007); and for WM, according to the sixth international workshop on WM, Owen, R.G. et al., Br J Haematol.160:171-176 (2013).
  • treating the B-cell malignancy using the methods and kits described herein reduces percent tumor burden from baseline (i.e., prior to administration of the combination of agents described herein) by about 25% - 100%. In some aspects, treating the B-cell malignancy using the methods and kits described herein reduces percent tumor burden from baseline by at least about 20%, by at least about 25%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%. In certain aspects, the methods and kits described herein promote B-cell malignancy regression to the point of eliminating the B-cell malignancy.
  • subjects can be assessed for tumor burden or evaluated for anti-tumor response by CT, PET-CT, and/or MRI.
  • “duration of the percent reduction in tumor burden” is the interval from the first documentation of complete response (CR) or partial response (PR) to the earlier of the first documentation of definitive disease progression or death from any cause.
  • the “duration of the reduction in percent tumor burden” can be observed and continue for a period of at least about 24 weeks to about 36 months.
  • Combination administration refers to administering more than one pharmaceutically active ingredient (including, but not limited to, TG-1701 or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody, and/or a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor as disclosed herein) to a patient.
  • Combination administration may refer to simultaneous administration, sequential administration, or both simultaneous and sequential administration of TG-1701, or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody and a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor, as disclosed herein.
  • administration of an anti-CD20 antibody preceding or following e.g., by hour(s), day(s), week(s), or month(s)
  • administration of a PI3K-delta selective inhibitor preceding or following (e.g., by hour(s), day(s), week(s), or month(s)) administration of BTK inhibitor TG-1701, constitutes administration of a combination of agents.
  • a “combination of agents” can also include a BTK inhibitor (e.g., TG-1701), an anti-CD20 antibody (e.g., ublituximab), a PI3K-delta selective inhibitor (e.g., umbralisib), and one or more additional therapeutic agents, as described herein.
  • the therapeutic agents can be administered in a single pharmaceutical formulation or are administered simultaneously in separate pharmaceutical formulations by either the same or different routes of administration.
  • the term “combination of agents” is intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.
  • TG- 1701 or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody and a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor as disclosed herein, to a patient at the same time, or at two different time points that are separated by no more than 2 hours.
  • the simultaneous administration of TG-1701, or a pharmaceutically acceptable salt thereof, and an anti- CD20 antibody, and a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor may be in a single dosage form or in separate dosage forms.
  • the terms “sequential” and “sequentially” refer to the administration of TG-1701, or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody and a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor, as disclosed herein, to a patient at two different time points that are separated by more than 2 hours, e.g., about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or even longer.
  • “U2” refers to the combination of Ublituximab + Umbralisib, as used in the methods or kits of the disclosure.
  • TG-1701 + U2 refers to the triple combination of the BTK inhibitor TG-1701 + Ublituximab + Umbralisib, as used in the methods or kits of the invention.
  • an "adverse event” is any unfavorable and generally unintended or undesirable sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical or pharmaceutical treatment.
  • a B-cell malignancy which "does not respond,” “responds poorly,” or is “refractory” to treatment does not show statistically significant improvement in response to that treatment when compared to no treatment or treatment with a placebo in a recognized animal model or human clinical trial, or which responds to an initial treatment, but grows as treatment continues.
  • a B-cell malignancy which has “relapsed” means that the tumor has returned following treatment.
  • R/R means that the B-cell malignancy is relapsed or refractory, or possibly both.
  • the term "synergistic effect” refers to a situation where the combination of two or more agents produces a greater effect than the sum of the effects of each of the individual agents.
  • the term encompasses not only a reduction in symptoms of the disorder to be treated, but also an improved side effect profile, improved tolerability, improved patient compliance, improved efficacy, or any other improved clinical outcome.
  • the illustrative terms "include”, “such as”, “for example” and the like (and variations thereof, e.g., “includes” and “including”, “examples”), unless otherwise specified, are intended to be non-limiting.
  • variable means including but not limited to.
  • the recitation of a numerical range for a variable is intended to convey that the disclosure may be practiced with the variable equal to any of the values within that range.
  • the variable can be equal to any integer value within the numerical range, including the end-points of the range.
  • the variable can be equal to any real value within the numerical range, including the end-points of the range.
  • a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values > 0 and ⁇ 2 if the variable is inherently continuous.
  • the term "Bruton's tyrosine kinase” also known as “BTK,” agammaglobulinemia tyrosine kinase (ATK), or B-cell progenitor kinase (BPK) refers to a non-receptor tyrosine kinase enzyme in the B-cell antigen receptor (BCR) signaling pathway.
  • BTK a member of the Tec family of protein tyrosine kinases, is predominantly expressed in B- lymphocytes at various stages of development (except in terminally differentiated plasma cells).
  • BTK is a signal transduction protein that regulates normal B-cell development, differentiation activation, proliferation, and survival (Kurosaki, T., Curr Op Imm 12:276- 281 (2000); Schaeffer, E.M. and Schwartzberg, P.L., Curr Op Imm 12: 282-288 (2000)).
  • BTK has also been implicated in initiation, survival, and progression of mature B-cell lymphoproliferative disorders, such as B-cell malignancies (Akinleye, A. et al., J. Hematol.
  • BTK is from homo sapiens, as disclosed in U.S. Patent No.6,326,469 (Gen Bank Acc. No. NP_000052). BTK is also a key signaling enzyme expressed in all hematopoietic cells types except T lymphocytes and natural killer (NK) cells and plays a role in hematopoietic cell signaling pathways such as, e.g., Toll like receptor (TLR) and cytokine receptor-mediated TNF-alpha production in macrophages, IgE receptor (FcepsilonRI) signaling in mast cells, inhibition of Fas/APO-1 apoptotic signaling in B-lineage lymphoid cells, and collagen-stimulated platelet aggregation (See, e.g., Jeffries, C.
  • TLR Toll like receptor
  • FcepsilonRI IgE receptor
  • BTK functions as an important regulator of cell proliferation and cell survival in various B-cell malignancies.
  • An "inhibitor of BTK” or a “BTK inhibitor” refers to a small molecule that targets BTK and either inhibits BTK tyrosine phosphorylation and/or B-cell activation and/or otherwise inhibits or diminishes or abolishes the biological activity of a BTK protein.
  • An “irreversible BTK inhibitor” refers to a molecule that upon contact with BTK, causes the formation of a new covalent bond with an amino acid residue of BTK.
  • the BTK inhibitor TG-1701 which is used in the methods and kits of the present invention and discussed further below, is an irreversible BTK inhibitor.
  • ibrutinib IMBRUVICA®
  • CALQUENCE® acalabrutinib
  • BTK inhibitors include, but are not limited to, zanubrutinib, acalabrutinib, evobrutinib, tirabrutinib, fenebrutinib, pirtobrutinib, GS-4059 (NCT02457598), spebrutinib, HM71224, SNS-062, ABBV-105, LCB 03-0110 dihydrochloride, LFM-A13, PCI 29732, PF 06465469, M7583 (NCI Code C129710), or (-)-Terreic acid.
  • an "anti-CD20 antibody” or “an antibody that binds to CD20” refers to an antibody that is capable of binding CD20 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD20.
  • the extent of binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is less than about 10% of the binding of the antibody to CD20 as measured, e.g., by a radioimmunoassay (RIA).
  • anti-CD20 antibodies include, but are not limited to, ublituximab, ofatumumab, ubinutuzumab, ibritumomab, tiuxetan, obinutuzumab, rituximab, rituximab- hyaluronidase, and ocrelizumab.
  • Ikaros refers to a human transcription factor belonging to the IKZF family of zinc-finger DNA-binding proteins associated with chromatin remodeling and regulation of hematopoietic cell development.
  • the IKZF family consists of five members: Ikaros (encoded by the gene IKZF1), Helios (IKZF2), Aiolos (IKZF3), Eos (IKZF4), and Pegasus (IKZF5). These factors contain N-terminal zinc finger (ZF) domains, which are responsible for mediating direct interactions with DNA, and C- terminal ZFs, which facilitate homo- and heterodimerization between IKZF family members. Powell, MD et al., Frontiers in Immunology 10, Article 1299 (2019).
  • the zinc- finger 1 transcription factor gene “IKZF1” encoding Ikaros can be found at HGNC: 13176 or NCBI Entrez Gene: 10320.
  • Ikaros-repressed gene or “Ikaros-repressed gene signature” refers to a gene or number of genes, the transcription of which is inhibited when Ikaros binds to the promoter region of the gene. This inhibition of transcription leads to a decreased level of the transcript (mRNA) that can be assessed by, e.g., PCR..
  • mRNA transcript
  • the transcripts of the Ikaros-repressed gene signature comprise one or more of TXNIP, CD36, CA2, YOD1, CFP, DENND3, YES1, NBEAL2, TMC8, PSTPIP2, CD97, DAAM1, NT5E, LYZ, SDK2, TSC22D4, GYPC, FAM129A, TPM3, GNAQ, and/or LUZP1.
  • Ikaros-enhanced gene or Ikaros-enhanced gene signature” refers to a gene or a number of genes, the transcription of which is increased when Ikaros binds to the promoter region of the gene.
  • the transcripts of an Ikaros-enhanced gene signature comprise one or more of TCL1A, CBX5, HNRNPA0, PDHB, BCL2, DYNLL1, SUPT16H, CAMK2D, ALDH6A1, PPP2R5C, ERGIC1, BUB3, SORD, SEPHS1, CTNNBL1, CCT5, and/or APOBEC3G.
  • the term “proteomic profile” or “proteomic analysis” refers to the compilation of all the changes (increase or decrease) of the level of all the proteins that can be detected by a given method (e.g., mass spectrometry).
  • the term “decreased quantity of transcripts of an Ikaros-enhanced gene signature” refers to a decrease of mRNA molecules as measured by, e.g., PCR. The significance of a decrease can be assessed when multiple repeats are available. If not, the accepted threshold is a 2-fold change (value of 1 in Log2 scale).
  • the quantity of a transcript of an Ikaros-repressed gene and/or a transcript of an Ikaros-enhanced gene is measured using various methods known in the art, including, e.g., amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.); hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.); and sequencing-based methods (e.g., next-generation sequencing (NGS) methods, for example, using the Illumina or Ion Torrent sequencing platforms).
  • amplification-based methods e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle a
  • the term “rolling circle amplification” refers to a process of unidirectional nucleic acid amplification that can rapidly synthesize multiple copies of circular DNA or RNA molecules.
  • the term “NanoString analysis” refers to a process that is a variation of DNA microarray and uses molecular “barcodes” and microscopic imaging to detect and count up to several hundred transcripts in hybridization reactions.
  • the term “next-generation sequencing” (NGS) refers to sequencing platforms that perform sequencing of millions of small fragments of DNA in parallel and employ bioinformatics analysis to piece together the individual fragments by mapping the individual reads to a reference genome.
  • branched DNA signal amplification refers to an assay that uses support-bound small single stranded DNA capture molecules bound at a free end to DNA extender molecules, which extender molecules bind to DNA and/or RNA in a sample and the sample DNA and/or RNA in turn is bound by a label extender, a pre-amplifier and an enzyme-linked amplifier molecule to detect and quantify small amounts of DNA and/or RNA without a reverse transcription and/or PCR step.
  • expression refers to a process by which a polynucleotide produces a gene product, e.g., RNA or a polypeptide.
  • RNA messenger RNA
  • RNA transcript messenger RNA
  • expression produces a "gene product.”
  • a “gene product” can be, e.g., a nucleic acid, such as an RNA produced by transcription of a gene.
  • a “gene product” can be either a nucleic acid, RNA, or miRNA produced by the transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post-translational modifications, e.g., phosphorylation, methylation, glycosylation, ubiquitination, acetylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • the term “phosphoproteomic profile” or “phosphoproteomic analysis” refers to a type of proteomic analysis that comprises identifying, cataloguing, and/or characterizing proteins containing a phosphate group, including phosphorylated serine, threonine, and/or tyrosine residues.
  • Phosphoproteomic analysis includes the quantitative measurement of changes in phosphorylation (phosphorylation patterns or phosphorylation maps). Phosphoproteomic analysis may be performed by, e.g., mass spectrometry (MS), including, e.g., MALDI-TOF, ICP-MS, DART-MS, Secondary ion mass spectrometry (SIMS), Gas chromatography mass spectrometry (GC-MS), Liquid chromatography mass spectrometry (LC-MS), Crosslinking mass spectrometry (XL-MS), and Hydrogen-exchange mass spectrometry (HX-MS).
  • MS mass spectrometry
  • MALDI-TOF MALDI-TOF
  • ICP-MS ICP-MS
  • DART-MS Secondary ion mass spectrometry
  • SIMS Secondary ion mass spectrometry
  • GC-MS Gas chromatography mass spectrometry
  • LC-MS Liquid chromatography mass spectrometry
  • phosphopeptide refers to a peptide (short chain of between two and fifty amino acids, linked by peptide bonds) that incorporates one or more phosphate groups as a result of phosphorylation and is detected as a pair comprising the unmodified peptide and the phospho-peptide with an added mass of 80 daltons for each phosphorylated residue.
  • phospho-flow or “phospho-flow cytometry” refers to a technology that measures the phosphorylation state of intracellular proteins at the single cell level using labeled antibodies that bind phosphorylated amino acids such as phosphoserine, phosphothreonine, and phosphotyrosine within proteins.
  • the binding of the antibodies can be detected by various methods, including, e.g., the use of fluorescently labeled antibodies.
  • the BTK inhibitor used in the methods and kits described herein is (R)-4-amino-1-(1-(but-2-ynoyl)pyrrolidin-3-yl)-3-(4-(2,6-difluorophenoxy)phenyl)- 1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (TG-1701), or an isomer, polymorph, enantiomer, pharmaceutically acceptable salt, solvate, or prodrug thereof.
  • the BTK inhibitor is (R)-4-amino-1-(1-(but-2- ynoyl)pyrrolidin-3-yl)-3-(4-(2,6-difluorophenoxy)phenyl)-1,6-dihydro-7H-pyrrolo[2,3- d]pyridazin-7-one, also known as TG-1701, SHR-1459, or EBI-1459.
  • TG-1701 The alternate chemical name for TG-1701 is (R)-1-(3-(4-amino-3-(4-(2,6-difluorophenoxy)phenyl)-7- hydroxy-1H-pyrrolo[2,3-d]pyridazin-1-yl)pyrrolidin-1-yl)but-2-yn-1-one.
  • the terms “TG-1701” or “BTK inhibitor TG-1701” are used interchangeably and will be used predominantly herein.
  • the chemical formula of TG-1701 is C 26 H 21 F 2 N 5 O 3 , and its molecular weight is 489.48 g/mol.
  • TG-1701 has the following chemical structure: [0087] TG-1701 is described in PCT Publication No.
  • TG-1701 is an orally available, covalently-bound, selectively irreversible inhibitor of BTK. TG-1701 has been shown to exhibit superior selectivity for BTK compared to other clinically available BTK inhibitors, such as ibrutinib and acalabrutinib. See, e.g., Normant, E.
  • TG-1701 was evaluated and compared to ibrutinib and/or acalabrutinib in numerous enzyme based, cell-based, and animal models. For example, TG-1701 was shown to be as active as ibrutinib (having comparable kinase inhibition IC50s against BTK - 3 nM and 1.5 nM, respectively), but with improved selectivity for BTK in an in vitro whole kinome screening. Id. In addition, TG-1701 was 90-fold less active on EGFR compared to BTK with an IC50 of 270 nM and 3 nM respectively.
  • TG-1701 inhibited the growth of the follicular lymphoma (FL) DOHH-2, mantle cell lymphoma (MCL) Mino, and DLBCL SU-DHL-6 cell lines with IC50s of 369, 449, and 313 nM, respectively.
  • TG-1701 inhibited the IgM-activated BCR pathway in DOHH-2 cells, in particular, the phosphorylation of BTK, PLCy2, and ERK1/2.
  • TG-1701 blocked IgM-dependent CD69 expression, adhesion of JEKO cells to VCAM-1, and CXCL12-dependent migration. Id.
  • a fluorescent BTK-occupancy assay was developed and validated in vivo, in the spleen of mice, where BTK was found to be completely occupied after administration of a single dose of TG-1701 at 12.5 mg/kg.
  • TG-1701 In vivo, the anti-tumor efficacy of TG-1701 was assessed in several lymphoma xenograft models, e.g., SU-DHL-6 (GCB-DLBCL), Mino (MCL), and OCI-Ly10 (ABC-DLBCL), where TG-1701 showed potent anti-tumor activity equivalent to or greater than ibrutinib and similar to the recently approved BTK inhibitor, acalabrutinib.
  • GCB-DLBCL SU-DHL-6
  • MCL Mino
  • ABSC-DLBCL OCI-Ly10
  • the pharmacokinetic profile of TG-1701 allows for a once a day dosing.
  • TG-1701 is a novel and highly selective, irreversible BTK inhibitor with potent in vitro and in vivo activity. Id.
  • B-NHL B-cell non-Hodgkin lymphoma
  • BTKi BTK inhibitor
  • the subject is a TG- 1701 responder.
  • a “responder of TG-1701” or a “TG-1701 responder” refers to a subject whose NHL cells contain at least one positive phosphopeptide on a sequence selected from SEQ ID NOs 1-95, in particular, SEQ ID NO: 1, as determined by Western blot and/or phospho-flow analysis.
  • a “responder of TG-1701” or a “TG-1701 responder” refers to a subject with a 50% or more tumor reduction from baseline following TG-1701 treatment, as determined by scan.
  • a “BTK inhibitor responder” or “BTKi responder” refers to the same definitions provided above for TG-1701, but for any non-TG-1701 BTK inhibitor, as described herein.
  • the B-NHL cells of a BTKi responder contain at least one phosphopeptide that the B-NHL cells of a non-responder do not contain.
  • the subject is a TG-1701 responder and the B-NHL cells of the TG-1701 responder contain at least one phosphopeptide that the B-NHL cells of a TG- 1701 non-responder do not contain.
  • the subject has been determined to be a BTKi responder by quantifying phosphopeptides and/or phosphopolypeptides in the B-NHL cells of the subject. In some aspects, the subject has been determined to be a TG-1701 responder by quantifying phosphopeptides and/or phosphopolypeptides in the B-NHL cells of the subject. [0098] In some aspects, the subject is determined to be a BTKi responder by quantifying at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspect, the at least one phosphopeptide is SEQ ID NO: 1.
  • the subject is determined to be a TG-1701 responder by quantifying at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1. [0100] In some aspects, the subject is determined to be a BTKi responder by quantifying a phosphopolypeptide that comprises at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1.
  • the subject is determined to be a TG-1701 responder by quantifying a phosphopolypeptide that comprises at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject.
  • the at least one phosphopeptide is SEQ ID NO: 1.
  • the phosphopeptide and/or phosphopolypeptide quantification is performed in B-NHL cells isolated from blood samples, biopsy samples and/or bone marrow aspirates. The percentage of circulating B-NHL cancer cells in the samples of the subject can be from about 40% to about 98%.
  • the subject prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. In some aspects, prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. [0104] In some aspects, after initiation of treatment with a BTKi, the subject is determined to be responsive to a BTKi according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject.
  • the subject after initiation of treatment with TG-1701, the subject is determined to be responsive to TG-1701 according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject.
  • the method comprises providing B-NHL cells of a subject comprising polypeptides, extracting the polypeptides and enriching phosphopolypeptides from the polypeptide extract.
  • peptides are prepared from the extracted phosphopolypeptides.
  • the peptides are prepared using enzyme digestion.
  • the phosphopolypeptides are enriched using immunoprecipitation, metal affinity chromatography, metal-oxide affinity chromatography, Phos-Tag chromatography, polymer-based metal ion affinity capture, hydroxyapatite chromatography, enrichment by chemical modification, and/or phosphopolypeptide precipitation.
  • the ion affinity chromatography comprises iron (Fe 3+ ), gallium (Ga 3+ ), aluminium (Al 3+ ), zirconium (Zr 4+ ) or titanium (Ti 4+ ) ion affinity chromatography or sequential iron (Fe 3+ ), gallium (Ga 3+ ), aluminium (Al 3+ ), zirconium (Zr 4+ ) or titanium (Ti 4+ ) ion affinity chromatography.
  • sequential ion affinity chromatography comprises phosphopolypeptide and/or phosphopeptide enrichment using TiO2 affinity chromatography followed by Fe-NTA chromatography of the TiO2 flow- through and wash fractions.
  • sequential ion affinity chromatography comprises phosphopolypeptide and/or phosphopeptide enrichment using Fe-NTA chromatography followed by TiO2 chromatography of the Fe-NTA flow through and wash fractions.
  • the phosphopolypeptides and/or phosphopeptides are enriched using either TiO 2 or Fe-NTA affinity chromatography.
  • the ion affinity chromatography is immobilized ion affinity chromatography.
  • the eluates obtained from any of the phosphopolypeptide and/or phosphopeptide enrichment procedures are fractionated before further processing.
  • the enriched phosphopolypeptides are subjected to western blot or flow cytometry analysis using antibodies that bind to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95.
  • the enriched phosphopolypeptides are subjected to further immunoaffinity purification steps prior to performance of western blots and/or flow cytometry.
  • the enriched phosphopolypeptides are subjected to immunoaffinity purification using at least one antibody that binds to a phosphorylated amino acid.
  • the enriched phosphopolypeptides are subjected to immunoaffinity purification using at least one antibody that binds to phosphorylated serine, phosphorylated threonine, or a phosphorylated tyrosine.
  • the phosphopolypeptide fractions eluted from the immunoaffinity purification step are subsequently subjected to western blot or flow cytometry analysis using at least one antibody that binds to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95.
  • the eluates that contain phosphoserine, phosphothreonine and/or phosphotyrosine consistent with the phosphopeptides of SEQ ID NO: 1-95 are further subjected to peptide sequencing.
  • peptide sequencing of the eluted phosphopolypeptides comprises enzyme digestion to generate peptides and subjection to mass spectrometry.
  • the samples that have been enriched for phosphopolypeptides and have been eluted from immunoaffinity purification steps as binding to at least one phosphorylated serine, at least one phosphorylated threonine, or at least one phosphorylated tyrosine and also bind to at least one antibody that binds to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95 are further subjected to method steps that quantify in said samples at least one transcript of an Ikaros-repressed gene and/or at least one transcript of an Ikaros-enhanced gene such that the subject from which the sample was obtained is determined to be a TG-1701 responder.
  • such combined determination of the presence of phosphopeptides selected from SEQ ID NO: 1-95 and determination of the presence of at least one transcript of an Ikaros-repressed gene and/or the absence of at least one transcript of an Ikaros-enhanced gene determines that the subject is a TG-1701 responder. In some aspects, such combined determination is performed when mass spectrometry is not available. [0109] In some aspects, the enriched phosphopolypeptides are subjected to separate immunoaffinity purification steps using at least one antibody that binds to phosphorylated serine, at least one antibody that binds to phosphorylated threonine, and at least one antibody that binds to a phosphorylated tyrosine separately.
  • the eluates of the separate immunoaffinity purification are subsequently subjected to western blot or flow cytometry analysis using at least one antibody that binds to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95.
  • the eluates that contain phosphoserine, phosphothreonine and/or phosphotyrosine consistent with the phosphopeptides of SEQ ID NO: 1-95 are further subjected to peptide sequencing.
  • the phosphopolypeptides comprise one or more phosphopeptides selected from SEQ ID NO: 1-95.
  • the B-NHL cells of the subject comprise 1 to about 95 phosphopeptides; or about 1 to 10, 11 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 72 to 80, 81 to 90 or 91-95 phosphopeptides. In some aspects, the B-NHL cells of the subject comprise 1 to 95, 2 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to 60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, or 91 to 95 phosphopeptides.
  • the B-NHL cells of the subject comprise 1 to about 95 phosphopeptides prior to a treatment with a BTKi and lack at least one of these phosphopeptide after treatment with a BTKi.
  • the B-NHL cells of the subject comprise 1 to about 95 phosphopeptides prior to a treatment with TG-1701 and lack at least one of these phosphopeptide after treatment with TG-1701.
  • the B-NHL cells of the BTKi responder contain at least one transcript of an Ikaros-repressed gene signature.
  • the subject is a TG-1701 responder and the B-NHL cells of the TG-1701 responder contain at least one transcript of an Ikaros-repressed gene signature.
  • the genes of an Ikaros-repressed gene signature comprise one or more of LUZP1, GYPC, BAALC, MAN1A1, RPS16, CPEB4, RAB1A, SERPINB6, TNK2, CD59, GOLGA9P, PRKCH, GNAQ, YPEL5, ZC3HAV1, KLF10, OLFML2A, GPR110, FAM160B1, C7orf41, BAALC, ARPC5L, MAP3K8, TIPARP, LRRFIP1, CLEC2B, MCL1, CD97, DNAJB6, ITSN2, YES1, SOCS5, USP15, SEPT6, LRRFIP1, CPEB4, ARPC5L, TPM3, SERPINB9, MAN1A1, CISH, SIK
  • the genes of an Ikaros-repressed gene signature comprise one or more of TXNIP, CD36, CA2, YOD1, CFP, DENND3, YES1, NBEAL2, TMC8, PSTPIP2, CD97, DAAM1, NT5E, LYZ, SDK2, TSC22D4, GYPC, FAM129A, TPM3, GNAQ, and/or LUZP1.
  • the B-NHL cells of the BTK inhibitor responder lack at least one transcript of an Ikaros-enhanced gene signature.
  • the B-NHL cells of a TG-1701 responder lack at least one transcript of an Ikaros-enhanced gene signature.
  • the genes of an Ikaros-enhanced gene signature comprise one or more of ZHX2, GLT8D1, FLI1, TCL1A, ZFP36L1, BDH2, HNRNPA0, EFTUD1, PLEKHA2, C14orf142, BUB3, YWHAB, QRSL1, CCND3, FMNL2, MGC3032, TTRAP, KIAA1430, CUTC, MRPL46, DBN1, CD22, BCORL1, GINS3, UBE2V2, AEBP1, BTK, HDDC2, PDHB, C21orf59, FAIM, CAMK2D, VTA1, EIF2S1, VRK1, UBLCP1, LOC93622, SEPHS1, PPID, C1orf59, ARPP-21, LMNB1, BACH2, LOC116236, DPY19L3, SDCCAG10, CCT5, PDIA6, PPP2R5C, CECR5, LOC90925, GINS3, PDE4A, AHCYL
  • the genes of an Ikaros-enhanced gene signature comprise one or more of TCL1A, CBX5, HNRNPA0, PDHB, BCL2, DYNLL1, SUPT16H, CAMK2D, ALDH6A1, PPP2R5C, ERGIC1, BUB3, SORD, SEPHS1, CTNNBL1, CCT5, and/or APOBEC3G.
  • standard gene symbols such as those listed above and in Tables 5 and 7, found in community databases specific to human proteins (e.g., www.genenames.org) have been utilized.
  • the quantity of a transcript of an Ikaros-repressed gene and/or a transcript of an Ikaros-enhanced gene is measured using various suitable methods known in the art, including, e.g., amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.); hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.); and sequencing- based methods (e.g., next-generation sequencing (NGS) methods, for example, using the Illumina or Ion Torrent sequencing platforms).
  • amplification-based methods e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle
  • the quantity of transcripts is determined by at least one amplification-based method.
  • the amplification-based method is Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification.
  • the amplification-based method is qPCR.
  • the quantity of transcripts is determined by at least one non- amplification-based method.
  • the non-amplification-based method is a hybridization-based method or a sequencing-based method.
  • the hybridization-based method is a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, or in situ hybridization.
  • the sequencing-based method is a next-generation sequencing (NGS) method.
  • NGS next-generation sequencing
  • the quantity of transcripts is determined by a combination of amplification-based and non-amplification-based methods.
  • the subject prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of B-NHL cells of the subject.
  • the subject prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of the B-NHL cells of the subject.
  • the subject prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring in a sample of B-NHL cells of the subject, the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature, wherein the at least one transcript is either not detectable in the B-NHL cells of the subject or is measured at levels lower than in the B-NHL cells of a subject that is not a BTKi responder.
  • the subject prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring in a sample of B-NHL cells of the subject, the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature, wherein the at least one transcript is either not detectable in the B-NHL cells of the subject or is measured at levels lower than in the B-NHL cells of a subject that is not a TG-1701 responder.
  • the subject prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring in a sample of B-NHL cells of the subject the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature and the amount of at least one transcript of a gene of an Ikaros-repressed gene signature.
  • the subject prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring in a sample of B-NHL cells of the subject the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature and the amount of at least one transcript of a gene of an Ikaros-repressed gene signature.
  • the subject has not been treated for B-NHL prior to quantifying the amount of at least one transcript of a gene of an Ikaros-repressed gene signature and at least one transcript of a gene of an Ikaros-enhanced gene signature in the B-NHL cells of the subject.
  • the subject has been treated for B-NHL prior to quantifying the amount of at least one transcript of a gene of an Ikaros-repressed gene signature and at least one transcript of a gene of an Ikaros-enhanced gene signature in the B-NHL cells of the subject.
  • the subject has been treated with a compound and/or a combination of compounds including, but not limited to, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) without or with a monoclonal antibody, including, but not limited to, rituximab (R-CHOP); dose-adjusted etoposide, doxorubicin and cyclophosphamide with vincristine, prednisone and rituximab (DA-EPOCH-R); cyclophosphamide, vincristine, prednisone (CVP); cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) alternating with rituximab and cytarabine; cyclophosphamide, vincristine, doxorubicin, and high-dose methotrexate (CODO)
  • the methods according to the invention comprise administering to a subject a therapeutically effective amount of a BTKi.
  • the BTKi is ibrutinib, zanubrutinib, acalabrutinib, evobrutinib, tirabrutinib, fenebrutinib, pirtobrutinib, GS-4059 (NCT02457598), spebrutinib, HM71224, SNS-062, ABBV-105, LCB 03-0110 dihydrochloride, LFM-A13, PCI 29732, PF 06465469, M7583 (NCI Code C129710), or (-)-Terreic acid or a BTK degrader including DD 03-171.
  • the methods according to the invention comprise administering to a subject a therapeutically effective amount of the BTK inhibitor TG-1701.
  • TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d.
  • TG-1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d.
  • TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d.
  • TG-1701 is administered in combination with an anti-CD20 antibody.
  • the anti-CD20 antibody is rituximab, obinutuzumab, ofatumumab, or ublituximab.
  • the anti-CD20 antibody is ublituximab.
  • TG-1701 is administered in combination with a dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor.
  • the dual PI3K ⁇ and casein kinase-1 ⁇ inhibitor is umbralisib.
  • TG-1701 is administered with a PI3K ⁇ inhibitor, such as, e.g., duvelisib, idelalisib, zandelisib, or copanlisib.
  • a PI3K ⁇ inhibitor such as, e.g., duvelisib, idelalisib, zandelisib, or copanlisib.
  • TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d in combination with an anti-CD20 antibody.
  • TG-1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d in combination with an anti-CD20 antibody. In some aspects, TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d in combination with an anti-CD20 antibody. [0142] In some aspects, TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d in combination with umbralisib.
  • TG-1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d in combination with umbralisib. In some aspects, TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d in combination with umbralisib.
  • TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d in combination with an anti-CD 20 antibody and umbralisib. In some aspects, TG- 1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d in combination with an anti-CD20 antibody and umbralisib.
  • TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d in combination with an anti-CD20 antibody and umbralisib.
  • umbralisib is administered at a dose from about 200 mg/d to about 1000 mg/d, about 250 mg/d to about 900 mg/d, about 300 mg/d to about 850 mg/d, about 350 mg/d to about 800 mg/d, about 400 mg/d to about 750 mg/d, about 450 mg/d to about 700 mg/d, about 500 mg/d to 650 mg/d, or about 400 mg/d, about 600 mg/d or about 800 mg/d.
  • the anti-CD20 antibody is administered at 500 mg/d to about 1200 mg/d, or about 600 mg/d to about 1000 mg/d, about 700 mg/d to about 900 mg/d, or about 650 mg/d, 700 mg/d, 750 mg/d, 800 mg/d, 850 mg/d, or 900 mg/d.
  • TG-1701 is administered on a daily schedule. In some aspects, TG-1701 is administered twice a day, three times a day, or four times a day. In some aspects, TG-1701 is administered every other day. In some aspects, TG-1701 is administered once every three days. In some aspects, TG-1701 is administered on a weekly schedule.
  • TG-1701 is administered on a once every two weeks schedule. In some aspects, TG-1701 is administered on a once every three weeks schedule. In some aspects, TG-1701 is administered on a once every four weeks schedule. [0147] In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered once a day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered twice a day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered three times a day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered four times a day.
  • TG-1701, the anti-CD20 antibody and/or umbralisib are administered every other day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered every three days. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered once a week. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered on a once every two weeks schedule. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered on a once every three weeks schedule.
  • TG-1701, the anti-CD20 antibody and/or umbralisib are administered on a once every four weeks schedule. [0148] In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered once every other day. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered once every three days. In some aspects, TG-1701 is administered every day and the anti- CD20 antibody and/or umbralisib are administered on a weekly schedule. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every two weeks schedule.
  • TG- 1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every three weeks schedule. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every four weeks schedule. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every eight weeks schedule. [0149] In some aspects, TG-1701 is administered every other day and the CD20 antibody and/or umbralisib are administered every day. In some aspects, TG-1701 is administered every other day and the CD20 antibody and/or umbralisib are administered once every three day.
  • TG-1701 is administered every other day and the anti-CD20 antibody and/or umbralisib are administered once a week. In some aspects, TG-1701 is administered every other day and the anti-CD20 antibody and/or umbralisib are administered once every two weeks. In some aspects, TG-1701 is administered every other day and the anti-CD20 antibody and/or umbralisib are administered once every three weeks. In some aspects, TG-1701 is administered every other day and the anti- CD20 antibody and/or umbralisib are administered once every four weeks. [0150] In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered every day.
  • TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered every other day. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once every three days. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once a week. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once every two weeks. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once every three weeks.
  • TG-1701 is administered every three days and the anti- CD20 antibody and/or umbralisib are administered once every four weeks. [0151] In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered every day. In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered every other day. In some aspects, TG-1701 is administered once a week and the anti- CD20 antibody and/or umbralisib are administered every three days. In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered once every two weeks.
  • TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered once every three weeks. In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered once every four weeks.
  • TG-1701 is administered in combination with an anti-CD20 antibody and umbralisib, wherein TG-1701 is administered daily, every other day, every three days, once a week, once every two weeks, once every three weeks or once every four weeks and the anti-CD20 antibody is administered daily, every other day, every three days, once a week, once every two weeks, once every three weeks or once every four weeks and umbralisib is administered daily, every other day, every three days, once a week, once every two weeks, once every three weeks or once every four weeks.
  • B-NHL B-cell non- Hodgkin lymphoma
  • the method comprises quantifying at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject.
  • the at least one phosphopeptide is SEQ ID NO: 1 (Ikaros).
  • the method comprises quantifying at least one protein that comprises a phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject.
  • the at least one protein comprises at least one phosphopeptide is SEQ ID NO: 1.
  • the presence of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis.
  • the phosphopeptide and/or phosphopolypeptide quantification is performed in B-NHL cells isolated from blood samples, biopsy samples and/or bone marrow aspirates. The percentage of circulating B-NHL cancer cells in the samples of the subject can be from about 40% to about 98%.
  • the method comprises, prior to initiation of treatment with a BTKi, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. In some aspects, the method comprises, prior to initiation of treatment with TG-1701, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. [0159] In some aspects, the method comprises, after initiation of treatment with a BTKi, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. In some aspects, the method comprise, after initiation of treatment with TG-1701, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject.
  • the method comprises providing B-NHL cells of a subject comprising polypeptides, extracting the polypeptides and enriching phosphopolypeptides from the polypeptide extract.
  • peptides are prepared from the extracted phosphopolypeptides.
  • the peptides are prepared using enzyme digestion.
  • the phosphopolypeptides are enriched using immunoprecipitation, metal affinity chromatography, metal-oxide affinity chromatography, Phos-Tag chromatography, polymer-based metal ion affinity capture, hydroxyapatite chromatography, enrichment by chemical modification, and/or phosphopolypeptide precipitation.
  • the enriched phosphopolypeptides are subjected to western blot or flow cytometry phosph-flow analysis using antibodies that bind to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95.
  • the method comprises quantifying at least one transcript of an Ikaros-repressed gene and/or at least one transcript of an Ikaros-enhanced gene by measured transcript quantity using various methods known to those skilled in the art, including, e.g., amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.); hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.); and sequencing- based methods (e.g., next-generation sequencing (NGS) methods, for example, using the Illumina or Ion Torrent sequencing platforms).
  • amplification-based methods e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain
  • the method comprises, prior to initiation of treatment with a BTKi, measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of B-NHL cells of the subject. In some aspects, the method comprises, prior to initiation of treatment with a BTKi, measuring the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature in a sample of the B-NHL cells of the subject. [0164] In some aspects, the method comprises, after initiation of treatment with a BTKi, measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of B-NHL cells of the subject.
  • the method comprises measuring prior to initiation of treatment with a BTKi, in a sample of B-NHL cells of the subject the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature, wherein the at least one transcript is either not detectable in the B-NHL cells of the subject or is measured at levels lower than in the B-NHL cells of a subject that is not a BTKi responder.
  • Kits [0165]
  • the present disclosure provides a kit comprising a combination of antibodies and, optionally, reagents packaged in a manner that facilitates their use to practice the methods of the present disclosure.
  • a kit comprises: (i) at least one antibody that binds to at least one phosphopeptide selected from SEQ ID NOs: 1-95; (ii) optionally, reagents to perform a western blot analysis; and/or reagents to perform a phospho-flow analysis; and (iii) instructions for treating B-NHL in a TG-1701 inhibitor responder according to any of the methods described herein.
  • the at least one phosphopeptide is SEQ ID NO: 1 (Ikaros).
  • the kit further comprises reagents to perform a Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification.
  • PCR Polymerase Chain Reaction
  • RT-PCR Real-Time Polymerase Chain Reaction
  • qPCR Quantitative Polymerase Chain Reaction
  • the kit further comprises reagents to perform a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, next-generation sequencing (NGS) method, or in situ hybridization.
  • NGS next-generation sequencing
  • WBC white blood cells
  • ALC absolute lymphocyte count
  • Electroporation was performed using Neon Transfection System (Thermo Fisher) at 1600 V, 20 ms, and 10 ⁇ L tip. Cells were then plated in a 24-well plate for 10 days, changing the culture medium every 3 days. Protein lysates were collected from REC-1 BTK KO cells to assess BTK levels by Western blot. REC-1 BTK C481S cells were resuspended in 96-well plates with a limiting dilution of 0.3 cells per well. After 20 days, REC-1 BTK C481S single clones were analyzed by PCR amplification with BTK primers and digestion with EaeI (NEB) restriction enzyme. Positive clones were validated by Sanger Sequencing.
  • Neon Transfection System Thermo Fisher
  • TG-1701 was provided by TG Therapeutics, Inc. Ibrutinib was purchased from Selleckchem. Xenograft mouse model and immunohistochemical staining [0176] In the Mino MCL xenograft model, tumor-bearing 6-7 week-old nude mice (Shanghai Ling Chang experimental animal Co., Ltd.) were dosed orally with TG-1701 (25, 50, or 100 mg/kg, orally, twice a day (bid)), ibrutinib (100 mg/kg, orally, bid), or vehicle for 21 days.
  • TG-1701 25, 50, or 100 mg/kg, orally, twice a day (bid)
  • ibrutinib 100 mg/kg, orally, bid
  • CB17-SCID mice (Janvier Labs) were inoculated subcutaneously with 10 7 REC-1 GFP+Luc+ cells or UPN-IbruR cells and monitored for tumor growth, bioluminescence signal, and vital parameters as previously described (Body, S et al., Sci Rep.7:13946 (2017)).
  • Tumor samples were snap-frozen in OCT medium (Sakura Tissue Tek) or formalin-fixed and paraffin-embedded prior to immunohistochemical staining with primary antibodies against Ikaros (Cell Signaling Technology), CD20 (Beckman Coulter), NCAM (also known as CD56), and granzyme B (Abcam). Preparations were evaluated using an Olympus microscope and MicroManager software.
  • Membrane-transferred proteins were revealed by incubating with primary antibodies against p-BTK-Tyr223, BTK, p-ERK1/2, p-PLC ⁇ 2, PLC ⁇ 2, p-AKT-Ser473, AKT, Ikaros, MYC (Cell Signaling Technology), ERK1/2 and IRF4 (Santa Cruz Biotechnology), and appropriate anti-rabbit (Cell Signaling) or anti-mouse (Sigma- Aldrich) secondary antibodies followed by chemiluminescence detection using the ECL system (Pierce) and a Fusion FX imaging system (Vilber Lourmat). Anti-GAPDH (Santa Cruz) detection was carried out to check protein loading.
  • PBMCs Peripheral blood mononuclear cells
  • PRE Peripheral blood mononuclear cells
  • POST 4 hour treatment
  • TG-1701 Table 3
  • Proteins were extracted by adding a Urea-based buffer (6M Urea, 100mM Tris-HCl pH 7.5), followed by sonication in a bioruptor (10 ON/OFF cycles, 30s each).
  • the supernatants were recovered by centrifugation (15,000g for 10min at 4oC) and the proteins precipitated by adding 100% trichloroacetic acid (TCA) for 1h at 4oC.
  • TCA trichloroacetic acid
  • the resulting proteins pellets were recovered by centrifugation at 15,000g for 5 min at 4oC and subsequently washed twice with chilled acetone (30 min each). The pellets were recovered and then resuspended in 6M Urea, 100mM Tris-HCl pH 7.5. Finally, the samples were quantified with the RCDC® Protein Assay Kit (Biorad). All the proteins samples were sequentially digested with Lys-C (1:25, enzyme-to-protein ratio for 18h at 30oC) and Trypsin (1:25 for 8 hours at 30oC). Prior to digestion, the samples were reduced and alkylated with dithiothreitol (DTT) and carbamydomethylated with chloroacetamide (CAA), respectively.
  • DTT dithiothreitol
  • CAA carbamydomethylated with chloroacetamide
  • proteomics quantitative analyses were performed using thirteen (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N and 132C) of the sixteen channels available in a 16plex - Tandem Mass Tag (TMT) system. The labelling was performed according to the manufacturer instructions (Thermo Fisher).
  • samples were dried in a speedvac and analyzed in an Orbitrap Fusion LumosTM Tribrid mass spectrometer.
  • samples were loaded to 300 ⁇ m ⁇ 5 mm C18 PepMap100, 5 ⁇ m, 100 ⁇ (Thermo Scientific) at a flow rate of 15 ⁇ L/min using a Thermo Scientific Dionex Ultimate 3000 chromatographic system (Thermo Scientific).
  • a C18 analytical column nanoEaseTM M/Z HSS C18 T3 (75 ⁇ m ⁇ 25 cm, 100 ⁇ , Waters) with a 150 min run, comprising three consecutive steps with linear gradients from 3% to 35 % B in 120 min, from 35 % to 50 % B in 5 min, from 50 % to 85% B in 2min, followed by isocratic elution
  • the column outlets were directly connected to an Advion TriVersa NanoMate (Advion) fitted on an OrbitrapFusion LumosTM Tribrid mass spectrometer (Thermo).
  • the mass spectrometer was operated in a data-dependent acquisition (DDA) mode.
  • Survey MS scans were acquired in the orbitrap with the resolution (defined at 200 m/z) set to 120,000.
  • the lock mass was user-defined at 445.12 m/z in each Orbitrap scan.
  • the top speed (most intense) ions per scan were fragmented by HCD.
  • the MSMS was detected in the Orbitrap (with 30,000 resolution).
  • the ion count target value was 400,000 for the survey scan and 10,000 (CID) for the MS/MS scan.
  • Target ions already selected for MS/MS were dynamically excluded for 15 s.
  • Spray voltage in the NanoMate source was set to 1.70 kV.
  • RF Lens were tuned to 30%.
  • Minimal signal required to trigger MS to MS/MS switch was set to 5000 and activation Q was 0.250.
  • the spectrometer was working in positive polarity mode and singly charge state precursors were rejected for fragmentation.
  • Data was acquired with Xcalibur software vs 4.0.27.10 (Thermo Scientific).
  • MaxQuant software 1.6.7.0
  • its built-in search engine Andromeda were used to search the .raw files against a Swisprot/Uniprot human database downloaded from the www.uniprot.org web site (October 2019).
  • FDR false discovery rate
  • the total proteome analysis was performed using only the proteins quantified in all the samples, while for phosphoproteome, we used the peptides quantified in more than 80% of the samples.
  • the differential analyses were performed by using the R package called “limma” (Ritchie, ME et al., Nucleic Acids Res 43(7):e47 (2015)) considering paired samples.
  • the normalization of both total proteome and phosphoproteome was done using the median. Additionally, the phosphoproteome was normalized using the R package called “phosphonormalizer” (Saraei, S et al., Bioinformatics 34(4):693-694 (2016)).
  • CID collision induced dissociation
  • RNA-Seq Analysis and Real-Time qPCR [0183] Total RNA was extracted using TRIZOL (Thermo Fisher) following manufacturer’s instructions and Poly-A-tailed enriched mRNA selected. Paired-end Stranded RNA libraries with 51 read-inward facing paired mates were prepared, following sequencing with Illumina’s NovaSeq6000 at the Centro Nacional de Analisis Genomico (CNAG). [0184] The reverse transcription (RT) reaction was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems).
  • RNA-Seq Data Analysis [0185] Quality Control (QC) of all samples was performed with the publicly available software FASTQC. For a more optimal visual inspection, these analyses were concatenated with MultiQC. Trimming of unwanted adapter sequences were filtered out using FASTP and CutAdapt.
  • TG-1701 plasma concentrations were assessed using a GLP LC/MS/MS method developed at North East Bio Analytical Laboratories.
  • Binding and Enzymatic Assays [0188] The binding of TG-1701 (1 ⁇ M) and ibrutinib (1 ⁇ M) to a panel of 453 kinases was determined using a quantitative binding assay (KINOMEscan, DiscoverX, Eurofins). TG-1701 and ibrutinib inhibitory activities were also tested on BTK wt and BTK C481S kinase activity at ReactionBio in an enzymatic filtration assay using 33 P-ATP.
  • the in vitro occupancy assay was developed and performed by Jiangsu Hengrui Medicine Co. at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences in Shanghai. Briefly, the DoHH2 BTK-expressing cells were incubated with ibrutinib or TG-1701 (0.1 to 100 nM) for 1 hour and cell lysates were subjected to SDS-PAGE, followed by incubation with a fluorescent-ibrutinib probe or an anti-BTK antibody. The BTK occupancy method for the detection of free BTK in patient lysates was developed and run at Cambridge Biomedical Laboratories (now Bioagilytix).
  • PBMC pellets (2 x 10 6 cells) were lysed in 100 ⁇ L ice cold lysis buffer PBS with 0.1% Nonidet P-40 supplemented with 1X protease inhibitor cocktail (Sigma-Aldrich). Samples were prepared in triplicate. Cell lysates were incubated with the BTK occupancy probe biotin-ibrutinib (MedChemExpress) for 1h and plated onto a streptavidin-coated plate. Two hours of incubation were followed by washing and an hour incubation with an anti- BTK antibody (Becton Dickinson) in PBS + 0.05% Tween-20 and 0.5% BSA.
  • BTK occupancy probe biotin-ibrutinib MedChemExpress
  • ADCC was evaluated by measuring the release of lactate dehydrogenase (LDH) into the medium after treatment using the Cytotoxicity Detection KitPLUS (Roche). Briefly, PBMCs were purified by standard Ficoll-Hypaque gradient centrifugation of healthy human peripheral blood. Target cells were opsonized with 2 ⁇ g/mL ublituximab or an irrelevant hIgG1 in combination with 1 ⁇ M ibrutinib/TG-1701, for 1 hour at 37°C. Next, 10 4 target cells were mixed with 10 5 PBMCs effector cells.
  • LDH lactate dehydrogenase
  • ADCC 100 x [(sample – non-specific lysis control) / (total lysis control – neg control)].
  • % ADCC 100 x [(sample – non-specific lysis control) / (total lysis control – neg control)].
  • Malignant B cells were then CFSE-labeled and opsonized with an anti-CD20 mAb (ublituximab or rituximab, 2 ⁇ g/ml) or with an irrelevant hIgG1, in combination with the indicated BTKi at 1 ⁇ M, for 1 hour at 37°C. Those cells were added to macrophages with an E:T ratio of 1:5. The cell mixture was incubated for 2.5 hours at 37°C and cells were analyzed on a Navios flow cytometer (Beckman Coulter) after macrophage staining with an anti-human CD14 antibody (Beckman Coulter).
  • an anti-CD20 mAb ublituximab or rituximab, 2 ⁇ g/ml
  • an irrelevant hIgG1 in combination with the indicated BTKi at 1 ⁇ M
  • the percentage of phagocytosis is defined as the percentage of macrophages that have engulfed at least one target malignant cell.
  • CCG CellTiter-Glo
  • CCG CellTiter-Glo
  • Cell viability was performed using a CellTiter-Glo luminescent cell viability assay (Promega), according to the manufacturer’s instructions. Briefly, 5 x 10 4 cells/well were cultured in sterile 96-well plates in the presence of increasing concentrations of TG-1701 or ibrutinib (100 nM to 100 ⁇ M) in RPMI 10% FBS medium. The plates were incubated for 72 h (unless otherwise specified) and 100 ⁇ L of CellTiter-Glo reagent were added.
  • Example 1 TG-1701 is a novel irreversible BTK inhibitor, more selective than ibrutinib [0195] This Example shows that TG-1701 exerts similar activity to the first-in-class BTK inhibitor, ibrutinib, but with greater selectivity, in in vitro and in vivo models of B-NHL.
  • TG-1701 was more selective than ibrutinib, with a comparable BTK Kd (3 nM vs 1.5 nM, respectively) and a lower binding to EGFR, ITK, TXK, and JAK3 (Kd 135-, >48-, 68- and >94-fold higher than those of ibrutinib, respectively, FIG.1A and Table 1).
  • Table 1 TG-1701 and ibrutinib Kds (nM) [0197] A BTK kinase activity assay revealed a TG-1701 EC 50 of 6.70 nM, slightly higher than ibrutinib IC50 (1.65 nM, FIG.1B and Table 2).
  • Table 2 Compared kinase and growth inhibitory activity of TG-1701 and ibrutinib
  • TG-1701 and ibrutinib showed a similar dose-dependent capacity to displace a BTK-specific fluorescent probe in the FL cell line DoHH-2, with complete BTK occupancy at 30 nM and 10 nM, respectively (FIG.1C). Consistently, BCR downstream signaling was impaired in a concentration- dependent manner in IgM-stimulated cells, with maximal effects observed at 100 nM for both BTKis (FIG.1D).
  • TGI tumor growth inhibition
  • a single oral gavage with 50 mg/kg TG-1701 further confirmed a rapid dephosphorylation of BTK and AKT as early as 2 hours and 4 hours, respectively, which was maintained at least for 24 hours due to the irreversible nature of TG-1701- mediated BTK inhibition (FIG.1F).
  • Example 2 Phosphoproteomic analysis differentiates early clinical response to TG-1701 and points to the inhibition of the Ikaros pathway as an important mechanism of TG-1701 activity
  • a mass spectrometry (MS)-based phosphoproteomic platform used to interrogate the effects of TG-1701 on CLL patients enrolled in the phase 1 dose- escalation study (NCT03671590) pointed to the transcription factor Ikaros as both a potential biomarker of clinical activity and an important transcription factor downstream of BTK in the BCR pathway.
  • Biomarker validation and signal transduction analysis were conducted through real-time PCR, western blot, immunofluorescence, immunostaining, and gene knock-out (KO) experiments. Patients, materials, and methods were described above. [0201] Results. To identify potential biomarkers of TG-1701 activity in B-NHL, PBMCs from six CLL patients enrolled in the TG-1701-101 phase 1 clinical trial were isolated at different time points for phosphoproteomic and RNA-seq analysis. In all patient samples but one, the percentage of circulating cancer cells was comprised between 60% and 98% (Table 3 and Table 4). All the patient samples harbored a wild type BTK gene, as confirmed by Sanger sequencing of BTK exon 11.
  • Table 3 Clinical and Biological Characteristics of 6 CLL Patients
  • Table 4 PBMC analysis of 6 CLL patients [0202] Near complete BTK occupancy was obtained in all samples tested (FIG.2A and FIG.8) and linear kinetics was observed with approximately dose proportional increases in Cmax and AUC0-8h from 100 mg to 400 mg, with a positive correlation between the daily dose and TG-1701 Cmax (FIG.2B). After three cycles of treatment, the best change in tumor burden as assessed by CT scan ranged from 38 % to 87%, with one tumor-free patient with a lymphocytosis that defined his best response as a stable disease (FIG.2C).
  • CCL3 and CCL4 chemokine genes two bona fide biomarkers for BCR pathway activation (Takahashi, K et al., Br J Haematol 171:726–735 (2015)), were more downregulated in responder patients (FIG. 3A).
  • a set of 118 phosphopeptides were differentially phosphorylated (7 down- and 111 up-regulated) after TG-1701 treatment in the responder subgroup only (adjusted p-value ⁇ 0.1, FIG.2E). These sites corresponded to the putative modulation of 14 protein kinases (Tables 5 and 6).
  • Table 6 List of Activated/Inhibited Kinases [0204] Predicted kinases in Table 6 were identified by NetPhos v3.1 Phosphorylation Sites Predictor (Blom, N. et al., J Mol Biol 294(5): 1351–1362 (1999)). [0205] Besides the 118 phosphosites described above and depicted in FIG.2E, another set of 95 phosphopeptides was present in pre-treatment samples and totally dephosphorylated upon treatment with TG-1701. The total absence of phosphorylation preempted the statistical analysis and incorporation of these 95 samples in the volcano plot, even though these sites were the most impacted by TG-1701 treatment.
  • Ikaros-Ser442/445 residue of Ikaros a zinc finger-containing DNA-binding protein that plays a pivotal role in B-cell homeostasis.
  • Ikaros-Ser442/445 dephosphorylation was indeed the strongest event associated with TG-1701 activity (Table 7).
  • Ikaros nuclear localization and transcriptional activity both depend on BTK- mediated phosphorylation at Ser214/215 residues (Ma, H et al., PLoS One 8:e71302.27 (2013)), it was investigated whether, analogously, Ikaros function was differentially affected by TG-1701 in responder versus non-responder CLL patient samples.
  • TG- 1701 treatment led to efficient BTK dephosphorylation, YES1 upregulation and IRF4 and MYC downregulation both at mRNA and protein levels (FIGS.4E and 4F).
  • IKZF1-target genes studied here YES1 reactivation was significantly higher in TG-1701-treated than in ibrutinib-exposed cells (FIG.4E).
  • FIG.4G shows that ibrutinib and TG-1701 elicited a 67% and 45% reduction in nuclear Ikaros, consistent with a ⁇ 50% decrease of Ikaros in REC-1 nuclear protein fraction (FIG.4H), suggesting that dual dephosphorylation of Ikaros at Ser442 and Ser445 was associated with the nuclear exclusion of this factor.
  • RNA-seq analysis revealed that TG-1701 treatment blunted the Ikaros gene signature, including YES1, MYC, and IRF4, in responder patients, as well as in BTKi- sensitive B-NHL cell lines and xenografts.
  • Ikaros nuclear activity and Ikaros- dependent gene regulation remained unaffected by the drug in non-responder patients, and in BTK C481S , BTK KO , and non-canonical NF ⁇ B models in vitro and in vivo.
  • ibrutinib, TG-1701 did not impair Fc ⁇ R-driven ADCC and ADCP triggered by the anti-CD20 antibodies rituximab and ublituximab in different B-NHL co-culture system, and cooperated with U2 in reducing the tumor growth in both ibrutinib-sensitive and ibrutinib-insensitive mouse models of B-NHL.
  • Example 3 Ikaros signature is a bona fide hallmark of BTKi mechanism of action [0210] To further explore TG-1701 mechanisms of action and potential mechanism of resistance, the REC-1 GFP+LUC+ cell line was CRISPR-engineered to express the BTK C481S mutation.
  • This mutation was associated with a 10.3-fold and a 54.8-fold decrease in ibrutinib and TG-1701 inhibitory kinase activity, respectively (FIG.7A and Table 2).
  • the REC-1-BTK C481S cell line was 4.2-fold and 2.8-fold less sensitive to ibrutinib and TG-1701 respectively, compared to parental REC-1 cells (FIG.5A and Table 2).
  • a washout experiment further showed that irreversible BTK inhibition - illustrated by kinase phosphorylation over 24 hours after BTKi removal in REC-1 cells - was mostly lost in REC-1-BTK C481S cells (FIG.5B).
  • Rec-1- BTK KO derivative characterized by an almost complete depletion of BTK (FIG.5E) was refractory to both ibrutinib and TG-1701 (Table 2) and did not undergo significant modulation of YES1 and MYC expression after exposure to TG-1701 (FIGS.5E-5F).
  • TG-1701 is a novel second-generation BTKi currently under clinical development. TG-1701 is more selective than ibrutinib, with a comparable BTK Kd and similar in vitro and in vivo characteristics.
  • TG-1701 is currently being tested in a phase 1 trial comprised of a single agent arm and a combination arm with ublituximab (a novel CD20 antibody) and umbralisib (a dual PI3Kd and CK1 ⁇ inhibitor).
  • ORR overall response rates
  • CR complete responses
  • Ikaros is a zinc finger protein involved in gene regulation and chromatin remodeling.
  • CKII was ranked #4 in the list of kinases with reduced activity in TG-1701 responder patients, while phosphorylation of the PP1 inhibitory subunit, PPP1R14A, and dephosphorylation of the PP1 inhibitor, PPP1R2, both associated with reduced PP1 activity (Verbinnen, I et al., Biochem. Soc. Trans.45: 583-584 (2017)) were among the top four modifications detected in TG-1701 responder patients (Table 5 and Table 6). Ikaros expression was affected by TG-1701 treatment, neither in CLL primary cells nor in BTKi-sensitive REC-1 models, indicating that the inhibition of Ikaros signature in responders was more likely due to a nuclear exclusion of the transcription factor.

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Abstract

Provided are methods of treating B-NHL by administering a BTK inhibitor (e.g., TG- 1701) to a subject in need thereof that has been determined to be a BTK inhibitor responder (e.g., a TG-1701 responder). The BTK inhibitor (e.g., TG-1701) responder is a subject whose B-NHL cells, prior to treatment, comprise at least one phosphopeptide (e.g., Ikaros), not comprised by B-NHL cells of a non-responder, which is dephosphorylated after treatment with the BTK inhibitor. The B-NHL cells of the BTK inhibitor (e.g., TG-1701) responder further comprise at least one transcript of a gene of an Ikaros-enhanced gene signature and/or lack at least one transcript of a gene of an Ikaros-enhanced gene signature. The methods further comprise treating B-NHL by administering TG-1701 to a TG-1701 responder, in combination with an anti-CD20 antibody (e.g., ublituximab) and/or a dual PI3K5 and casein kinase- 1ε inhibitor (e.g., umbralisib).

Description

DISRUPTED IKAROS SIGNALING AS BIOMARKER FOR BTK INHIBITION CROSS REFERENCE TO RELATED APPLICATION [0001] The present application claims benefit of U.S. Provisional Application No. 63/208,344, filed June 8, 2021, the disclosure of which is hereby incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] The content of the electronically submitted sequence listing in ASCII text file (Name: 3261_018PC01_Seqlisting_ST25; Size: 54,470 bytes; and Date of Creation: June 7, 2022) filed with the application is incorporated herein by reference in its entirety. FIELD [0003] The present disclosure relates to methods of treating hematologic malignancies, such as, e.g., B-cell non-Hodgkin lymphomas (B-NHLs). In particular, the present disclosure provides methods for treating various B-NHLs by administering a BTK inhibitor (e.g., TG-1701) to a subject that has been determined to be a responder to a BTK inhibitor (e.g., TG-1701) through, e.g., phosphoproteomic analyses. The present disclosure also provides methods and kits for identifying if a subject is a responder to a BTK inhibitor. BACKGROUND [0004] B-cell non-Hodgkin lymphomas (B-NHLs) account for up to 4% of globally diagnosed cancers (Fisher et al., Oncogene 23:6524–6534 (2004)). B-NHLs are divided into low and high grades, typically corresponding to indolent (slow-growing) lymphomas, such as chronic lymphocytic leukemia (CLL), and aggressive lymphomas, such as mantle cell lymphoma (MCL), respectively (Quintanilla-ML, Hematol Oncol.35:37-45 (2017)). Targeting of the B-cell receptor (BCR) pathway through the use of covalent Bruton's tyrosine kinase inhibitors (BTKi’s) have transformed the treatment of B-NHL. The first- in-class BTKi, ibrutinib, has demonstrated exceptional clinical activity as a monotherapy for various subtypes of B-NHL, most notably CLL (Byrd, JC et al., N Engl J Med 369: 32-42 (2013)), MCL (Wang, ML et al., N Engl J Med 369: 507-516 (2013)), but also Waldenström's macroglobulinemia (WM) (Treon, SP et al., N Engl J Med 372:1430-1440 (2015)), and activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) (Wilson WH et al., Nat Med 21:922-926 (2015)). [0005] Nonetheless, the activity of BTK inhibitors, such as ibrutinib, has often been limited due to 1) off-target activity that precluded their use in combination with anti- CD20 antibodies; and 2) acquired resistance due to the development of mutations that either effect the irreversible binding of the BTKi to BTK, or activate the PLCγ2 enzyme, a downstream enzyme in the BTK pathway (Woyach, JA et al., N Engl J Med 370:2286- 2294 (2014)). In the case of BTK, a cysteine-to-serine mutation at the BTK catalytic site (BTKC481S) abrogates the covalent binding of ibrutinib to BTK, and has been detected in up to 86% of relapsing CLL patients, but only anecdotally in MCL relapsing patients (Id.; Furman, RR et al., N Engl J Med 370:2352-2354 (2014); Chiron, D et al., Cancer Discov. 4:1022-1035 (2014)). Additional intrinsic mechanisms of resistance involve activation of the non-canonical NIK-NFκB signaling in MCL (Wu, J et al., J. Hematol. Oncol.9:80 (2016); Rahal, R et al., Nat Med.20:87-92 (2013)). [0006] The use of distinct next-generation sequencing (NGS)-based genomic techniques, including whole exome and targeted deep sequencing, have been instrumental in identifying the BTKC481S mutation as a genetic cause of BTKi resistances (Doostparast, TA and Wang. K., Drug Discov Today 23: 1776-1783 (2018); Wacker, SA et al., Nat Chem Biol 8:235-237 (2012)). More recently, global drug profiling using mass spectrometry (MS)-based phosphoproteomics has been successfully employed to characterize drug mechanisms of action in single-agent therapy or multidrug combinations in solid cancers (Carvalho, AS and Matthiesen, R., Methods Mol Biol 1449: 469-479 (2016)). In addition, phosphoproteomic profiling can help understand the role of BTKi in CLL patients; in one report, CLL cells from patients with an unmutated IGVH status showed higher basal phosphorylation than patients with IGVH-mutated status (Beckmann, L et al., “MARCKS affects cell motility and response to BTK inhibitors in CLL,” Blood (2021)). [0007] TG-1701 is a novel, orally available, irreversible, and highly specific BTKi that exhibits improved selectivity when compared to ibrutinib (Normant, E. et al., “TG-1701 A Novel, Orally Available, and Covalently-Bound BTK Inhibitor,” EHA Library, 215080, Abstr. No.638 (June 152018)), and shows activity in various in vitro and in vivo models of B-NHL. TG-1701 is currently under study in patients with relapsed/refractory (R/R) B-NHL, alone and in combination with ublituximab, a glycoengineered anti-CD20 antibody, and umbralisib, a dual PI3Kδ and casein kinase-1ε inhibitor (also referred to as the “U2” regimen). (NCT03671590) (Cheah, CY et al., “Clinical Activity of TG-1701, As Monotherapy and in Combination with Ublituximab and Umbralisib (U2), in Patients with B-Cell Malignancies,” Poster Abstract #1130, 62nd ASH Annual Meeting and Exposition, Blood (2020); Cheah, CY et al., HemaSphere.4:309-309 (2020); Cheah, CY et al., Blood 134 (Supplement_1): 4001 (2019)). [0008] Despite the excellent activity of BTK inhibitors in relapsed B-NHL, not all patients respond to BTK inhibitor therapy. Accordingly, clinically-relevant biomarkers to identify BTK inhibitor responders before therapy, and/or monitor treatment efficacy during therapy, are critically needed. BRIEF SUMMARY OF THE INVENTION [0009] Provided are methods of treating B-cell non-Hodgkin lymphoma (B-NHL) in a subject in need thereof, the methods comprising administering to a subject that is a TG- 1701 responder a therapeutically effective amount of BTK inhibitor TG-1701, wherein prior to said administration, B-NHL cells of the TG-1701 responder contain at least one phosphopeptide selected from SEQ ID NOS: 1-95. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1. In some aspects, the presence of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis. [0010] In some aspects, the B-NHL cells of the TG-1701 responder lack at least one phosphopeptide selected from SEQ ID NOS: 1-95 after administration of TG-1701 to the subject compared to before administration of TG-1701. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1. In some aspects, the lack of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis. [0011] In some aspects, the B-NHL cells of the TG-1701 responder comprise an increased quantity of transcripts of an Ikaros-repressed gene signature after the administration of TG-1701 to the subject compared to the quantity of transcripts before the administration of TG-1701. [0012] In some aspects, the transcripts of the Ikaros-repressed gene signature comprise one or more of TXNIP, CD36, CA2, YOD1, CFP, DENND3, YES1, NBEAL2, TMC8, PSTPIP2, CD97, DAAM1, NT5E, LYZ, SDK2, TSC22D4, GYPC, FAM129A, TPM3, GNAQ, and/or LUZP1. [0013] In some aspects, the B-NHL cells of the TG-1701 responder comprise a decreased quantity of transcripts of an Ikaros-enhanced gene signature after the administration of TG-1701 to the subject compared to the quantity of transcripts before the administration of TG-1701. [0014] In some aspects, the transcripts of an Ikaros-enhanced gene signature comprise one or more of TCL1A, CBX5, HNRNPA0, PDHB, BCL2, DYNLL1, SUPT16H, CAMK2D, ALDH6A1, PPP2R5C, ERGIC1, BUB3, SORD, SEPHS1, CTNNBL1, CCT5, and/or APOBEC3G. [0015] In some aspects, the quantity of transcripts is determined by at least one amplification-based method. In some aspects, the amplification-based method is Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification. In some aspects, the quantity of transcripts is determined by qPCR. [0016] In some aspects, the quantity of transcripts is determined by at least one non- amplification-based method. In some aspects, the non-amplification-based method is a hybridization-based method or a sequencing-based method. In some aspects, the hybridization-based method is a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, or in situ hybridization. In some aspects, the sequencing-based method is a next-generation sequencing (NGS) method. [0017] In some aspects, the quantity of transcripts is determined by a combination of amplification-based and non-amplification-based methods. [0018] In some aspects, the B-NHL is a chronic lymphocytic leukemia, a mantle cell lymphoma, a follicular lymphoma, a diffuse large B-cell lymphoma, a marginal zone B- cell lymphoma, a Burkitt lymphoma, or a lymphoplasmacytic lymphoma. [0019] In some aspects, the method further comprises administering to a subject that is a TG-1701 responder a therapeutically effective amount of an anti-CD20 antibody. [0020] In some aspects, the method further comprises administering to a subject that is a TG-1701 responder a therapeutically effective amount of a dual PI3Kδ and casein kinase- 1ε inhibitor. [0021] In some aspects, the therapeutically effective amount of TG-1701 is between about 100 mg/day and about 400 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 100 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 200 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 300 mg/day. In some aspects, the therapeutically effective amount of TG-1701 is about 400 mg/day. [0022] In some aspects, the subject is a mammal. In some aspects, the subject is a human. [0023] Also provided are kits comprising: (i) at least one antibody that binds to at least one phosphopeptide selected from SEQ ID NOs: 1-95; (ii) optionally, reagents to perform a western blot analysis; and/or reagents to perform a phospho-flow analysis; and (iii) instructions for treating B-NHL in a TG-1701 responder according to the methods disclosed herein. [0024] In some aspects, the at least one phosphopeptide is SEQ ID NO: 1. [0025] In some aspects, the kit further comprises reagents to perform a Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification. [0026] In some aspects, the kit further comprises reagents to perform a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, next-generation sequencing (NGS) method, or in situ hybridization. BRIEF DESCRIPTION OF THE FIGURES [0027] FIGS.1A-1F show that TG-1701 is a novel irreversible BTK inhibitor as active as ibrutinib in in vitro and in vivo models of B-NHL. FIG.1A shows binding of TG-1701 and ibrutinib 1 µM against a panel of 441 kinases using the DiscoverX technology. The size of each dark grey circle is proportional to the strength of the binding. FIG.1B: TG- 1701 and ibrutinib BTKwt anti-kinase activities were tested using a 33P-ATP filtration assay. FIG.1C: DoHH2 BTK-expressing cells were incubated with ibrutinib or TG- 1701, lysed, and lysates were incubated with a fluorescent ibrutinib probe. Total BTK was assessed by Western blot. FIG.1D: increasing concentrations of ibrutinib or TG- 1701 were incubated with DoHH-2 cells. The BCR pathway is then activated with 10 µg/ml goat F(ab’)2 anti-IgM for 18h and levels of different downstream enzymes assessed using Western blot. FIG.1E: TG-1701 or ibrutinib were dosed orally in the Mino MCL xenograft model. FIG.1F: intratumor levels of several BCR-related kinases were assessed by Western blot. [0028] FIGS.2A-2F show that phosphoproteomic analysis of six CLL patients treated with TG-1701 can segregate TG-1701 responders and non-responders. FIG.2A: BTK occupancy was assessed in all patients in the study at 9 different times points. The occupancy of the 6 CLL patients are shown at 4 hours after treatment with TG-1701. FIG.2B: Correlation of TG-1701 Cmax and the daily dose received by the 6 patients. FIG.2C: Best tumor reduction in all six patients at cycle 3, day 1 (C3D1). * Patient AIK- 0003 lymphocytosis at C3D1 ranks the response as a stable disease (SD). FIG.2D: Phosphoproteomic profiling and principal components (PC) analysis were performed on all 6 CLL patients (left panel), three responders (middle panel), and three non-responders (right panel). Pre-treatment samples (PRE) are designated and the 4 hour post- treatment samples are designated in white (POST). Percentages refer to the total variance explained for each component. FIG.2E: Quantified phosphopeptides in responders. Volcano plot of the responder-only samples. The non-responders do not exhibit any TG- 1701-driven changes. FIG.2F: The phosphosites that are up- or down- regulated are shown for each single three responder patients. The non-responders did not show any changes in the phosphoproteomic analysis. [0029] FIGS.3A-3C: FIG.3A: Regulation of representative inflammatory and BCR- regulated genes upon TG-1701 treatment evaluated by qPCR in TG-1701 responders (circles) and non-responders (squares). Data shown are Log(2) fold change (POST vs PRE) n=3. FIG.3B: Comparative multi-dimensional (MDS) analysis of RNA-seq data. The closer the samples, the more similar their RNA-Seq signature. FIG.3C: Immunoblot evaluation of p-BTK, Ikaros and Ikaros downstream factors MYC and IRF4, in PBMC lysates from two CLL patients with distinct responses to TG-1701. [0030] FIGS.4A-4H show that impairment of Ikaros signaling is associated with B-NHL response to TG-1701 in both clinical and preclinical settings. Change of Ikaros-regulated factors upon TG-1701 treatment in responders (R) and non-responders (NR), according to total proteome data (FIG.4A) and RNA-seq analysis (FIG.4B) of the same samples. For each category, the average of the three patients is displayed. FIG.4C shows YES1 (an Ikaros-repressed gene) and MYC (an Ikaros-enhanced gene) mRNA changes after treatment with TG-1701. FIG.4D depicts immunoblot evaluation of p-BTK, Ikaros, and Ikaros downstream factors, MYC and IRF4, using one representative responder and one non-responder PBMC lysates. P-BTK detection was assessed to confirm on-target activity at 4 hours post-treatment. MCL REC-1 cells were treated for 24 hours with 1 µM ibrutinib or TG-1701, and variations in Ikaros-regulated factors were quantified using qPCR (FIG.4E) and Western blot (FIG.4F). Subcellular localization of Ikaros was determined by immunofluorescence staining (FIG.4G) and immunoblot analysis of nuclear protein fraction (FIG.4H), in REC-1 cells treated as above with ibrutinib or TG- 1701. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = non-significant. [0031] FIGS.5A-5F show that Ikaros modulation is associated with TG-1701 efficacy in distinct in vitro and in vivo MCL models of ibrutinib resistance. FIG.5A: Viability of BTKwt and BTKC481S REC-1 cells exposed to increasing doses of ibrutinib and TG-1701 were evaluated by CellTiter Glo assay. FIG.5B: REC-1 and REC-1-BTKC481S cells were exposed for 1h to 1µM TG-1701, washed-out for the indicated times, and levels of phospho-BTK and loading control GADPH were assessed using immunoblotting. Values below immunoblot correspond to the densitometric quantification of p-BTK/GADPH ratio. FIG.5C: Regulation of Ikaros-regulated factors after 4 hours of treatment with TG- 1701 or ibrutinib (1 µM) in REC-1 and REC-1-BTKC481S cells according to total proteome data. FIG.5D: Ikaros gene signatures were evaluated by qPCR in REC-1 and REC-1-BTKC481S cells exposed for 24 h to 1 µM ibrutinib or TG-1701. In REC-1- BTKC481S cells, values were referred to untreated REC-1 cells (control). Ikaros transcriptional (FIG.5E) and protein (FIG.5F) signatures were evaluated in REC-1- BTKKO cells as previously, using untreated REC-1 cells as a reference control. [0032] FIGS.6A-6D show in vivo activity of TG-1701 on BTKi-sensitive and BTKi- resistant MCL mouse models. FIG.6A: TG-1701 was dosed orally in BKTi-sensitive (REC-1-GFP+LUC+) and BTKi-resistant (UPN-IbruR) MCL xenograft models and tumor volumes were recorded at the endpoint (17 days) by bioluminescence signal recording (REC-1) or external calipers (UPN-IbruR). Ikaros signature was evaluated by western blot (FIG.6B) and qPCR (FIG.6C) in two/three representative tumors from each treatment group. FIG.6D: Immunohistochemical labeling of CD20 and Ikaros in tissue sections from four representative BTKi-sensitive (REC-1) and BTKi-resistant (UPN- IbruR) tumor specimens (scale bar: 25µm). *p < 0.05, **p < 0.01, ***p < 0.001, and ns = non-significant. [0033] FIGS.7A-7D: FIG.7A: Inhibition of BTK and BTKC481S kinase activity by TG- 1701 and ibrutinib were assessed using a 33P-ATP filtration assay. FIG.7B: Proteomic profiling and principal components analysis were performed on REC-1 and BTKC481S derivative after treatment with ibrutinib or TG-1701 (1 µM) for 4 hours. FIG.7C: Western blot examination of REC-1-BTKC481 whole cell extracts following a 24-hour treatment with ibrutinib or TG-1701. FIG.7D: Viability of BTKi-sensitive (UPN-wt) and insensitive (UPN-IbruR) UPN-1 derived cell lines exposed to increasing doses of ibrutinib and TG-1701 was evaluated by a CellTiter Glo assay. [0034] FIG.8 depicts a schematic of the BTK occupancy assay developed using a MSD chemoluminescent platform at Bioagilytix. The % occupancy was calculated as shown in the figure. DETAILED DESCRIPTION [0035] Provided herein are methods of treating B-NHL by administering a BTK inhibitor (e.g., TG-1701) to a subject in need thereof that is a BTK inhibitor responder (e.g., a TG- 1701 responder). The BTK inhibitor (e.g., TG-1701) responder is a subject whose B-NHL cells, prior to treatment, comprise at least one phosphopeptide (e.g., Ikaros), not comprised by B-NHL cells of a non-responder, which is dephosphorylated after treatment with the BTK inhibitor (e.g., TG-1701). The B-NHL cells of the BTK inhibitor (e.g., TG- 1701) responder further comprise at least one transcript of a gene of an Ikaros-enhanced gene signature and/or lack at least one transcript of a gene of an Ikaros-enhanced gene signature. The methods further comprise treating B-NHL by administering TG-1701 to a TG-1701 responder, in combination with an anti-CD20 antibody (e.g., ublituximab) and/or umbrasilib. [0036] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. Terms [0037] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. [0038] As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "either/or." [0039] Furthermore, “and/or,” where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). [0040] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound, or combination of one or more compounds that, when administered (either sequentially or simultaneously) elicits the desired biological or medicinal response, e.g., either destroys the target cancer cells or slows or arrests the progression of the cancer in a patient. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the patient and disease condition being treated, e.g., the weight and age of the patient, the severity of the disease condition, the manner of administration and the like, which may readily be determined by one skilled in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of cell proliferation and/or cell migration. For example, in some aspects, the "therapeutically effective amount" as used herein refers to the amount of TG-1701, or a pharmaceutically acceptable salt thereof, and/or the amount of an anti-CD20 antibody and/or of a dual PI3Kδ and casein kinase-1ε inhibitor that, when administered separately or in combination, have a beneficial effect. In some aspects, the "therapeutically effective amount" as used herein refers to the amount of TG- 1701, or a pharmaceutically acceptable salt thereof, and the amount of an anti-CD20 antibody and a dual PI3Kδ and casein kinase-1ε inhibitor that, when administered separately or in combination, have a beneficial effect. In some aspects, the combined effect is additive. In some aspects, the combined effect is synergistic. Further, it will be recognized by one skilled in the art that in the case of combination therapy, the amount of TG-1701, or a pharmaceutically acceptable salt thereof, and/or the amount of the CD20 antibody and/or the dual PI3Kδ and casein kinase-1ε inhibitor may be used in a "sub- therapeutic amount", i.e., less than the therapeutically effective amounts of each compound when used alone. [0041] The term "about" refers to approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a number or a numerical range, it means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of ±10%. [0042] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by uncontrolled or unregulated cell growth. Examples of cancer include, e.g., carcinoma, lymphoma, blastoma, sarcoma, and leukemia. [0043] As used herein, the terms "B-cell cancer" or "B-cell malignancy" refers to an uncontrolled or unregulated growth of B-cells in the blood, bone marrow, or lymph node. One skilled in the art would understand that a B-cell malignancy is a type of hematological malignancy (or hematological cancer) that includes lymphomas, leukemias, and myelomas. The B-cell malignancy may be indolent or aggressive. [0044] Non-limiting examples of B-cell malignancies that may be treated with the methods or kits disclosed herein include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) (referred to herein as “B-cell NHL” or “B-NHL”), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), which includes extranodal MZL, nodal MZL, and splenic MZL, hairy cell leukemia (HCL), Burkitt's lymphoma (BL), and Richter's transformation. In some aspects, the DLBCL is an activated B-cell DLBCL (ABC-DLBCL), a germinal center B-cell like DLBCL (GBC-DLBCL), a double hit DLBCL (DH-DLBCL), or a triple hit DLBCL (TH-DLBCL). [0045] In some aspects, certain CLLs (or other leukemias, such as the ones described herein) are considered "high risk" due to the presence of one of more genetic mutations. As used herein, "high risk" CLL, for example, means CLL characterized by at least one of the following genetic mutations: 17p del; 11q del; p53; unmutated IgVH together with ZAP-70+ and/or CD38+; and trisomy 12, and complex karyotype. [0046] In some aspects, the B-NHLs that may be treated with the methods or kits disclosed herein include the following B-NHL subtypes: chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, or a lymphoplasmacytic lymphoma. Other B- NHL subtypes that may be treated with the methods or kits disclosed herein are known to those skilled in the art. See, Quintanilla-ML, Hematol Oncol.35:37-45 (2017)). [0047] "Tumor" and "neoplasm" refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions. [0048] The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" will be modified by the term "non-tumorigenic" when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells. [0049] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment (e.g., TG-1701). Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject. Generally, the "patient" or “subject” who has been diagnosed with a disease, disorder, or condition (e.g., a B-cell malignancy as described herein), exhibits symptoms of, or is otherwise believed to be afflicted with a disease, disorder, or condition (such as, e.g., a B-cell malignancy). [0050] An "effective amount" of an antibody or an agent as disclosed herein, is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner by those skilled in the art, in relation to the stated purpose. [0051] The term "therapeutically effective amount" refers to the amount of an agent (e.g., monoclonal antibody, small molecule, chemotherapeutic drug, etc…), as disclosed herein, that is effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the agent or drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of "treating." To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. [0052] Terms such as "treating," "treatment," "to treat," "having a therapeutic effect," alleviating," "to alleviate," or "slowing the progression of" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder, such as a B-cell malignancy, and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. [0053] In certain aspects, a subject is successfully "treated" for a B-cell malignancy according to the methods of the present invention if the patient shows one or more of the following: reduction in cachexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass (e.g., by scan), reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrence or progressive disease, tumor response, complete response (CR), partial response (PR), stable disease (SD), progression free survival (PFS), overall survival (OS), each as measured by standards set by the National Cancer Institute and the U.S. Food and Drug Administration for the approval of new drugs. See, Johnson et al., J. Clin. Oncol.21:1404-1411 (2003). In some aspects, the "therapeutic effect," as defined above, also encompasses a reduction in toxicity or adverse side effects, and/or an improvement in tolerability. [0054] In certain aspects, guidelines provided by standard international workshops for particular B-cell malignancies are used to assess tumor response, such as, for CLL, as set forth in Hallek, M. et al., Blood 111:5446-5456 (2008); for NHL, as set forth in Cheson, B.D. et al., J Clin Oncol 25:579-586 (2007); and for WM, according to the sixth international workshop on WM, Owen, R.G. et al., Br J Haematol.160:171-176 (2013). [0055] In some aspects, treating the B-cell malignancy using the methods and kits described herein reduces percent tumor burden from baseline (i.e., prior to administration of the combination of agents described herein) by about 25% - 100%. In some aspects, treating the B-cell malignancy using the methods and kits described herein reduces percent tumor burden from baseline by at least about 20%, by at least about 25%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%. In certain aspects, the methods and kits described herein promote B-cell malignancy regression to the point of eliminating the B-cell malignancy. In some aspects, subjects can be assessed for tumor burden or evaluated for anti-tumor response by CT, PET-CT, and/or MRI. [0056] As used herein, “duration of the percent reduction in tumor burden” is the interval from the first documentation of complete response (CR) or partial response (PR) to the earlier of the first documentation of definitive disease progression or death from any cause. In certain aspects, the “duration of the reduction in percent tumor burden” can be observed and continue for a period of at least about 24 weeks to about 36 months. [0057] The term "combination administration," "administered in combination," and "administering a combination" refers to administering more than one pharmaceutically active ingredient (including, but not limited to, TG-1701 or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody, and/or a dual PI3Kδ and casein kinase-1ε inhibitor as disclosed herein) to a patient. Combination administration may refer to simultaneous administration, sequential administration, or both simultaneous and sequential administration of TG-1701, or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody and a dual PI3Kδ and casein kinase-1ε inhibitor, as disclosed herein. Thus, by way of example, administration of an anti-CD20 antibody preceding or following (e.g., by hour(s), day(s), week(s), or month(s)) administration of a PI3K-delta selective inhibitor, preceding or following (e.g., by hour(s), day(s), week(s), or month(s)) administration of BTK inhibitor TG-1701, constitutes administration of a combination of agents. [0058] As will be apparent to one skilled in the art from the context, a "combination of agents" can also include a BTK inhibitor (e.g., TG-1701), an anti-CD20 antibody (e.g., ublituximab), a PI3K-delta selective inhibitor (e.g., umbralisib), and one or more additional therapeutic agents, as described herein. The therapeutic agents can be administered in a single pharmaceutical formulation or are administered simultaneously in separate pharmaceutical formulations by either the same or different routes of administration. Further, the term "combination of agents" is intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time. [0059] The terms "simultaneous" and "simultaneously" refer to the administration of TG- 1701, or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody and a dual PI3Kδ and casein kinase-1ε inhibitor as disclosed herein, to a patient at the same time, or at two different time points that are separated by no more than 2 hours. The simultaneous administration of TG-1701, or a pharmaceutically acceptable salt thereof, and an anti- CD20 antibody, and a dual PI3Kδ and casein kinase-1ε inhibitor may be in a single dosage form or in separate dosage forms. [0060] The terms "sequential" and "sequentially" refer to the administration of TG-1701, or a pharmaceutically acceptable salt thereof, and an anti-CD20 antibody and a dual PI3Kδ and casein kinase-1ε inhibitor, as disclosed herein, to a patient at two different time points that are separated by more than 2 hours, e.g., about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or even longer. [0061] As used herein, "U2" refers to the combination of Ublituximab + Umbralisib, as used in the methods or kits of the disclosure. [0062] As used herein, the term "TG-1701 + U2" refers to the triple combination of the BTK inhibitor TG-1701 + Ublituximab + Umbralisib, as used in the methods or kits of the invention. [0063] As used herein, an "adverse event" (AE) is any unfavorable and generally unintended or undesirable sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical or pharmaceutical treatment. [0064] A B-cell malignancy which "does not respond," "responds poorly," or is "refractory" to treatment (with, for example, an anti-CD20 antibody) does not show statistically significant improvement in response to that treatment when compared to no treatment or treatment with a placebo in a recognized animal model or human clinical trial, or which responds to an initial treatment, but grows as treatment continues. [0065] A B-cell malignancy which has “relapsed” means that the tumor has returned following treatment. The term “R/R” means that the B-cell malignancy is relapsed or refractory, or possibly both. [0066] The term "synergistic effect" refers to a situation where the combination of two or more agents produces a greater effect than the sum of the effects of each of the individual agents. The term encompasses not only a reduction in symptoms of the disorder to be treated, but also an improved side effect profile, improved tolerability, improved patient compliance, improved efficacy, or any other improved clinical outcome. [0067] As used herein, the illustrative terms "include", "such as", "for example" and the like (and variations thereof, e.g., "includes" and "including", "examples"), unless otherwise specified, are intended to be non-limiting. That is, unless explicitly stated otherwise, such terms are intended to imply "but not limited to", e.g., "including" means including but not limited to. [0068] As used herein, the recitation of a numerical range for a variable is intended to convey that the disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable, which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable, which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values > 0 and < 2 if the variable is inherently continuous. [0069] The term "Bruton's tyrosine kinase" (also known as "BTK," agammaglobulinemia tyrosine kinase (ATK), or B-cell progenitor kinase (BPK)) refers to a non-receptor tyrosine kinase enzyme in the B-cell antigen receptor (BCR) signaling pathway. BTK, a member of the Tec family of protein tyrosine kinases, is predominantly expressed in B- lymphocytes at various stages of development (except in terminally differentiated plasma cells). BTK is a signal transduction protein that regulates normal B-cell development, differentiation activation, proliferation, and survival (Kurosaki, T., Curr Op Imm 12:276- 281 (2000); Schaeffer, E.M. and Schwartzberg, P.L., Curr Op Imm 12: 282-288 (2000)). BTK has also been implicated in initiation, survival, and progression of mature B-cell lymphoproliferative disorders, such as B-cell malignancies (Akinleye, A. et al., J. Hematol. Oncol.6:59 (2013)). As used herein, BTK is from homo sapiens, as disclosed in U.S. Patent No.6,326,469 (Gen Bank Acc. No. NP_000052). BTK is also a key signaling enzyme expressed in all hematopoietic cells types except T lymphocytes and natural killer (NK) cells and plays a role in hematopoietic cell signaling pathways such as, e.g., Toll like receptor (TLR) and cytokine receptor-mediated TNF-alpha production in macrophages, IgE receptor (FcepsilonRI) signaling in mast cells, inhibition of Fas/APO-1 apoptotic signaling in B-lineage lymphoid cells, and collagen-stimulated platelet aggregation (See, e.g., Jeffries, C. A. et al., J. Biol. Chem.278:26258-26264 (2003); Horwood, N. J. et al., The Journal of Experimental Medicine 197:1603-1611 (2003); Iwaki, S. et al., J. Biol. Chem.280: 40261-40270 (2005)). BTK functions as an important regulator of cell proliferation and cell survival in various B-cell malignancies. [0070] An "inhibitor of BTK" or a "BTK inhibitor" (or BTKi for short) refers to a small molecule that targets BTK and either inhibits BTK tyrosine phosphorylation and/or B-cell activation and/or otherwise inhibits or diminishes or abolishes the biological activity of a BTK protein. An "irreversible BTK inhibitor" refers to a molecule that upon contact with BTK, causes the formation of a new covalent bond with an amino acid residue of BTK. The BTK inhibitor TG-1701, which is used in the methods and kits of the present invention and discussed further below, is an irreversible BTK inhibitor. Several BTK inhibitors, such as, e.g., ibrutinib (IMBRUVICA®) and acalabrutinib (CALQUENCE®) have been FDA-approved for the treatment of e.g., CLL and MCL. Other BTK inhibitors include, but are not limited to, zanubrutinib, acalabrutinib, evobrutinib, tirabrutinib, fenebrutinib, pirtobrutinib, GS-4059 (NCT02457598), spebrutinib, HM71224, SNS-062, ABBV-105, LCB 03-0110 dihydrochloride, LFM-A13, PCI 29732, PF 06465469, M7583 (NCI Code C129710), or (-)-Terreic acid. [0071] An "anti-CD20 antibody" or "an antibody that binds to CD20" refers to an antibody that is capable of binding CD20 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD20. The extent of binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is less than about 10% of the binding of the antibody to CD20 as measured, e.g., by a radioimmunoassay (RIA). Exemplary anti-CD20 antibodies include, but are not limited to, ublituximab, ofatumumab, ubinutuzumab, ibritumomab, tiuxetan, obinutuzumab, rituximab, rituximab- hyaluronidase, and ocrelizumab. [0072] As used herein, “Ikaros” refers to a human transcription factor belonging to the IKZF family of zinc-finger DNA-binding proteins associated with chromatin remodeling and regulation of hematopoietic cell development. The IKZF family consists of five members: Ikaros (encoded by the gene IKZF1), Helios (IKZF2), Aiolos (IKZF3), Eos (IKZF4), and Pegasus (IKZF5). These factors contain N-terminal zinc finger (ZF) domains, which are responsible for mediating direct interactions with DNA, and C- terminal ZFs, which facilitate homo- and heterodimerization between IKZF family members. Powell, MD et al., Frontiers in Immunology 10, Article 1299 (2019). The zinc- finger 1 transcription factor gene “IKZF1” encoding Ikaros can be found at HGNC: 13176 or NCBI Entrez Gene: 10320. [0073] As used herein, the term “Ikaros-repressed gene” or “Ikaros-repressed gene signature” refers to a gene or number of genes, the transcription of which is inhibited when Ikaros binds to the promoter region of the gene. This inhibition of transcription leads to a decreased level of the transcript (mRNA) that can be assessed by, e.g., PCR.. For example, in some aspects, the transcripts of the Ikaros-repressed gene signature comprise one or more of TXNIP, CD36, CA2, YOD1, CFP, DENND3, YES1, NBEAL2, TMC8, PSTPIP2, CD97, DAAM1, NT5E, LYZ, SDK2, TSC22D4, GYPC, FAM129A, TPM3, GNAQ, and/or LUZP1. [0074] As used herein, the term “Ikaros-enhanced gene” or Ikaros-enhanced gene signature” refers to a gene or a number of genes, the transcription of which is increased when Ikaros binds to the promoter region of the gene. This activation of transcription leads to an increased level of the transcript (mRNA) that can be assessed by, e.g., PCR. For example, in some aspects, the transcripts of an Ikaros-enhanced gene signature comprise one or more of TCL1A, CBX5, HNRNPA0, PDHB, BCL2, DYNLL1, SUPT16H, CAMK2D, ALDH6A1, PPP2R5C, ERGIC1, BUB3, SORD, SEPHS1, CTNNBL1, CCT5, and/or APOBEC3G. [0075] As used herein, the term “proteomic profile” or “proteomic analysis” refers to the compilation of all the changes (increase or decrease) of the level of all the proteins that can be detected by a given method (e.g., mass spectrometry). [0076] As used herein, the term “decreased quantity of transcripts of an Ikaros-enhanced gene signature” refers to a decrease of mRNA molecules as measured by, e.g., PCR. The significance of a decrease can be assessed when multiple repeats are available. If not, the accepted threshold is a 2-fold change (value of 1 in Log2 scale). [0077] The quantity of a transcript of an Ikaros-repressed gene and/or a transcript of an Ikaros-enhanced gene is measured using various methods known in the art, including, e.g., amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.); hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.); and sequencing-based methods (e.g., next-generation sequencing (NGS) methods, for example, using the Illumina or Ion Torrent sequencing platforms). As used herein, the term ”rolling circle amplification” refers to a process of unidirectional nucleic acid amplification that can rapidly synthesize multiple copies of circular DNA or RNA molecules. As used herein, the term “NanoString analysis” refers to a process that is a variation of DNA microarray and uses molecular “barcodes” and microscopic imaging to detect and count up to several hundred transcripts in hybridization reactions. As used herein, the term “next-generation sequencing” (NGS) refers to sequencing platforms that perform sequencing of millions of small fragments of DNA in parallel and employ bioinformatics analysis to piece together the individual fragments by mapping the individual reads to a reference genome. As used herein, the term “branched DNA signal amplification” refers to an assay that uses support-bound small single stranded DNA capture molecules bound at a free end to DNA extender molecules, which extender molecules bind to DNA and/or RNA in a sample and the sample DNA and/or RNA in turn is bound by a label extender, a pre-amplifier and an enzyme-linked amplifier molecule to detect and quantify small amounts of DNA and/or RNA without a reverse transcription and/or PCR step. [0078] As used herein, the term “expression” refers to a process by which a polynucleotide produces a gene product, e.g., RNA or a polypeptide. It includes, without limitation, transcription of the polynucleotide into messenger RNA (mRNA) (also known as the “RNA transcript”), and the translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a “gene product” can be, e.g., a nucleic acid, such as an RNA produced by transcription of a gene. As used herein, a “gene product” can be either a nucleic acid, RNA, or miRNA produced by the transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post-translational modifications, e.g., phosphorylation, methylation, glycosylation, ubiquitination, acetylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage. [0079] As used herein, the term “phosphoproteomic profile” or “phosphoproteomic analysis” refers to a type of proteomic analysis that comprises identifying, cataloguing, and/or characterizing proteins containing a phosphate group, including phosphorylated serine, threonine, and/or tyrosine residues. Phosphoproteomic analysis includes the quantitative measurement of changes in phosphorylation (phosphorylation patterns or phosphorylation maps). Phosphoproteomic analysis may be performed by, e.g., mass spectrometry (MS), including, e.g., MALDI-TOF, ICP-MS, DART-MS, Secondary ion mass spectrometry (SIMS), Gas chromatography mass spectrometry (GC-MS), Liquid chromatography mass spectrometry (LC-MS), Crosslinking mass spectrometry (XL-MS), and Hydrogen-exchange mass spectrometry (HX-MS). [0080] As used herein, the term “phosphopeptide” refers to a peptide (short chain of between two and fifty amino acids, linked by peptide bonds) that incorporates one or more phosphate groups as a result of phosphorylation and is detected as a pair comprising the unmodified peptide and the phospho-peptide with an added mass of 80 daltons for each phosphorylated residue. [0081] As used herein, the term “phospho-flow” or “phospho-flow cytometry” refers to a technology that measures the phosphorylation state of intracellular proteins at the single cell level using labeled antibodies that bind phosphorylated amino acids such as phosphoserine, phosphothreonine, and phosphotyrosine within proteins. See, Krutzik, PO et al., Methods Mol Biol.699: 179-202 (2011). With different fluorescent labels attached to the antibodies, the relative quantity of each of phosphoserine, phosphothreonine, and phosphotyrosine within proteins of a single cell can be determined. [0082] "Western blot" is a well-known and widely used analytical technique in molecular biology to identify the presence of a specific single protein within a complex mixture of proteins. Generally, the proteins are run on gels to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized proteins are then exposed to antibodies with reactivity against an antigen of interest. The binding of the antibodies can be detected by various methods, including, e.g., the use of fluorescently labeled antibodies. [0083] Various aspects described herein are described in further detail in the following subsections. TG-1701 [0084] In some aspects, the BTK inhibitor used in the methods and kits described herein is (R)-4-amino-1-(1-(but-2-ynoyl)pyrrolidin-3-yl)-3-(4-(2,6-difluorophenoxy)phenyl)- 1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (TG-1701), or an isomer, polymorph, enantiomer, pharmaceutically acceptable salt, solvate, or prodrug thereof. [0085] In a preferred aspect, the BTK inhibitor is (R)-4-amino-1-(1-(but-2- ynoyl)pyrrolidin-3-yl)-3-(4-(2,6-difluorophenoxy)phenyl)-1,6-dihydro-7H-pyrrolo[2,3- d]pyridazin-7-one, also known as TG-1701, SHR-1459, or EBI-1459. The alternate chemical name for TG-1701 is (R)-1-(3-(4-amino-3-(4-(2,6-difluorophenoxy)phenyl)-7- hydroxy-1H-pyrrolo[2,3-d]pyridazin-1-yl)pyrrolidin-1-yl)but-2-yn-1-one. The terms “TG-1701” or “BTK inhibitor TG-1701” are used interchangeably and will be used predominantly herein. [0086] The chemical formula of TG-1701 is C26H21F2N5O3, and its molecular weight is 489.48 g/mol. TG-1701 has the following chemical structure:
Figure imgf000021_0001
[0087] TG-1701 is described in PCT Publication No. WO2016/007185 and U.S. Patent Nos.9,951,077 and 10,323,037, which are all incorporated by reference in their entireties. Crystalline forms of TG-1701 are described in PCT Publication No. WO2017/118277 and U.S. Patent No.10,626,116, and are also incorporated by reference in their entireties. [0088] TG-1701 is an orally available, covalently-bound, selectively irreversible inhibitor of BTK. TG-1701 has been shown to exhibit superior selectivity for BTK compared to other clinically available BTK inhibitors, such as ibrutinib and acalabrutinib. See, e.g., Normant, E. et al., Abstract 3973, European Hematology Association Annual Meeting, Stockholm, Sweden (June 14, 2018). TG-1701 was evaluated and compared to ibrutinib and/or acalabrutinib in numerous enzyme based, cell-based, and animal models. For example, TG-1701 was shown to be as active as ibrutinib (having comparable kinase inhibition IC50s against BTK - 3 nM and 1.5 nM, respectively), but with improved selectivity for BTK in an in vitro whole kinome screening. Id. In addition, TG-1701 was 90-fold less active on EGFR compared to BTK with an IC50 of 270 nM and 3 nM respectively. Ibrutinib, however, was only 4.3-fold less active on EGFR compared to BTK with an IC50 of 6.4 nM and 1.5 nM respectively. Id. [0089] The inhibitory effect of TG-1701 on cell proliferation was measured in several cell lines (B-cell lymphomas). TG-1701 inhibited the growth of the follicular lymphoma (FL) DOHH-2, mantle cell lymphoma (MCL) Mino, and DLBCL SU-DHL-6 cell lines with IC50s of 369, 449, and 313 nM, respectively. TG-1701 inhibited the IgM-activated BCR pathway in DOHH-2 cells, in particular, the phosphorylation of BTK, PLCy2, and ERK1/2. In a cell-based assay, TG-1701 blocked IgM-dependent CD69 expression, adhesion of JEKO cells to VCAM-1, and CXCL12-dependent migration. Id. [0090] A fluorescent BTK-occupancy assay was developed and validated in vivo, in the spleen of mice, where BTK was found to be completely occupied after administration of a single dose of TG-1701 at 12.5 mg/kg. In vivo, the anti-tumor efficacy of TG-1701 was assessed in several lymphoma xenograft models, e.g., SU-DHL-6 (GCB-DLBCL), Mino (MCL), and OCI-Ly10 (ABC-DLBCL), where TG-1701 showed potent anti-tumor activity equivalent to or greater than ibrutinib and similar to the recently approved BTK inhibitor, acalabrutinib. In addition, the pharmacokinetic profile of TG-1701 allows for a once a day dosing. TG-1701 is a novel and highly selective, irreversible BTK inhibitor with potent in vitro and in vivo activity. Id. Methods of Treating B-NHL in a TG-1701 Responder [0091] Provided are methods of treating B-cell non-Hodgkin lymphoma (B-NHL) in a subject comprising administering a BTK inhibitor (BTKi) to a subject that is a BTK inhibitor responder. In a preferred aspect, the BTKi is TG-1701 and the subject is a TG- 1701 responder. [0092] As used herein, if prior to administration of TG-1701, a “responder of TG-1701” or a “TG-1701 responder” refers to a subject whose NHL cells contain at least one positive phosphopeptide on a sequence selected from SEQ ID NOs 1-95, in particular, SEQ ID NO: 1, as determined by Western blot and/or phospho-flow analysis. [0093] As used herein, if after administration of TG-1701, a “responder of TG-1701” or a “TG-1701 responder” refers to a subject with a 50% or more tumor reduction from baseline following TG-1701 treatment, as determined by scan. [0094] As used herein, a “BTK inhibitor responder” or “BTKi responder” refers to the same definitions provided above for TG-1701, but for any non-TG-1701 BTK inhibitor, as described herein. [0095] In some aspects, the B-NHL cells of a BTKi responder contain at least one phosphopeptide that the B-NHL cells of a non-responder do not contain. [0096] In some aspects, the subject is a TG-1701 responder and the B-NHL cells of the TG-1701 responder contain at least one phosphopeptide that the B-NHL cells of a TG- 1701 non-responder do not contain. [0097] In some aspects, the subject has been determined to be a BTKi responder by quantifying phosphopeptides and/or phosphopolypeptides in the B-NHL cells of the subject. In some aspects, the subject has been determined to be a TG-1701 responder by quantifying phosphopeptides and/or phosphopolypeptides in the B-NHL cells of the subject. [0098] In some aspects, the subject is determined to be a BTKi responder by quantifying at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspect, the at least one phosphopeptide is SEQ ID NO: 1. [0099] In some aspects, the subject is determined to be a TG-1701 responder by quantifying at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1. [0100] In some aspects, the subject is determined to be a BTKi responder by quantifying a phosphopolypeptide that comprises at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1. [0101] In some aspects, the subject is determined to be a TG-1701 responder by quantifying a phosphopolypeptide that comprises at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1. [0102] In some aspects, the phosphopeptide and/or phosphopolypeptide quantification is performed in B-NHL cells isolated from blood samples, biopsy samples and/or bone marrow aspirates. The percentage of circulating B-NHL cancer cells in the samples of the subject can be from about 40% to about 98%. [0103] In some aspects, prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. In some aspects, prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. [0104] In some aspects, after initiation of treatment with a BTKi, the subject is determined to be responsive to a BTKi according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. In some aspects, after initiation of treatment with TG-1701, the subject is determined to be responsive to TG-1701 according to a method comprising measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. [0105] In some aspects, the method comprises providing B-NHL cells of a subject comprising polypeptides, extracting the polypeptides and enriching phosphopolypeptides from the polypeptide extract. In some aspects, peptides are prepared from the extracted phosphopolypeptides. In some aspects, the peptides are prepared using enzyme digestion. In some aspects, the phosphopolypeptides are enriched using immunoprecipitation, metal affinity chromatography, metal-oxide affinity chromatography, Phos-Tag chromatography, polymer-based metal ion affinity capture, hydroxyapatite chromatography, enrichment by chemical modification, and/or phosphopolypeptide precipitation. [0106] In some aspects, the ion affinity chromatography comprises iron (Fe3+), gallium (Ga3+), aluminium (Al3+), zirconium (Zr4+) or titanium (Ti4+) ion affinity chromatography or sequential iron (Fe3+), gallium (Ga3+), aluminium (Al3+), zirconium (Zr4+) or titanium (Ti4+) ion affinity chromatography. In some aspects, sequential ion affinity chromatography comprises phosphopolypeptide and/or phosphopeptide enrichment using TiO2 affinity chromatography followed by Fe-NTA chromatography of the TiO2 flow- through and wash fractions. In some aspects, sequential ion affinity chromatography comprises phosphopolypeptide and/or phosphopeptide enrichment using Fe-NTA chromatography followed by TiO2 chromatography of the Fe-NTA flow through and wash fractions. In some aspects, the phosphopolypeptides and/or phosphopeptides are enriched using either TiO2 or Fe-NTA affinity chromatography. In some aspects, the ion affinity chromatography is immobilized ion affinity chromatography. In some aspects, the eluates obtained from any of the phosphopolypeptide and/or phosphopeptide enrichment procedures are fractionated before further processing. [0107] In some aspects, the enriched phosphopolypeptides are subjected to western blot or flow cytometry analysis using antibodies that bind to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95. [0108] In some aspects, the enriched phosphopolypeptides are subjected to further immunoaffinity purification steps prior to performance of western blots and/or flow cytometry. In some aspects, the enriched phosphopolypeptides are subjected to immunoaffinity purification using at least one antibody that binds to a phosphorylated amino acid. In some aspects, the enriched phosphopolypeptides are subjected to immunoaffinity purification using at least one antibody that binds to phosphorylated serine, phosphorylated threonine, or a phosphorylated tyrosine. In some aspects, the phosphopolypeptide fractions eluted from the immunoaffinity purification step are subsequently subjected to western blot or flow cytometry analysis using at least one antibody that binds to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95. In some aspects, the eluates that contain phosphoserine, phosphothreonine and/or phosphotyrosine consistent with the phosphopeptides of SEQ ID NO: 1-95 are further subjected to peptide sequencing. In some aspects, peptide sequencing of the eluted phosphopolypeptides comprises enzyme digestion to generate peptides and subjection to mass spectrometry. In some aspects, the samples that have been enriched for phosphopolypeptides and have been eluted from immunoaffinity purification steps as binding to at least one phosphorylated serine, at least one phosphorylated threonine, or at least one phosphorylated tyrosine and also bind to at least one antibody that binds to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95 are further subjected to method steps that quantify in said samples at least one transcript of an Ikaros-repressed gene and/or at least one transcript of an Ikaros-enhanced gene such that the subject from which the sample was obtained is determined to be a TG-1701 responder. In some aspects, such combined determination of the presence of phosphopeptides selected from SEQ ID NO: 1-95 and determination of the presence of at least one transcript of an Ikaros-repressed gene and/or the absence of at least one transcript of an Ikaros-enhanced gene determines that the subject is a TG-1701 responder. In some aspects, such combined determination is performed when mass spectrometry is not available. [0109] In some aspects, the enriched phosphopolypeptides are subjected to separate immunoaffinity purification steps using at least one antibody that binds to phosphorylated serine, at least one antibody that binds to phosphorylated threonine, and at least one antibody that binds to a phosphorylated tyrosine separately. In some aspects, the eluates of the separate immunoaffinity purification are subsequently subjected to western blot or flow cytometry analysis using at least one antibody that binds to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95. In some aspects, the eluates that contain phosphoserine, phosphothreonine and/or phosphotyrosine consistent with the phosphopeptides of SEQ ID NO: 1-95 are further subjected to peptide sequencing. [0110] In some aspects, the phosphopolypeptides comprise one or more phosphopeptides selected from SEQ ID NO: 1-95. In some aspects, the B-NHL cells of the subject comprise 1 to about 95 phosphopeptides; or about 1 to 10, 11 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 72 to 80, 81 to 90 or 91-95 phosphopeptides. In some aspects, the B-NHL cells of the subject comprise 1 to 95, 2 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to 60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, or 91 to 95 phosphopeptides. [0111] In some aspects, the B-NHL cells of the subject comprise 1 to about 95 phosphopeptides prior to a treatment with a BTKi and lack at least one of these phosphopeptide after treatment with a BTKi. [0112] In some aspects, the B-NHL cells of the subject comprise 1 to about 95 phosphopeptides prior to a treatment with TG-1701 and lack at least one of these phosphopeptide after treatment with TG-1701. [0113] In some aspects, the B-NHL cells of the BTKi responder contain at least one transcript of an Ikaros-repressed gene signature. [0114] In some aspects, the subject is a TG-1701 responder and the B-NHL cells of the TG-1701 responder contain at least one transcript of an Ikaros-repressed gene signature. [0115] In some aspects, the genes of an Ikaros-repressed gene signature comprise one or more of LUZP1, GYPC, BAALC, MAN1A1, RPS16, CPEB4, RAB1A, SERPINB6, TNK2, CD59, GOLGA9P, PRKCH, GNAQ, YPEL5, ZC3HAV1, KLF10, OLFML2A, GPR110, FAM160B1, C7orf41, BAALC, ARPC5L, MAP3K8, TIPARP, LRRFIP1, CLEC2B, MCL1, CD97, DNAJB6, ITSN2, YES1, SOCS5, USP15, SEPT6, LRRFIP1, CPEB4, ARPC5L, TPM3, SERPINB9, MAN1A1, CISH, SIK1, KIF16B, NET1, synleurin, ETV6, AXUD1, YOD1, MSI2, HLA-DMA, PRKCH, SNORD89, RAB11FIP1, YPEL5, TIAM2, CCNL1, MXI1, STAT3, CYLD, GNAQ, RNF19A, BMPR1B, TMEM63A, CCNL1, CD302, PER1, N4BP2L1, IER5, MLL, FBXW7, ST18, ARPC5L, HINT1, KIAA1466, AHR, PFKFB3, LATS2, FLT1, SOCS3, DUSP26, ARID3B, CTDSP2, CFP, CPEB4, BMPR1B, TTYH2, NECAP2, KLF9, ETV6, KLF7, STX3, TMC8, PHTF2, SEC14L1, LRRC8C, SOCS1, ASB13, IL1RAP, HNRNPD, CUGBP2, EIF1, S100Z, RIPK2, SV2A, ANKRD28, NFKBIZ, CMTM7, SPON1, FGFR1OP2, SDK2, STX3, TSPYL2, PSTPIP2, CLEC2B, ZBTB8, MARCKS, DDX3X, LRRFIP1, DENND4A, C6orf204, ATXN1L, GBP5, SPTAN1, CD36, FLJ43663, KBTBD8, C1orf71, ID2, IRAK3, WDR1, C1QTNF4, MMP28, ARPC5L, TFEC, PDE4B, KLF9, HEMGN, FAM129A, RBM39, RNF103, ARHGAP24, DNAJB6, USP15, SMEK2, PER1, HEMGN, CNN3, LRP5L, CD99, CDADC1, PELI1, FLJ46875, PRDM2, RBM14, BHLHE40, NUDT4, PPP1R15A, JDP2, FGFR1OP2, MLL, CISH, MBNL1, RNF10, PDE4B, RC3H2, FKBP4, CDKN1A, ADNP2, RAB31, SIAH2, PPP1R16B, REL, SBDS, KLHL15, CDADC1, STK4, C17orf91, ID2B, ZFAND5, SEMA6A, REV3L, MAFF, FAM13A1, NRXN3, GREM1, GBP5, MSI2, INPP5D, CLK1, PLXND1, RYK, TMEM70, NPDC1, MARCKS, RGPD4, RGPD5, RGPD6, RGPD8, CUL1, TLE4, MKNK1, ATXN1L, LATS2, NFKBIA, SDHA, SDHALP1, SDHALP2, SPON1, MLLT3, H2AFY, KLF13, PPP1R15A, RNF12, SEMA6A, CSF3R, MAP4K3, ARHGAP24, MMP28, HBS1L, AUTS2, C4orf15, RC3H2, NUDT4P1, ARF1, STAM, TP53INP1, STK17B, TLE4, GRB10, GADD45B, IFITM1, SBDS, PVRL2, C10orf18, NBEAL2, CREB5, IL8, NRXN3, FNIP1, MUC4, SERPINB9, UBE2D3, CD36, PCF11, ZFP36L2, UBE2H, AGPS, LRRFIP1, IFITM1, C5orf41, BCLAF1, FAM65B, CYTIP, RANBP2, MLLT3, PDE4B, C10orf54, DENND3, RANBP2, RGPD1, RGPD2, RGPD3, RGPD4, RGPD5, RGPD6, RGPD7, RGPD8, TP53INP1, RRAS2, MGLL, SLC2A3, NUDT4, H1F0, SOCS2, CYGB, H2AFY, AHCTF1, ARHGAP24, BIN2, MYO10, LYZ, ARF6, CYYR1, JUND, AGAP3, GADD45B, KLF7, LST1, UBE2E3, IGJ, GADD45A, HHIP, DUSP5, ARHGAP24, RAB31, TP53INP2, GLIPR1, USP53, PLXND1, RUNX3, SLC37A3, TTC39C, SHANK3, MXD1, IREB2, CD55, TRA2A, ARF1, NDFIP1, LST1, MDFIC, XIST, RUFY3, ID1, RAPGEF3, RTN4, HBS1L, FRMD8, MUC4, ALDH2, SAMSN1, GRAP, ICAM3, SF1, SMCHD1, WNT9A, STRAP, TARP, TRGC2, UPP1, TRAK2, CA1, UBE2H, MARCH8, TSC22D4, AGPS, CDKN2C, XIST, ANKRD28, ZFAND5, DUSP6, RYK, HBG1, HBG2, ZFP36L2, ATF3, SERTAD1, RAB31, DUSP6, RANBP2, LPIN1, NT5E, TXNIP, DNAJC1, G0S2, GREM1, RNF125, CTDSPL, RGPD5, RGPD6, RGPD8, AFF4, CD200, WHDC1, CCNH, RIN3, TUBB2A, HLA- DQA1, ARRDC3, FAM46C, CA2, TRBC1, TRBC2, FLJ14213, and/or CREG1. [0116] In some aspects, the genes of an Ikaros-repressed gene signature comprise one or more of TXNIP, CD36, CA2, YOD1, CFP, DENND3, YES1, NBEAL2, TMC8, PSTPIP2, CD97, DAAM1, NT5E, LYZ, SDK2, TSC22D4, GYPC, FAM129A, TPM3, GNAQ, and/or LUZP1. [0117] In some aspects, the B-NHL cells of the BTK inhibitor responder lack at least one transcript of an Ikaros-enhanced gene signature. [0118] In some aspects, the B-NHL cells of a TG-1701 responder lack at least one transcript of an Ikaros-enhanced gene signature. [0119] In some aspects, the genes of an Ikaros-enhanced gene signature comprise one or more of ZHX2, GLT8D1, FLI1, TCL1A, ZFP36L1, BDH2, HNRNPA0, EFTUD1, PLEKHA2, C14orf142, BUB3, YWHAB, QRSL1, CCND3, FMNL2, MGC3032, TTRAP, KIAA1430, CUTC, MRPL46, DBN1, CD22, BCORL1, GINS3, UBE2V2, AEBP1, BTK, HDDC2, PDHB, C21orf59, FAIM, CAMK2D, VTA1, EIF2S1, VRK1, UBLCP1, LOC93622, SEPHS1, PPID, C1orf59, ARPP-21, LMNB1, BACH2, LOC116236, DPY19L3, SDCCAG10, CCT5, PDIA6, PPP2R5C, CECR5, LOC90925, GINS3, PDE4A, AHCYL1, BID, MRFAP1L1, ARL11, FAM98B, ALDH6A1, ZFP36L1, PTGER4, PFDN6, IGLL3, SLC35F2, NCF4, ZEB2, OPN3, ECHDC1, BACH2, DAAM1, ECHDC1, PFDN6, PLCG2, APOBEC3G, MSH2, IL28RA, ALDH18A1, EIF2S1, EEF1E1, NCF4, MSH6, OPN3, SLC16A1, C5orf33, DYNLL1, RALA, MAP3K1, SAC3D1, KIAA0802, SUPT16H, PHYH, MRPL13, VPS35, SFRS7, EHD3, C21orf45, TBC1D4, MRPS31, AFF3, FMNL2, C5orf13, DHFR, ERGIC1, MAGEH1, BCL2, PGM2, FADS3, ZFX, RASGRP1, MARCKSL1, TRAC, CLTC, P4HA2, MSH6, C14orf119, TNS3, KIAA0746, SERINC2, SORD, PRAGMIN, HS6ST2, IGLL1, FABP5, FABP5L2, FABP5L7, SMARCC1, BAHCC1, HMGB3, RSRC1, CAPN3, BLK, CBX5, HS6ST2, CTNNBL1, CDCA7, MTHFD2, RASSF4, and/or QDPR. [0120] In some aspects, the genes of an Ikaros-enhanced gene signature comprise one or more of TCL1A, CBX5, HNRNPA0, PDHB, BCL2, DYNLL1, SUPT16H, CAMK2D, ALDH6A1, PPP2R5C, ERGIC1, BUB3, SORD, SEPHS1, CTNNBL1, CCT5, and/or APOBEC3G. [0121] Throughout the present disclosure, unless otherwise indicated, standard gene symbols, such as those listed above and in Tables 5 and 7, found in community databases specific to human proteins (e.g., www.genenames.org) have been utilized. One skilled in the art would be familiar with these databases in order to identify a particular standard gene name (or the protein it encodes) from its standard gene symbol. [0122] In some aspects, the quantity of a transcript of an Ikaros-repressed gene and/or a transcript of an Ikaros-enhanced gene is measured using various suitable methods known in the art, including, e.g., amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.); hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.); and sequencing- based methods (e.g., next-generation sequencing (NGS) methods, for example, using the Illumina or Ion Torrent sequencing platforms). [0123] In some aspects, the quantity of transcripts is determined by at least one amplification-based method. In some aspects, the amplification-based method is Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification. In a preferred aspect, the amplification-based method is qPCR. [0124] In some aspects, the quantity of transcripts is determined by at least one non- amplification-based method. In some aspects, the non-amplification-based method is a hybridization-based method or a sequencing-based method. In some aspects, the hybridization-based method is a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, or in situ hybridization. In some aspects, the sequencing-based method is a next-generation sequencing (NGS) method. [0125] In some aspects, the quantity of transcripts is determined by a combination of amplification-based and non-amplification-based methods. [0126] In some aspects, prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of B-NHL cells of the subject. In some aspects, prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of the B-NHL cells of the subject. [0127] In some aspects, prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring in a sample of B-NHL cells of the subject, the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature, wherein the at least one transcript is either not detectable in the B-NHL cells of the subject or is measured at levels lower than in the B-NHL cells of a subject that is not a BTKi responder. [0128] In some aspects, prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring in a sample of B-NHL cells of the subject, the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature, wherein the at least one transcript is either not detectable in the B-NHL cells of the subject or is measured at levels lower than in the B-NHL cells of a subject that is not a TG-1701 responder. [0129] In some aspects, prior to initiation of treatment with a BTKi, the subject has been determined to be responsive to a BTKi according to a method comprising measuring in a sample of B-NHL cells of the subject the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature and the amount of at least one transcript of a gene of an Ikaros-repressed gene signature. [0130] In some aspects, prior to initiation of treatment with TG-1701, the subject has been determined to be responsive to TG-1701 according to a method comprising measuring in a sample of B-NHL cells of the subject the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature and the amount of at least one transcript of a gene of an Ikaros-repressed gene signature. [0131] In some aspects, the subject has not been treated for B-NHL prior to quantifying the amount of at least one transcript of a gene of an Ikaros-repressed gene signature and at least one transcript of a gene of an Ikaros-enhanced gene signature in the B-NHL cells of the subject. [0132] In some aspects, the subject has been treated for B-NHL prior to quantifying the amount of at least one transcript of a gene of an Ikaros-repressed gene signature and at least one transcript of a gene of an Ikaros-enhanced gene signature in the B-NHL cells of the subject. [0133] In some aspects, the subject has been treated with a compound and/or a combination of compounds including, but not limited to, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) without or with a monoclonal antibody, including, but not limited to, rituximab (R-CHOP); dose-adjusted etoposide, doxorubicin and cyclophosphamide with vincristine, prednisone and rituximab (DA-EPOCH-R); cyclophosphamide, vincristine, prednisone (CVP); cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) alternating with rituximab and cytarabine; cyclophosphamide, vincristine, doxorubicin, and high-dose methotrexate (CODOX-M) alternating with ifosfamide, etoposide, and cytarabine; etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH); chlorambucil or cyclophosphamide with rituximab; lenalidomide with rituximab; chlorambucil, fludarabine, bendamustine without or with rituximab or obinutuzumab; ibrutinib without or with rituximab; bortezomib; carfilzomib; ixazomib; oprozomib; cladribine; fludarabine; pentostatin; lenalidomide; ibrutinib; acalabrutinib; zanubrutinib; ibrutinib, acalabrutinib, zanubrutinib; ACP-196; AVL-292; venetoclax; idelalisib; buparlisib; alisertib; duvelisib; rituximab; pembrolizumab; obinutuzumab; as ulocuplumab; radioactive monoclonal antibody ibritumomab; selinexor; everolimus; temsirolimus; panobinostat; romidepsin; belinostat; radiation; stem cell transplant; or CAR T-cell therapy including, but not limited to, brexucabtagene autoleucel. [0134] In some aspects, the methods according to the invention comprise administering to a subject a therapeutically effective amount of a BTKi. [0135] In some aspects, the BTKi is ibrutinib, zanubrutinib, acalabrutinib, evobrutinib, tirabrutinib, fenebrutinib, pirtobrutinib, GS-4059 (NCT02457598), spebrutinib, HM71224, SNS-062, ABBV-105, LCB 03-0110 dihydrochloride, LFM-A13, PCI 29732, PF 06465469, M7583 (NCI Code C129710), or (-)-Terreic acid or a BTK degrader including DD 03-171. [0136] In some aspects, the methods according to the invention comprise administering to a subject a therapeutically effective amount of the BTK inhibitor TG-1701. [0137] In some aspects, TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d. In some aspects, TG-1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d. In some aspects, TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d. [0138] In some aspects, TG-1701 is administered in combination with an anti-CD20 antibody. In some aspects, the anti-CD20 antibody is rituximab, obinutuzumab, ofatumumab, or ublituximab. In some aspects, the anti-CD20 antibody is ublituximab. [0139] In some aspects, TG-1701 is administered in combination with a dual PI3Kδ and casein kinase-1ε inhibitor. In some aspects, the dual PI3Kδ and casein kinase-1ε inhibitor is umbralisib. [0140] In some aspects, TG-1701 is administered with a PI3Kδ inhibitor, such as, e.g., duvelisib, idelalisib, zandelisib, or copanlisib. [0141] In some aspects, TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d in combination with an anti-CD20 antibody. In some aspects, TG-1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d in combination with an anti-CD20 antibody. In some aspects, TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d in combination with an anti-CD20 antibody. [0142] In some aspects, TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d in combination with umbralisib. In some aspects, TG-1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d in combination with umbralisib. In some aspects, TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d in combination with umbralisib. [0143] In some aspects, TG-1701 is administered at a dose of about 50 mg/d to about 800 mg/d in combination with an anti-CD 20 antibody and umbralisib. In some aspects, TG- 1701 is administered at a dose from about 60 mg/d to about 700 mg/d; about 70 mg/d to about 600 mg/d; about 80 mg/d to about 500 mg/d; about 90 mg/d to about 450 mg/d or about 100 mg/d to about 400 mg/d in combination with an anti-CD20 antibody and umbralisib. In some aspects, TG-1701 is administered at about 100 mg/d, about 150 mg/d, about 200 mg/d; about 250 mg/d, about 300 mg/d, about 350 mg /d, or about 400 mg/d in combination with an anti-CD20 antibody and umbralisib. [0144] In some aspects, umbralisib is administered at a dose from about 200 mg/d to about 1000 mg/d, about 250 mg/d to about 900 mg/d, about 300 mg/d to about 850 mg/d, about 350 mg/d to about 800 mg/d, about 400 mg/d to about 750 mg/d, about 450 mg/d to about 700 mg/d, about 500 mg/d to 650 mg/d, or about 400 mg/d, about 600 mg/d or about 800 mg/d. [0145] In some aspects, the anti-CD20 antibody is administered at 500 mg/d to about 1200 mg/d, or about 600 mg/d to about 1000 mg/d, about 700 mg/d to about 900 mg/d, or about 650 mg/d, 700 mg/d, 750 mg/d, 800 mg/d, 850 mg/d, or 900 mg/d. [0146] In some aspects, TG-1701 is administered on a daily schedule. In some aspects, TG-1701 is administered twice a day, three times a day, or four times a day. In some aspects, TG-1701 is administered every other day. In some aspects, TG-1701 is administered once every three days. In some aspects, TG-1701 is administered on a weekly schedule. In some aspects, TG-1701 is administered on a once every two weeks schedule. In some aspects, TG-1701 is administered on a once every three weeks schedule. In some aspects, TG-1701 is administered on a once every four weeks schedule. [0147] In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered once a day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered twice a day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered three times a day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered four times a day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered every other day. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered every three days. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered once a week. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered on a once every two weeks schedule. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered on a once every three weeks schedule. In some aspects, TG-1701, the anti-CD20 antibody and/or umbralisib are administered on a once every four weeks schedule. [0148] In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered once every other day. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered once every three days. In some aspects, TG-1701 is administered every day and the anti- CD20 antibody and/or umbralisib are administered on a weekly schedule. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every two weeks schedule. In some aspects, TG- 1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every three weeks schedule. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every four weeks schedule. In some aspects, TG-1701 is administered every day and the anti-CD20 antibody and/or umbralisib are administered on a once every eight weeks schedule. [0149] In some aspects, TG-1701 is administered every other day and the CD20 antibody and/or umbralisib are administered every day. In some aspects, TG-1701 is administered every other day and the CD20 antibody and/or umbralisib are administered once every three day. In some aspects, TG-1701 is administered every other day and the anti-CD20 antibody and/or umbralisib are administered once a week. In some aspects, TG-1701 is administered every other day and the anti-CD20 antibody and/or umbralisib are administered once every two weeks. In some aspects, TG-1701 is administered every other day and the anti-CD20 antibody and/or umbralisib are administered once every three weeks. In some aspects, TG-1701 is administered every other day and the anti- CD20 antibody and/or umbralisib are administered once every four weeks. [0150] In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered every day. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered every other day. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once every three days. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once a week. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once every two weeks. In some aspects, TG-1701 is administered every three days and the anti-CD20 antibody and/or umbralisib are administered once every three weeks. In some aspects, TG-1701 is administered every three days and the anti- CD20 antibody and/or umbralisib are administered once every four weeks. [0151] In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered every day. In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered every other day. In some aspects, TG-1701 is administered once a week and the anti- CD20 antibody and/or umbralisib are administered every three days. In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered once every two weeks. In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered once every three weeks. In some aspects, TG-1701 is administered once a week and the anti-CD20 antibody and/or umbralisib are administered once every four weeks. [0152] In some aspects, TG-1701 is administered in combination with an anti-CD20 antibody and umbralisib, wherein TG-1701 is administered daily, every other day, every three days, once a week, once every two weeks, once every three weeks or once every four weeks and the anti-CD20 antibody is administered daily, every other day, every three days, once a week, once every two weeks, once every three weeks or once every four weeks and umbralisib is administered daily, every other day, every three days, once a week, once every two weeks, once every three weeks or once every four weeks. Methods of Identifying a BTKi Responder [0153] Also provided are methods of identifying a subject that suffers from B-cell non- Hodgkin lymphoma (B-NHL) as a BTKi responder. In a preferred aspect, the BTKi is TG-1701 and the subject is a TG-1701 responder. [0154] In some aspects, the method comprises quantifying at least one phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1 (Ikaros). [0155] In some aspects, the method comprises quantifying at least one protein that comprises a phosphopeptide selected from SEQ ID NO: 1-95 in the B-NHL cells of the subject. In some aspects, the at least one protein comprises at least one phosphopeptide is SEQ ID NO: 1. [0156] In some aspects, the presence of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis. [0157] In some aspects, the phosphopeptide and/or phosphopolypeptide quantification is performed in B-NHL cells isolated from blood samples, biopsy samples and/or bone marrow aspirates. The percentage of circulating B-NHL cancer cells in the samples of the subject can be from about 40% to about 98%. [0158] In some aspects, the method comprises, prior to initiation of treatment with a BTKi, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. In some aspects, the method comprises, prior to initiation of treatment with TG-1701, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. [0159] In some aspects, the method comprises, after initiation of treatment with a BTKi, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. In some aspects, the method comprise, after initiation of treatment with TG-1701, measuring the amount of phosphopeptides present in a sample of B-NHL cells of the subject. [0160] In some aspects, the method comprises providing B-NHL cells of a subject comprising polypeptides, extracting the polypeptides and enriching phosphopolypeptides from the polypeptide extract. In some aspects, peptides are prepared from the extracted phosphopolypeptides. In some aspects, the peptides are prepared using enzyme digestion. In some aspects, the phosphopolypeptides are enriched using immunoprecipitation, metal affinity chromatography, metal-oxide affinity chromatography, Phos-Tag chromatography, polymer-based metal ion affinity capture, hydroxyapatite chromatography, enrichment by chemical modification, and/or phosphopolypeptide precipitation. [0161] In some aspects, the enriched phosphopolypeptides are subjected to western blot or flow cytometry phosph-flow analysis using antibodies that bind to at least one polypeptide comprising at least one phosphopeptide selected from SEQ ID NO: 1-95. [0162] In some aspects, the method comprises quantifying at least one transcript of an Ikaros-repressed gene and/or at least one transcript of an Ikaros-enhanced gene by measured transcript quantity using various methods known to those skilled in the art, including, e.g., amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.); hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.); and sequencing- based methods (e.g., next-generation sequencing (NGS) methods, for example, using the Illumina or Ion Torrent sequencing platforms). [0163] In some aspects, the method comprises, prior to initiation of treatment with a BTKi, measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of B-NHL cells of the subject. In some aspects, the method comprises, prior to initiation of treatment with a BTKi, measuring the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature in a sample of the B-NHL cells of the subject. [0164] In some aspects, the method comprises, after initiation of treatment with a BTKi, measuring the amount of at least one transcript of a gene of an Ikaros-repressed gene signature in a sample of B-NHL cells of the subject. In some aspects, the method comprises measuring prior to initiation of treatment with a BTKi, in a sample of B-NHL cells of the subject the amount of at least one transcript of a gene of an Ikaros-enhanced gene signature, wherein the at least one transcript is either not detectable in the B-NHL cells of the subject or is measured at levels lower than in the B-NHL cells of a subject that is not a BTKi responder. Kits [0165] In some aspects, the present disclosure provides a kit comprising a combination of antibodies and, optionally, reagents packaged in a manner that facilitates their use to practice the methods of the present disclosure. [0166] In some aspects, a kit comprises: (i) at least one antibody that binds to at least one phosphopeptide selected from SEQ ID NOs: 1-95; (ii) optionally, reagents to perform a western blot analysis; and/or reagents to perform a phospho-flow analysis; and (iii) instructions for treating B-NHL in a TG-1701 inhibitor responder according to any of the methods described herein. In some aspects, the at least one phosphopeptide is SEQ ID NO: 1 (Ikaros). [0167] In some aspects, the kit further comprises reagents to perform a Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification. [0168] In some aspects, the kit further comprises reagents to perform a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, next-generation sequencing (NGS) method, or in situ hybridization. [0169] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES PATIENTS, MATERIALS AND METHODS Patients [0170] Blood samples were obtained from six CLL patients treated in the NCT03671590 phase I clinical trial (Table 3). Relapsed/refractory patients with either del(17p) or TP53 mutations, or over the age of 65 years were eligible for the trial and treated with TG- 1701, orally once daily until disease progression or the occurrence of intolerable side effects. Patients received either 200 mg, 300 mg, or 400 mg TG-1701 daily (Table 3). [0171] Peripheral blood mononuclear cells (PBMC) from the six CLL patients were obtained at cycle 1, day 1 (C1D1) pretreatment and 4 hours post treatment. [0172] On the first day of treatment, white blood cells were counted (WBC), the absolute lymphocyte count (ALC) calculated, and the individual normal lower and upper limits for ALC were calculated (Table 4). The percentage of CLL cells in each sample was the ratio of ALC subtracted from highest normal ALC and divided by total ALC. Cell Lines [0173] Seven MCL (REC-1GFP+LUC+, Jeko-1, UPN-1, UPN-IbruR, Granta-519, Z-138 and Mino-1), two ABC-DLBCL (OCI-Ly3 and HBL-1), and two FL (DoHH2 and RL) cell lines were cultured as described elsewhere (Balsas, P et al., J Hematol Oncol 10:80 (2017); Body, S. et al., Sci. Rep.7: 13946 (2017)). All cultures were routinely tested for Mycoplasma infection by PCR and the identity of all cell lines was verified by using an AmpFISTR identifier kit (Thermo Fisher). Generation of BTKC481S and BTKKO cell lines by CRISPR-Cas9 [0174] The generation of a CRISPR-Cas9 gene editing tool was employed to edit the REC-1GFP+LUC+ cell line to create either a BTK knockout and also to insert a C to S point mutation at 481 residue in BTK protein.0.5 x 106 REC-1GFP+LUC+ cells were electroporated with 36 pmol SpCas9 Nuclease V3, 44 pmol CRISPR-Cas9 tracRNA, ATTO 550, 44 pmol Alt-R CRISPR-Cas9 crRNA Hs.Cas9.BTK.1.AC (BTKKO 5´- ATGAGTATGACTTTGAACGTGGG-3`) (SEQ ID NO: 214), 44 pmol CRISPR-Cas9 crRNA XT BTK (BTKC481S 5’-GUAGUUCAGGAGGCAGCCAU-3’) (SEQ ID NO: 215), 44 pmols Alt-R Cas9 Electroporation Enhancer, and 36 pmols BTK Ultramer DNA Oligo (BTKC481S
Figure imgf000039_0001
(SEQ ID NO: 216) (IDT-Integrated DNA Technologies). Electroporation was performed using Neon Transfection System (Thermo Fisher) at 1600 V, 20 ms, and 10 μL tip. Cells were then plated in a 24-well plate for 10 days, changing the culture medium every 3 days. Protein lysates were collected from REC-1 BTKKO cells to assess BTK levels by Western blot. REC-1 BTKC481S cells were resuspended in 96-well plates with a limiting dilution of 0.3 cells per well. After 20 days, REC-1 BTKC481S single clones were analyzed by PCR amplification with BTK primers
Figure imgf000039_0002
Figure imgf000039_0003
Figure imgf000039_0004
and digestion with EaeI (NEB) restriction enzyme. Positive clones were validated by Sanger Sequencing. Drugs [0175] TG-1701 was provided by TG Therapeutics, Inc. Ibrutinib was purchased from Selleckchem. Xenograft mouse model and immunohistochemical staining [0176] In the Mino MCL xenograft model, tumor-bearing 6-7 week-old nude mice (Shanghai Ling Chang experimental animal Co., Ltd.) were dosed orally with TG-1701 (25, 50, or 100 mg/kg, orally, twice a day (bid)), ibrutinib (100 mg/kg, orally, bid), or vehicle for 21 days. In REC-1 and UPN-IbruR xenografts, CB17-SCID mice (Janvier Labs) were inoculated subcutaneously with 107 REC-1GFP+Luc+ cells or UPN-IbruR cells and monitored for tumor growth, bioluminescence signal, and vital parameters as previously described (Body, S et al., Sci Rep.7:13946 (2017)). Tumor-bearing mice received either TG-1701 (25 mg/kg, qw/qd) or ibrutinib (25 mg/kg, qd) for 17 days (qd = each day; qw = each week). Tumor samples were snap-frozen in OCT medium (Sakura Tissue Tek) or formalin-fixed and paraffin-embedded prior to immunohistochemical staining with primary antibodies against Ikaros (Cell Signaling Technology), CD20 (Beckman Coulter), NCAM (also known as CD56), and granzyme B (Abcam). Preparations were evaluated using an Olympus microscope and MicroManager software. Western Blot [0177] Total and nuclear protein extracts were obtained from MCL cell lines and tumor specimens using RIPA (Sigma-Aldrich), the Nuclear/Cytosol Fractionation Kit (BioVision), and T-PER (Thermo Scientific) buffers, respectively, and subjected to SDS- PAGE, as previously described (Pérez-Galán, P et al., Blood 117:542-552 (2011); Esteve-Arenys, A et al., Oncogene 37:1830-1844 (2018)). [0178] Membrane-transferred proteins were revealed by incubating with primary antibodies against p-BTK-Tyr223, BTK, p-ERK1/2, p-PLCγ2, PLCγ2, p-AKT-Ser473, AKT, Ikaros, MYC (Cell Signaling Technology), ERK1/2 and IRF4 (Santa Cruz Biotechnology), and appropriate anti-rabbit (Cell Signaling) or anti-mouse (Sigma- Aldrich) secondary antibodies followed by chemiluminescence detection using the ECL system (Pierce) and a Fusion FX imaging system (Vilber Lourmat). Anti-GAPDH (Santa Cruz) detection was carried out to check protein loading. Proteomic and Phosphoproteomic Profiling in Patients [0179] Peripheral blood mononuclear cells (PBMCs) were obtained from a total of six CLL patients before (PRE) and after 4 hour treatment (POST) with TG-1701 (Table 3) using standard Ficoll-Hypaque density gradient. Proteins were extracted by adding a Urea-based buffer (6M Urea, 100mM Tris-HCl pH 7.5), followed by sonication in a bioruptor (10 ON/OFF cycles, 30s each). The supernatants were recovered by centrifugation (15,000g for 10min at 4ºC) and the proteins precipitated by adding 100% trichloroacetic acid (TCA) for 1h at 4ºC. The resulting proteins pellets were recovered by centrifugation at 15,000g for 5 min at 4ºC and subsequently washed twice with chilled acetone (30 min each). The pellets were recovered and then resuspended in 6M Urea, 100mM Tris-HCl pH 7.5. Finally, the samples were quantified with the RCDC® Protein Assay Kit (Biorad). All the proteins samples were sequentially digested with Lys-C (1:25, enzyme-to-protein ratio for 18h at 30ºC) and Trypsin (1:25 for 8 hours at 30ºC). Prior to digestion, the samples were reduced and alkylated with dithiothreitol (DTT) and carbamydomethylated with chloroacetamide (CAA), respectively. The enzymatic digestions were stopped with formic acid (FA) (10%, final concentration). Finally, the peptides were desalted with a reverse phase C18 chromatography, dried in a speedvac and kept at -80ºC until further use. [0180] The proteomics quantitative analyses were performed using thirteen (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N and 132C) of the sixteen channels available in a 16plex - Tandem Mass Tag (TMT) system. The labelling was performed according to the manufacturer instructions (Thermo Fisher). Finally, all the channels were mixed in one single tube obtaining a total of 2.21 mg of TMT-labeled peptides. A small part of the sample (260 µg) was used for total proteome analysis. The remaining proteins were subjected to phosphoproteome enrichment using the High- Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher) according to the manufacturer instructions. Before injecting the samples into the mass spectrometer, both the total proteome and phosphopeptide fractions were fractionated in 24 and 9 fractions using a Zorbax Extent-C18 (2.1 x 150 mm 3.5 µm 300Å) column and the High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher, MA) kit, respectively. Finally, the samples were dried in a speedvac and analyzed in an Orbitrap Fusion Lumos™ Tribrid mass spectrometer. For this aim, samples were loaded to 300 µm × 5 mm C18 PepMap100, 5µm, 100Å (Thermo Scientific) at a flow rate of 15 µL/min using a Thermo Scientific Dionex Ultimate 3000 chromatographic system (Thermo Scientific). Peptides were separated using a C18 analytical column (nanoEaseTM M/Z HSS C18 T3 (75 µm × 25 cm, 100Å, Waters) with a 150 min run, comprising three consecutive steps with linear gradients from 3% to 35 % B in 120 min, from 35 % to 50 % B in 5 min, from 50 % to 85% B in 2min, followed by isocratic elution at 85 % B in 5 min and stabilization to initial conditions (A= 0.1% FA in water, B= 0.1% FA in CH3CN) at 250 nL/min flow rate. The column outlets were directly connected to an Advion TriVersa NanoMate (Advion) fitted on an OrbitrapFusion Lumos™ Tribrid mass spectrometer (Thermo). The mass spectrometer was operated in a data-dependent acquisition (DDA) mode. Survey MS scans were acquired in the orbitrap with the resolution (defined at 200 m/z) set to 120,000. The lock mass was user-defined at 445.12 m/z in each Orbitrap scan. The top speed (most intense) ions per scan were fragmented by HCD. The MSMS was detected in the Orbitrap (with 30,000 resolution). The ion count target value was 400,000 for the survey scan and 10,000 (CID) for the MS/MS scan. Target ions already selected for MS/MS were dynamically excluded for 15 s. Spray voltage in the NanoMate source was set to 1.70 kV. RF Lens were tuned to 30%. Minimal signal required to trigger MS to MS/MS switch was set to 5000 and activation Q was 0.250. The spectrometer was working in positive polarity mode and singly charge state precursors were rejected for fragmentation. Data was acquired with Xcalibur software vs 4.0.27.10 (Thermo Scientific). [0181] For data analysis, MaxQuant software (1.6.7.0) and its built-in search engine Andromeda were used to search the .raw files against a Swisprot/Uniprot human database downloaded from the www.uniprot.org web site (October 2019). A target and decoy database were used to assess the false discovery rate (FDR) which was set to 1% at both peptide and protein level. Trypsin was chosen as enzyme and a maximum of two missed cleavages were allowed. Carbamidomethylation (C) was set as fixed modification, whereas oxidation (M), acetylation (N-terminal) and phosphorylation (STY) were used as variable modifications. The reporter ions were quantified at MS2. Searches were performed using a peptide tolerance of 7 ppm and a product ion tolerance of 0.5 Da. Proteins and peptides classified as “Identified Only by Site”, “Potential Contaminant” and “Reverse” were removed from the final list. The total proteome analysis was performed using only the proteins quantified in all the samples, while for phosphoproteome, we used the peptides quantified in more than 80% of the samples. The missing values were imputated by using the kNN algorithm (k = 10). The differential analyses were performed by using the R package called “limma” (Ritchie, ME et al., Nucleic Acids Res 43(7):e47 (2015)) considering paired samples. The normalization of both total proteome and phosphoproteome was done using the median. Additionally, the phosphoproteome was normalized using the R package called “phosphonormalizer” (Saraei, S et al., Bioinformatics 34(4):693-694 (2018)). Mass Spectrometer Data Acquisition in Cell Lines [0182] REC-1 and REC-BTKC481S protein extracts were labelled in triplicates with two TMT10plex, using a single channel (131N) to label a pool of all the samples. TMT fractionation and MS analysis were performed as above. Raw data were processed in MaxQuant and analyzed. The mass spectrometer was operated in a data-dependent acquisition (DDA) mode. In each data collection cycle, one full MS scan (350-1800 m/z) was acquired in the Orbitrap (1.2 x 105 resolution setting and automatic gain control (AGC) of 2 x 105). The following MS2-MS3 analysis was conducted with a top speed approach. The most abundant ions were selected for fragmentation by collision induced dissociation (CID). CID was performed with a collision energy of 35%, 0.25 activation Q, an AGC target of 1 x 104, an isolation window of 0.7 Da, a maximum ion accumulation time of 50 ms and turbo ion scan rate. Previously analysed precursor ions were dynamically excluded for 30 s. For the MS3 analyses for TMT quantification, multiple fragment ions from the previous MS2 scan (SPS ions) were coselected and fragmented by HCD using a 65 % collision energy and a precursor isolation window of 2 Da. Reporter ions were detected using the Orbitrap with a resolution of 30,000, an AGC of 1 x 105 and a maximum ion accumulation time of 120 ms. RNA-Seq Analysis and Real-Time qPCR [0183] Total RNA was extracted using TRIZOL (Thermo Fisher) following manufacturer’s instructions and Poly-A-tailed enriched mRNA selected. Paired-end Stranded RNA libraries with 51 read-inward facing paired mates were prepared, following sequencing with Illumina’s NovaSeq6000 at the Centro Nacional de Analisis Genomico (CNAG). [0184] The reverse transcription (RT) reaction was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). The mRNA expression was analyzed in triplicate by quantitative real-time PCR. Amplification was performed using SYBR Green-based detection (GoTaqPCR Master Mix; Promega). The relative expression of each gene was quantified by the comparative cycle threshold method (ΔΔCt). β-actin, GAPDH, and B2M were used as endogenous controls. RNA-Seq Data Analysis [0185] Quality Control (QC) of all samples was performed with the publicly available software FASTQC. For a more optimal visual inspection, these analyses were concatenated with MultiQC. Trimming of unwanted adapter sequences were filtered out using FASTP and CutAdapt. Gene-transcript level quantification of all samples was performed using Salmon (Version1.3.0) in the pseudoalignment mode and with default settings (hg19). DESeq2 (Version 1.24.0) was used for Salmon’s raw count normalization. To estimate the effect of (Post- vs Pre-) Treatment in Responders (Rs) vs. Non Responders (NRs), a suitable design matrix was built, where we made use of a complex interaction term with nested groups, following a DESeq2 tutorial. Finally, differential gene expression (DGE) between the above described groups (Rs vs. NRs) was calculated. Heatmaps representing both enhanced and repressed IKAROS/IKZF1 gene signatures were built as previously described (Díaz, T et al., Haematologica 102 (2017)). All the statistical analyses were performed in Responders (R). Immunofluorescence [0186] BTKi-treated REC-1 cells (2–3 × 105) were seeded on poly-L-lysine-coated glass coverslips and stained as previously described (Esteve-Arenys, A et al., Oncogene 37:1830-1844 (2018)) with anti-Ikaros antibody (Cell Signaling Technology). Fluorescence signal was acquired on a Leica microscope and quantified using the LAS X (Leica) and Image J softwares. Pharmacokinetic (PK) Analysis [0187] TG-1701 plasma concentrations were assessed using a GLP LC/MS/MS method developed at North East Bio Analytical Laboratories. Binding and Enzymatic Assays [0188] The binding of TG-1701 (1 µM) and ibrutinib (1 µM) to a panel of 453 kinases was determined using a quantitative binding assay (KINOMEscan, DiscoverX, Eurofins). TG-1701 and ibrutinib inhibitory activities were also tested on BTKwt and BTKC481S kinase activity at ReactionBio in an enzymatic filtration assay using 33P-ATP. Occupancy Assay [0189] The in vitro occupancy assay was developed and performed by Jiangsu Hengrui Medicine Co. at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences in Shanghai. Briefly, the DoHH2 BTK-expressing cells were incubated with ibrutinib or TG-1701 (0.1 to 100 nM) for 1 hour and cell lysates were subjected to SDS-PAGE, followed by incubation with a fluorescent-ibrutinib probe or an anti-BTK antibody. The BTK occupancy method for the detection of free BTK in patient lysates was developed and run at Cambridge Biomedical Laboratories (now Bioagilytix). For all time points, PBMC pellets (2 x 106 cells) were lysed in 100 μL ice cold lysis buffer PBS with 0.1% Nonidet P-40 supplemented with 1X protease inhibitor cocktail (Sigma-Aldrich). Samples were prepared in triplicate. Cell lysates were incubated with the BTK occupancy probe biotin-ibrutinib (MedChemExpress) for 1h and plated onto a streptavidin-coated plate. Two hours of incubation were followed by washing and an hour incubation with an anti- BTK antibody (Becton Dickinson) in PBS + 0.05% Tween-20 and 0.5% BSA. After washing, plates are incubated for an 1h with a goat secondary antibody-sulfoTAG (Meso Scale Discovery, MSD). [0190] The percentage of BTK protein bound by TG-1701 was calculated as indicated in FIG.8. Total BTK was assessed using the biotinylated rabbit anti-human BTK monoclonal antibody (D3H5, Cell Signaling Technologies) bound to the streptavidin plate, in a classic sandwich ELISA. Both free and total BTK was assessed using the MSD electrochemoluminescent signal on a MESOquickplex SQ120 device. ADCC and ADCP Assays [0191] ADCC was evaluated by measuring the release of lactate dehydrogenase (LDH) into the medium after treatment using the Cytotoxicity Detection KitPLUS (Roche). Briefly, PBMCs were purified by standard Ficoll-Hypaque gradient centrifugation of healthy human peripheral blood. Target cells were opsonized with 2 µg/mL ublituximab or an irrelevant hIgG1 in combination with 1 µM ibrutinib/TG-1701, for 1 hour at 37°C. Next, 104 target cells were mixed with 105 PBMCs effector cells. The cell mixture was incubated for 4 h at 37°C.100 µL supernatant was then incubated with LDH substrate reagent for 30 min in the dark. LDH release from target cells was quantified by measuring absorbance at 490 nm. Negative controls (spontaneous LDH release) consisted of target cells incubated with medium in the absence of effector cells. Total lysis control consisted of target cells incubated with 5 µL of a cell lysis solution (Triton X-100). Nonspecific lysis control (baseline) consisted of target cells incubated with effector cells, without any antibody addition. The ADCC percentage was calculated using the following formula: % ADCC = 100 x [(sample – non-specific lysis control) / (total lysis control – neg control)]. [0192] ADCP assays were evaluated by flow cytometry. Briefly, initially, macrophages were differentiated from healthy donors’ PBMCs (2 x 106 cells/mL) cultured in complete RPMI medium supplemented 20 ng/mL recombinant human M-CSF (Peprotech) for 8 days. The density and shape of the macrophages was assessed by microscopic analysis (long and stretched shape). Malignant B cells were then CFSE-labeled and opsonized with an anti-CD20 mAb (ublituximab or rituximab, 2 µg/ml) or with an irrelevant hIgG1, in combination with the indicated BTKi at 1 µM, for 1 hour at 37°C. Those cells were added to macrophages with an E:T ratio of 1:5. The cell mixture was incubated for 2.5 hours at 37°C and cells were analyzed on a Navios flow cytometer (Beckman Coulter) after macrophage staining with an anti-human CD14 antibody (Beckman Coulter). The percentage of phagocytosis is defined as the percentage of macrophages that have engulfed at least one target malignant cell. CellTiter-Glo (CTG) Viability Assay [0193] Cell viability was performed using a CellTiter-Glo luminescent cell viability assay (Promega), according to the manufacturer’s instructions. Briefly, 5 x 104 cells/well were cultured in sterile 96-well plates in the presence of increasing concentrations of TG-1701 or ibrutinib (100 nM to 100 µM) in RPMI 10% FBS medium. The plates were incubated for 72 h (unless otherwise specified) and 100 µL of CellTiter-Glo reagent were added. After a 10-minute incubation at room temperature, luminescence was recorded with an integration time of 1 second per well. DMSO was used as control. Statistical Analysis [0194] Presented data in the Examples below are the means ± SD or SEM of three independent experiments. All statistical analyses were done using GraphPad Prism 4.0 software (GraphPad Software). Comparison between two groups of samples was evaluated by a nonparametric Mann–Whitney test to determine how response is affected by two factors. A Pearson test was used to assess statistical significance of correlation. Results were considered statistically significant when p value < 0.05. Example 1: TG-1701 is a novel irreversible BTK inhibitor, more selective than ibrutinib [0195] This Example shows that TG-1701 exerts similar activity to the first-in-class BTK inhibitor, ibrutinib, but with greater selectivity, in in vitro and in vivo models of B-NHL. [0196] In a binding assay on a panel of 441 human kinases, TG-1701 was more selective than ibrutinib, with a comparable BTK Kd (3 nM vs 1.5 nM, respectively) and a lower binding to EGFR, ITK, TXK, and JAK3 (Kd 135-, >48-, 68- and >94-fold higher than those of ibrutinib, respectively, FIG.1A and Table 1). Table 1: TG-1701 and ibrutinib Kds (nM)
Figure imgf000047_0001
[0197] A BTK kinase activity assay revealed a TG-1701 EC50 of 6.70 nM, slightly higher than ibrutinib IC50 (1.65 nM, FIG.1B and Table 2). Table 2: Compared kinase and growth inhibitory activity of TG-1701 and ibrutinib
Figure imgf000047_0002
Figure imgf000048_0001
[0198] Accordingly, in an in vitro BTK occupancy assay, TG-1701 and ibrutinib showed a similar dose-dependent capacity to displace a BTK-specific fluorescent probe in the FL cell line DoHH-2, with complete BTK occupancy at 30 nM and 10 nM, respectively (FIG.1C). Consistently, BCR downstream signaling was impaired in a concentration- dependent manner in IgM-stimulated cells, with maximal effects observed at 100 nM for both BTKis (FIG.1D). These effects were associated with a mean TG-1701 GI50 of 6.6 µM at 72 hours, in a set of 9 parental B-NHL cell lines, which was slightly inferior to mean ibrutinib GI50 (14.9 µM), especially in MCL cell lines (4.4 µM vs.11.65 µM, respectively) (Table 2). The tumor growth inhibition (TGI) achieved by a 16-day treatment in a MCL xenograft model, with 25, 50 and 100 mg/kg TG-1701 (56%, 72% and 78%, respectively), was comparable to the 70% TGI observed in the ibrutinib (100 mg/kg) arm (FIG.1E). A single oral gavage with 50 mg/kg TG-1701 further confirmed a rapid dephosphorylation of BTK and AKT as early as 2 hours and 4 hours, respectively, which was maintained at least for 24 hours due to the irreversible nature of TG-1701- mediated BTK inhibition (FIG.1F). Example 2: Phosphoproteomic analysis differentiates early clinical response to TG-1701 and points to the inhibition of the Ikaros pathway as an important mechanism of TG-1701 activity [0199] In this Example, a mass spectrometry (MS)-based phosphoproteomic platform used to interrogate the effects of TG-1701 on CLL patients enrolled in the phase 1 dose- escalation study (NCT03671590) pointed to the transcription factor Ikaros as both a potential biomarker of clinical activity and an important transcription factor downstream of BTK in the BCR pathway. [0200] Overview of Experimental Design: A set of six B-NHL clinical samples from the TG-1701 phase 1 clinical trial (NCT03671590) were characterized by phosphoproteomic and RNA-seq analysis, followed by biomarker validations using real-time PCR and Western blot. The activity of TG-1701, either alone or in combination with the U2 regimen, was evaluated by proliferation, antibody-dependent cell cytotoxicity (ADCC), and phagocytosis (ADCP) assays in a panel of n=10 B-NHL co-cultures and in two mouse xenografts, including non-canonical NFκB- and BTKC481S BTKi resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, western blot, immunofluorescence, immunostaining, and gene knock-out (KO) experiments. Patients, materials, and methods were described above. [0201] Results. To identify potential biomarkers of TG-1701 activity in B-NHL, PBMCs from six CLL patients enrolled in the TG-1701-101 phase 1 clinical trial were isolated at different time points for phosphoproteomic and RNA-seq analysis. In all patient samples but one, the percentage of circulating cancer cells was comprised between 60% and 98% (Table 3 and Table 4). All the patient samples harbored a wild type BTK gene, as confirmed by Sanger sequencing of BTK exon 11. IGVH mutational status was also assessed, as shown in Table 3 (UM= unmutated and M = mutated). Table 3: Clinical and Biological Characteristics of 6 CLL Patients
Figure imgf000049_0001
Table 4: PBMC analysis of 6 CLL patients
Figure imgf000049_0002
Figure imgf000050_0001
[0202] Near complete BTK occupancy was obtained in all samples tested (FIG.2A and FIG.8) and linear kinetics was observed with approximately dose proportional increases in Cmax and AUC0-8h from 100 mg to 400 mg, with a positive correlation between the daily dose and TG-1701 Cmax (FIG.2B). After three cycles of treatment, the best change in tumor burden as assessed by CT scan ranged from 38 % to 87%, with one tumor-free patient with a lymphocytosis that defined his best response as a stable disease (FIG.2C). [0203] In PBMC protein extracts from the six pre-dose and the six 4 hour post-dose samples, a total of 5585 proteins, and 2438 phospho-sites were identified. An initial principal component analysis (PCA) did not discriminate the pre-treatment (PRE) from the post-treatment (POST) profiles. However, a specific unsupervised clustering of the data into two subgroups of three patients allowed a clear differentiation between the PRE and POST samples (FIG.2D). These two subgroups, blindly selected to uncover changes due to TG-1701 treatment, clearly fitted the clinical outcome of the patients (FIG.2C), separating de facto a group encompassing the three best responses (called the “responders”) and a group gathering the three other patients with lower responses (called the “non-responders”). Supporting this partition of the patients, the responders exhibited a stronger IL-10 de-repression and a stronger, although not significant, decrease in IL-2 and IL-6 expression (FIG.3A), suggesting that they developed a more potent early anti- inflammatory response, a common feature of BTKi activity (Purvis, GSD et al., Br J Pharmacol 177: 4416-4432 (2020)). Additionally, CCL3 and CCL4 chemokine genes, two bona fide biomarkers for BCR pathway activation (Takahashi, K et al., Br J Haematol 171:726–735 (2015)), were more downregulated in responder patients (FIG. 3A). A set of 118 phosphopeptides were differentially phosphorylated (7 down- and 111 up-regulated) after TG-1701 treatment in the responder subgroup only (adjusted p-value < 0.1, FIG.2E). These sites corresponded to the putative modulation of 14 protein kinases (Tables 5 and 6). Importantly, these quantitative phosphoproteomic changes showed a strong homogeneity in the three responder patients and enabled a clear distinction between PRE and POST samples (FIG.2F). Only one single phosphosite was found significantly upregulated in the non-responder patients after TG-1701 treatment. Table 5: Phosphopeptides Significantly Up- and Down-Regulated Following TG-1701 Treatment in Responding CLL Patients (Parenthetical numerical values included in the SEQ ID NOS. below indicate the probability of phosphorylation of the preceding amino acid (1 = 100%), which are dephosphorylated after TG- 1701 treatment in Responders.)
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Table 6: List of Activated/Inhibited Kinases
Figure imgf000055_0002
Figure imgf000056_0002
[0204] Predicted kinases in Table 6 were identified by NetPhos v3.1 Phosphorylation Sites Predictor (Blom, N. et al., J Mol Biol 294(5): 1351–1362 (1999)). [0205] Besides the 118 phosphosites described above and depicted in FIG.2E, another set of 95 phosphopeptides was present in pre-treatment samples and totally dephosphorylated upon treatment with TG-1701. The total absence of phosphorylation preempted the statistical analysis and incorporation of these 95 samples in the volcano plot, even though these sites were the most impacted by TG-1701 treatment. Of special interest, the corresponding list of phosphosites comprised the p-Ser442/445 residue of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in B-cell homeostasis. Ikaros-Ser442/445 dephosphorylation was indeed the strongest event associated with TG-1701 activity (Table 7). Table 7: Phosphosites Totally Dephosphorylated by TG-1701 Treatment in Responder and Not in Non-Responder Patients (Parenthetical numerical values included in the SEQ ID NOS. below indicate the probability of phosphorylation of the preceding amino acid (1 = 100%), which are dephosphorylated after TG- 1701 treatment in Responders)
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
[0206] Since Ikaros nuclear localization and transcriptional activity both depend on BTK- mediated phosphorylation at Ser214/215 residues (Ma, H et al., PLoS One 8:e71302.27 (2013)), it was investigated whether, analogously, Ikaros function was differentially affected by TG-1701 in responder versus non-responder CLL patient samples. Using previously validated Ikaros-repressed and Ikaros-enhanced gene signatures (Díaz, T, et al., Haematologica 102:1776-1784 (2017)), 21 proteins of the repressed signature that were upregulated were identified, whereas another set of 17 factors from the Ikaros- enhanced gene signature were depleted only in responder patients, suggesting that Ikaros was functionally impaired after TG-1701 treatment (FIG.4A). Although a comparative multidimensional (MDS) analysis of RNA-seq data obtained from the same subset of samples failed to show a clear difference between responsive and non-responsive patient clusters (FIG.4B), a clear trend in the upregulation of Ikaros-repressed genes and downregulation of Ikaros-enhanced genes was seen in TG-1701 responsive patients only (FIG.4B). Accordingly, the IKZF1-repressed gene, YES1, was significantly upregulated, while the IKZF1-enhanced gene MYC was downregulated in responders but not in non- responders (FIG.4C). Importantly, this effect was not due to Ikaros protein destabilization, as its level of expression did not vary upon TG-1701 treatment in responder patients, whereas the expression of p-BTK, MYC, and IRF4 proteins underwent a 70%, 48%, and 74% downregulation, respectively (FIG.4D and FIG.3C). Such modifications were not seen in non-responder patients, although BTK was notably dephosphorylated (FIG.4D and FIG.3C). [0207] These data were confirmed in vitro using the ibrutinib-sensitive MCL cell line REC-1 characterized by ibrutinib and TG-1701 GI50 values of 5.82 and 3.83 µM at 72 hours, respectively (Table 2). In these cells, as observed in CLL responder patients, TG- 1701 treatment led to efficient BTK dephosphorylation, YES1 upregulation and IRF4 and MYC downregulation both at mRNA and protein levels (FIGS.4E and 4F). Of note, among the IKZF1-target genes studied here, YES1 reactivation was significantly higher in TG-1701-treated than in ibrutinib-exposed cells (FIG.4E). Finally, FIG.4G shows that ibrutinib and TG-1701 elicited a 67% and 45% reduction in nuclear Ikaros, consistent with a ~50% decrease of Ikaros in REC-1 nuclear protein fraction (FIG.4H), suggesting that dual dephosphorylation of Ikaros at Ser442 and Ser445 was associated with the nuclear exclusion of this factor. [0208] Results and Discussion. This Example reports that a phosphoproteomic-based analysis can discriminate between TG-1701 responders and non-responders in B-NHL patients. Phosphoproteomic analysis of tumoral lymphocytes from B-NHL patients receiving TG-1701 led to a non-supervised clustering that matched the early clinical outcomes of N-BHL patients, and separated a group of early “responders” from a group of “non-responders.” This clustering was based on a selected list of 95 phosphosites, with Ikaros-Ser442/445 phosphorylation as a potential biomarker for TG-1701 efficacy. [0209] RNA-seq analysis revealed that TG-1701 treatment blunted the Ikaros gene signature, including YES1, MYC, and IRF4, in responder patients, as well as in BTKi- sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and Ikaros- dependent gene regulation remained unaffected by the drug in non-responder patients, and in BTKC481S, BTKKO, and non-canonical NFκB models in vitro and in vivo. Interestingly, and in contrast with the first-in-class BTKI, ibrutinib, TG-1701 did not impair FcγR-driven ADCC and ADCP triggered by the anti-CD20 antibodies rituximab and ublituximab in different B-NHL co-culture system, and cooperated with U2 in reducing the tumor growth in both ibrutinib-sensitive and ibrutinib-insensitive mouse models of B-NHL. Therefore, these data validate a phosphoproteomic approach as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of a drug mechanism of action. Further, results in this Example support the use of TG-1701-U2 combination in R/R B-NHL patients, irrespective of prior response to ibrutinib. Example 3: Ikaros signature is a bona fide hallmark of BTKi mechanism of action [0210] To further explore TG-1701 mechanisms of action and potential mechanism of resistance, the REC-1GFP+LUC+ cell line was CRISPR-engineered to express the BTKC481S mutation. This mutation was associated with a 10.3-fold and a 54.8-fold decrease in ibrutinib and TG-1701 inhibitory kinase activity, respectively (FIG.7A and Table 2). The REC-1-BTKC481S cell line was 4.2-fold and 2.8-fold less sensitive to ibrutinib and TG-1701 respectively, compared to parental REC-1 cells (FIG.5A and Table 2). A washout experiment further showed that irreversible BTK inhibition - illustrated by kinase phosphorylation over 24 hours after BTKi removal in REC-1 cells - was mostly lost in REC-1-BTKC481S cells (FIG.5B). While total proteome composition was modified solely in the parental cells treated with either ibrutinib or TG-1701, as assessed by PCA analysis (FIG.7B), a set of 16 Ikaros-repressed proteins were upregulated and another set of 14 Ikaros-upregulated proteins were downregulated in REC-1, but not in REC-1- BTKC481S cells exposed to TG-1701 (FIG.5C). Despite the short drug exposure, transcriptional upregulation of YES1, and repression of MYC and IRF4 were observed in BTKwt cells treated with both BTKis. In REC-1-BTKC481S cells, a pronounced basal Ikaros-repressed gene signature was observed in the absence of any treatment (black bar, FIG.5D), including YES1 expression and IRF4 repression, and this pattern was not modified upon BTKi exposure (FIG.5D and FIG.7C). [0211] To confirm the role of BTK as an upstream regulator of Ikaros signaling in BTKi- exposed cells, a BTK knock-out (KO) model derived from the Rec-1 cell line was generated, using a CRISPR-Cas9 method, as described above. The obtained Rec-1- BTKKO derivative, characterized by an almost complete depletion of BTK (FIG.5E) was refractory to both ibrutinib and TG-1701 (Table 2) and did not undergo significant modulation of YES1 and MYC expression after exposure to TG-1701 (FIGS.5E-5F). These data thus suggest that TG-1701 treatment leads to an impaired Ikaros signaling in MCL cells by a BTK-dependent process. [0212] Given the low recurrence of BTKC481S mutation in MCL patients, the UPN-IbruR non-canonical NF-κB-driven ibrutinib resistance model was also studied (Balsas, P et al., J Hematol Oncol.10: 80 (2017)). This subclone is characterized by the absence of mutations in the BTK and PLCG2 genes and the constitutive activation of p52-dependent signaling, driving a 2-3-fold increase in ibrutinib and TG-1701 GI50 at 72h (FIG.7D, Table 2; and Balsas, P et al., J Hematol Oncol.10: 80 (2017)). When compared with the BTKi-sensitive REC-1 xenograft model in which a 17-day dosing with TG-1701 achieved a 53% tumor TGI vs vehicle, UPN-IbruR tumors were almost insensitive to TG-1701 (FIG.6A). Accordingly, p-BTK was efficiently downregulated in representative REC-1 tumor specimens, but not in UPN-IbruR xenografts treated with TG-1701 (FIG.6B). In agreement with the in vitro results, MYC and IRF4 were downregulated, and YES1 upregulated at protein and/or mRNA levels, in association with Ikaros nuclear exnuxlwE PROTEINlusion and a decrease in CD20+ malignant B cells, in BTKi-sensitive, but not in BTK-insensitive MCL xenografts (FIGS.6B-6D). DISCUSSION [0213] During the past decade, BTK inhibitors have increasingly replaced chemotherapy- based regimen in patients with CLL and MCL. TG-1701 is a novel second-generation BTKi currently under clinical development. TG-1701 is more selective than ibrutinib, with a comparable BTK Kd and similar in vitro and in vivo characteristics. TG-1701 is currently being tested in a phase 1 trial comprised of a single agent arm and a combination arm with ublituximab (a novel CD20 antibody) and umbralisib (a dual PI3Kd and CK1ɛ inhibitor). With a median follow up of 7 months in a 200 mg daily monotherapy expansion cohort, preliminary overall response rates (ORR) are 95% (19/20) in CLL, 50% (9/18) in MCL, and 95% (18/19) in WM. No complete responses (CR) are confirmed on TG-1701 monotherapy (Cheah, CY et al., ” Poster Abstract #1130, 62nd ASH Annual Meeting and Exposition, Blood (2020)). [0214] In this Example, it has been shown that phosphoproteomic analysis of pre- and post-treatment samples clustered B-NHL patients according to their early clinical responses (responders vs. non-responders) and helped decipher the mechanisms underlying drug responsiveness. According to the results, the response from these patients did not depend on the pharmacokinetic or pharmacodynamic properties of TG-1701, but rather on differences in BTK downstream signaling. In responder patients, Ikaros-p- Ser442/445 were the phosphopeptides the most impacted (dephosphorylated) by TG-1701 treatment and several Ikaros-dependent factors were transcriptionally deregulated, suggesting that Ikaros may represent a biomarker for early response, and/or an important new node downstream BTK inhibition. Interestingly, a recent study in acalabrutinib- treated CLL patients (Beckmann, L et al., “MARCKS affects cell motility and response to BTK inhibitors in CLL,” Blood (2021)) has shown that the unmutated IGVH cells displayed a higher basal phosphorylation level compared to the mutated IGVH cells, showing that the phosphoproteomic profile of BTKi-treated patients can cluster specific subgroups of patients. [0215] Ikaros is a zinc finger protein involved in gene regulation and chromatin remodeling. Its nuclear localization, stability and transcriptional activity depend on its phosphorylation status which is regulated by BTK, casein kinase II (CKII) and protein phosphatase 1 (PP1) interplay (Ma, H et al., PLoS One 8:e71302.27 (2013); Popescu, M et al., J Biol Chem 284:13869-80 (2009)). Although the exact role of serine residues at position 442 and 445 is still unknown, their juxtaposition to the conserved PP1 binding motif in the C-terminal end of Ikaros protein (Georgopoulos, K, Genes Dev.31: 439-450 (2017)), might confer them a role in the PP1-mediated regulation of Ikaros stability and pericentromeric localization (Popescu, M et al., J Biol Chem 284:13869-80 (2009)). Interestingly, CKII was ranked #4 in the list of kinases with reduced activity in TG-1701 responder patients, while phosphorylation of the PP1 inhibitory subunit, PPP1R14A, and dephosphorylation of the PP1 inhibitor, PPP1R2, both associated with reduced PP1 activity (Verbinnen, I et al., Biochem. Soc. Trans.45: 583-584 (2017)) were among the top four modifications detected in TG-1701 responder patients (Table 5 and Table 6). Ikaros expression was affected by TG-1701 treatment, neither in CLL primary cells nor in BTKi-sensitive REC-1 models, indicating that the inhibition of Ikaros signature in responders was more likely due to a nuclear exclusion of the transcription factor. Importantly, the results obtained in REC-1-BTKC481S and REC-1-BTKKO subclones demonstrated that both the cell proliferation blockade and the modulation of Ikaros activity upon TG-1701 treatment are tightly dependent on the presence of a wild type BTK protein, supporting the theory that Ikaros phosphorylation at Ser442/445 is controlled by the kinase, and discarding a potential off-target effect of the compound. [0216] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. [0217] While various specific aspects have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

WHAT IS CLAIMED IS: 1. A method of treating B-cell non-Hodgkin lymphoma (B-NHL) in a subject in need thereof, comprising: administering to a subject that is a TG-1701 responder a therapeutically effective amount of BTK inhibitor TG-1701; wherein prior to said administration, B-NHL cells of the TG-1701 responder contain at least one phosphopeptide selected from SEQ ID NOs: 1-95.
2. The method of claim 1, wherein the at least one phosphopeptide is SEQ ID NO: 1.
3. The method of claim 1 or 2, wherein the presence of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis.
4. The method of claim 3, wherein, after administration of TG-1701 to the subject, the B- NHL cells of the TG-1701 responder lack at least one phosphopeptide selected from SEQ ID NOs: 1-95, compared to before administration of TG-1701.
5. The method of claim 4, wherein the at least one phosphopeptide is SEQ ID NO: 1.
6. The method of claim 4 or 5, wherein the lack of the at least one phosphopeptide is determined by western blot and/or phospho-flow analysis.
7. The method of any one of claims 1-6, wherein the B-NHL cells of the TG-1701 responder comprise an increased quantity of transcripts of an Ikaros-repressed gene signature after the administration of TG-1701 to the subject compared to the quantity of transcripts before the administration of TG-1701.
8. The method of claim 7, wherein the transcripts of the Ikaros-repressed gene signature comprise one or more of TXNIP, CD36, CA2, YOD1, CFP, DENND3, YES1, NBEAL2, TMC8, PSTPIP2, CD97, DAAM1, NT5E, LYZ, SDK2, TSC22D4, GYPC, FAM129A, TPM3, GNAQ, and/or LUZP1.
9. The method of any one of claims 1-8, wherein the B-NHL cells of the TG-1701 responder comprise a decreased quantity of transcripts of an Ikaros-enhanced gene signature after the administration of TG-1701 to the subject compared to the quantity of transcripts before the administration of TG-1701.
10. The method of claim 9, wherein the transcripts of an Ikaros-enhanced gene signature comprise one or more of TCL1A, CBX5, HNRNPA0, PDHB, BCL2, DYNLL1, SUPT16H, CAMK2D, ALDH6A1, PPP2R5C, ERGIC1, BUB3, SORD, SEPHS1, CTNNBL1, CCT5, and/or APOBEC3G.
11. The method of any one of claims 7-10, wherein the quantity of transcripts is determined by at least one amplification-based method.
12. The method of claim 11, wherein the amplification-based method is Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification.
13. The method of claim 12, wherein the amplification-based method is qPCR.
14. The method of any one of claims 7-10, wherein the quantity of transcripts is determined by at least one non-amplification-based method.
15. The method of claim 14, wherein the non-amplification-based method is a hybridization- based method or a sequencing-based method.
16. The method of claim 15, wherein the non-amplification-based method is a hybridization- based method.
17. The method of claim 15, wherein the hybridization-based method is a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, or in situ hybridization.
18. The method of claim 15, wherein the non-amplification-based method is a sequence- based method.
19. The method of claim 18, wherein the sequence-based method is a next-generation sequencing (NGS) method.
20. The method of any one of claims 7-10, wherein the quantity of transcripts is determined by a combination of amplification-based and non-amplification-based methods.
21. The method of any one of claims 1-20, wherein the B-NHL is a chronic lymphocytic leukemia, a mantle cell lymphoma, a follicular lymphoma, a diffuse large B-cell lymphoma, a marginal zone B-cell lymphoma, a Burkitt lymphoma, or a lymphoplasmacytic lymphoma.
22. The method of any one of claims 1-21, further comprising administering to a subject that is a TG-1701 responder a therapeutically effective amount of an anti-CD20 antibody.
23. The method of claim 22, further comprising administering to a subject that is a TG-1701 responder a therapeutically effective amount of a dual PI3Kδ and casein kinase-1ε inhibitor.
24. The method of any one of claims 1-23 wherein the therapeutically effective amount of TG-1701 is between about 100 mg/day and about 400 mg/day.
25. The method of claim 24, wherein the therapeutically effective amount of TG-1701 is about 100 mg/day.
26. The method of claim 24, wherein the therapeutically effective amount of TG-1701 is about 200 mg/day.
27. The method of claim 24, wherein the therapeutically effective amount of TG-1701 is about 300 mg/day.
28. The method of claim 14, wherein the therapeutically effective amount of TG-1701 is about 400 mg/day.
29. The method of any one of claims 1-28, wherein the subject is a mammal.
30. The method of claim 29, wherein the subject is a human.
31. A kit comprising: (i) at least one antibody that binds to at least one phosphopeptide selected from SEQ ID NOs: 1-95; (ii) optionally, reagents to perform a western blot analysis; and/or reagents to perform a phospho-flow analysis; and (iii) instructions for treating B-NHL in a TG-1701 responder according to the methods of any one of claims 1- 30.
32. The kit of claim 31, further comprising reagents to perform a Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), or rolling circle amplification.
33. The kit of claim 31, further comprising reagents to perform a microarray, Nanostring analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, next- generation sequencing (NGS) method, or in situ hybridization.
34. The kit of any one of claims 31-33, wherein the at least one phosphopeptide is SEQ ID NO: 1.
PCT/US2022/032563 2021-06-08 2022-06-07 Disrupted ikaros signaling as biomarker for btk inhibition WO2022261138A1 (en)

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