WO2018067946A1 - Methods of treating patients with a retinoic acid receptor-alpha agonist and an anti-cd38 antibody - Google Patents

Abstract

The invention provides methods that define cellular populations that are sensitive to RARA agonists and identify patient subgroups that will benefit from treatment with RARA agonists in combination with another therapy, more specifically an anti-CD38 therapy. The invention also provides packaged pharmaceutical compositions that comprise a RARA agonist and instructions for determining if such combination therapy is suitable for use in treatment.

Description

METHODS OF TREATING PATIENTS WITH A RETINOIC ACID RECEPTOR-a AGONIST AND AN ANTI-CD38 ANTIBODY

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of and priority to U.S. Provisional Patent

Application No. 62/405,164, filed October 6, 2016, U.S. Provisional Patent Application No. 62/449,450, filed January 23, 2017, and U.S. Provisional Patent Application No. 62/564,690, filed September 28, 2017, each of which is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION

[002] Retinoids are a class of compounds structurally related to vitamin A, comprising natural and synthetic compounds. Several series of retinoids have been found clinically useful in the treatment of dermatological and oncological diseases. Retinoic acid and its other naturally occurring retinoid analogs (9-cis retinoic acid, all-trans 3,4-didehydro retinoic acid, 4-oxo retinoic acid and retinol) are pleiotropic regulatory compounds that modulate the structure and function of a wide variety of inflammatory, immune and structural cells. They are important regulators of epithelial cell proliferation, differentiation and morphogenesis in lungs. Retinoids exert their biological effects through a series of hormone nuclear receptors that are ligand inducible transcription factors belonging to the steroid/thyroid receptor super family.

[003] The retinoid receptors are classified into two families, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), each consisting of three distinct subtypes (α, β, and γ). Each subtype of the RAR gene family encodes a variable number of isoforms arising from differential splicing of two primary RNA transcripts. All-trans retinoic acid is the physiological hormone for the retinoic acid receptors and binds with approximately equal affinity to all the three RAR subtypes, but does not bind to the RXR receptors for which 9-cis retinoic acid is the natural ligand. Retinoids have anti-inflammatory effects, alter the progression of epithelial cell differentiation, and inhibit stromal cell matrix production. These properties have led to the development of topical and systemic retinoid therapeutics for dermatological disorders such as psoriasis, acne, and hypertrophic cutaneous scars. Other applications include the control of acute promyelocytic leukemia, adeno- and squamous cell carcinoma, and hepatic fibrosis. [004] A limitation in the therapeutic use of retinoids has stemmed from the relative toxicity observed with the naturally occurring retinoids, all-trans retinoic acid and 9-cis retinoic acid. These natural ligands are non-selective in terms of RAR subtype and therefore have pleiotropic effects throughout the body, which are often toxic.

[005] Various retinoids have been described that interact selectively or specifically with the RAR or RXR receptors or with specific subtypes (α, β, γ) within a class. RARA specific agonists have held high promise for the treatment of cancers and many have entered human clinical trials. However, only one RARA specific agonist, tamibarotene, has ever been approved for the treatment of cancer. Moreover, tamibarotene is only approved in Japan and only for the treatment of acute promyelocytic leukemia, despite trials in the US and Europe. The disconnect between the theoretical efficacy of RARA agonists in cancer and the dearth of regulatory approvals for such agents raises the question of why such agonists are not effective and safe in humans. Therefore, there is a need to better understand why RARA agonists have not met their therapeutic potential.

[006] Cluster of differentiation 38 (CD38) is a protein expressed on the surface, primarily on white blood cells, and considered a cell surface marker indicative of differentiation initiation. It functions as a cyclic ADP-ribose hydrolase which plays a role in cell signaling (Mehta, K. & Cheema, S., Leuk. Lymphoma, 32, 441-449 (1999)). It is generally found to be highly expressed in cells of the B-cell and plasma cell lineage. In multiple myeloma a subset of patients have high CD38 expression, which has led to the development of anti-CD38 therapeutic antibodies, such as daratumumab (Lokhorst, H. M. et al., N. Engl. J. Med., 373, 1207-1219 (2015); de Weers, M. et al., /. Immunol., 186, 1840-1848 (2011)). Thus, cancer cells which express CD38 can be selectively targeted for elimination by the immune system using these therapeutic antibodies. In multiple myeloma daratumumab is most effective in high CD38- expressing tumor cells (CD38^hl) while lower levels of CD38 expression cause the therapeutic antibody to have less to no effect (Nijhof, I. S. et al., Blood, 128, 959-970 (2016)). This is why normal white blood cells or multiple myeloma cells that are initially low in CD38 expression (or becomes low as a mechanism of resistance) do not get efficiently cleared by anti-CD38 treatment.

[007] CD38 expression in AML is considered to be generally low (CD38^lD), but moderate in expression in a subset of patients (CD38^dim). Naturally-occurring CD38^hi AML cells are not typical and the percent positivity in AML is generally low. Thus, AML is believed unlikely to respond to anti-CD38 treatment, a finding that has been demonstrated with daratumumab monotherapy (Dos Santos, C. et al., Blood, 124, 2312-2312 (2014). [008] Recent advances in genomic technology and the understanding of gene regulatory circuits has led to the discovery of super enhancers. Whereas many genes in a given tissue or cancer type may be regulated by the presence of enhancers in proximity to the gene coding region, a small minority of these represent a highly asymmetric and disproportionately large loading of transcriptional marks and machinery relative to all other active genes. Recent discoveries suggest that such enhancers are tied to genes of special relevance to the function and survival of the cell harboring them. As such, an association of a super enhancer with a gene indicates the relative significance of said gene to the survival of that cell.

SUMMARY OF THE INVENTION

[009] The present disclosure provides methods for treating a subject suffering from a hematological cancer (e.g., multiple myeloma (MM), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), anaplastic large cell lymphoma (ALCL), B-cell acute lymphoblastic leukemia (BALL), B-cell non-Hodgkin lymphoma (BNHL), Burkitt's lymphoma, chronic myelogenous lymphoma (CML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, or T-cell acute lymphoblastic leukemia (TALL)) by administering to the subject a combination of a retinoid (e.g., a retinoic acid receptor alpha-specific agonist, e.g., tamibarotene) and an anti-CD38 antibody (e.g., daratumumab), wherein the hematological cancer is: a) unresponsive to a CD38 inhibitor as a monotherapy; and/or b) characterized as CD38^", CD38^lD, or CD38^dim.

[0010] The disclosure also provides methods that comprise detecting a RARA biomarker or IRF8 biomarker and administering a RARA agonist in combination with an antibody specific for CD38 to treat acute myeloid leukemia that is not of the acute promyelocytic leukemia type ("non-APL AML"), myelodysplastic syndrome ("MDS"), or multiple myeloma ("MM"), for example, when a RARA biomarker or IRF8 biomarker is present above a predetermined threshold level. Detection of a RARA biomarker or IRF8 biomarker includes determination of the presence, level, form, and/or activity of one or more RARA or IRF8 gene components or products, including for example RARA or IRF8 super enhancer strength, ordinal rank, or prevalence rank and RARA or IRF8 mRNA level or prevalence rank. The present disclosure demonstrates that cells (e.g., cancer cells or cells from a subject suffering from non- APL AML, MDS, or MM) containing one or more of a super enhancer associated with a RARA or IRF8 gene are more susceptible to the effects of a RARA agonist, such as tamibarotene, in combination with an antibody specific for CD38.

[0011] The various embodiments, aspects and alternatives of this invention solve the problem of defining which cellular populations are sensitive to a combination of agonists of retinoic acid receptor alpha ("RARA") and an antibody specific for CD38, identifying patient subgroups that will benefit from treatment with RARA agonist an antibody specific for CD38 (e.g., stratifying patients for treatment; separating responders from non-responders) and providing treatment therapies directed at such patient subgroups. The solution is based, at least in part, upon our discovery that elevated expression of a RARA biomarker or an IRF8 biomarker in non-APL AML, MDS or MM is indicative that such cell will respond to treatment with a RARA agonist and an antibody specific for CD38.

[0012] In a first embodiment, the invention relates to a method of diagnosing and treating a subject suffering from non-APL AML, MDS or MM comprising: a) diagnosing whether the subject has a RARA agonist sensitive (e.g., tamibarotene-sensitive) form of the disease based on: i) a level of retinoic acid receptor alpha mRNA previously determined to be equal to or above a pre-determined threshold in a sample of diseased cells from the subject; and/or ii) a level of IRF8 mRNA previously determined to be equal to or above a pre-determined threshold in a sample of diseased cells from the subject; b) administering to the subject an amount of a RARA agonist (e.g., tamibarotene) effective to treat the disease; and c) coadministering to the subject an antibody specific for CD38.

[0013] In some aspects of the first embodiment, the RARA agonist is tamibarotene. In some aspects of the first embodiment, the antibody specific for CD38 is daratumumab. In some aspects of the first embodiment, the subject is administered tamibarotene for a period of time prior to administration of the antibody specific for CD38; and is co-administered the antibody specific for CD38 only when the CD38 level in the subject has been determined to be CD38^hl following initial administration of tamibarotene. In some aspects of the first embodiment, the CD38 level in the subject was determined between 6 and 72 hours following the initial administration of tamibarotene.

[0014] In a second embodiment, the invention provides a method of treating a human subject suffering from non-APL AML, MDS or MM, wherein the level of retinoic acid receptor alpha mRNA and/or the level of IRF8 mRNA in diseased cells in the subject has been determined to have a level of retinoic acid receptor alpha mRNA equal to or above a pre- determined threshold, or a level of IRF8 mRNA equal to or above a pre-determined threshold, the method comprising a step of administering to the subject an amount of a RARA agonist (e.g., tamibarotene) effective to treat the disease; and co- administering to the subject an antibody specific for CD38.

[0015] In some aspects of the second embodiment, the RARA agonist is tamibarotene. In some aspects of the second embodiment, the antibody specific for CD38 is daratumumab. In some aspects of the second embodiment, the subject is administered tamibarotene for a period of time prior to administration of the antibody specific for CD38; and is co-administered the antibody specific for CD38 only when the CD38 level in the subject has been determined to be CD38^hl following initial administration of tamibarotene. In some aspects of the second embodiment, the CD38 level in the subject was determined between 6 and 72 hours following the initial administration of tamibarotene.

[0016] In a third embodiment, the invention provides a method of diagnosing and treating a human subject suffering from non-APL AML, MDS or MM comprising: a) diagnosing whether the subject has a RARA agonist- sensitive (e.g., tamibarotene- sensitive) form of the disease based on a level of retinoic acid receptor alpha mRNA and/or a level of IRF8 mRNA, either or both which were previously determined to be present in a sample of diseased cells from the subject; and b) administering therapy to the subject, wherein i) the therapy comprises administering an amount of a RARA agonist (e.g., tamibarotene) effective to treat the disease and co- administering an antibody specific for CD38 if the level indicates that the disease is tamibarotene-sensitive; and ii) the therapy comprises administering an agent other than a RARA agonist (e.g., other than tamibarotene) if the level indicates that the disease is not RARA agonist sensitive (e.g., not tamibarotene-sensitive).

[0017] In some aspects of the third embodiment, the retinoic acid receptor alpha mRNA and/or the IRF8 mRNA level indicates that the disease is RARA agonist- sensitive (e.g., tamibarotene-sensitive) if it is above a pre-determined threshold and that the disease is not RARA agonist- sensitive (e.g., not tamibarotene-sensitive) if it is below the pre-determined threshold. In some aspects of the third embodiment, the RARA agonist is tamibarotene. In some aspects of the third embodiment, the antibody specific for CD38 is daratumumab. In some aspects of the third embodiment, if the level of retinoic acid receptor alpha mRNA or IRF8 mRNA indicates that the disease is RARA agonist-sensitive (e.g., tamibarotene-sensitive) the subject is administered a RARA agonist (e.g., tamibarotene) for a period of time prior to administration of the antibody specific for CD38; and is co-administered the antibody specific for CD38 only when the CD38 level in the subject is determined to be CD38^hl following initial administration of a RARA agonist (e.g., tamibarotene). In some aspects of the third

embodiment, the CD38 level in the subject is determined between 6 and 72 hours following the initial administration of a RARA agonist (e.g., tamibarotene).

[0018] In a fourth embodiment, the invention provides a method for treating a subject having a hematological cancer that is unresponsive to a treatment with an anti-CD38 antibody in the absence of a retinoid, wherein the method comprises co-administering to the subject a retinoid and an anti-CD38 antibody. In some aspects of the fourth embodiment, the subject is suffering from a hematological cancer selected from multiple myeloma (MM), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), anaplastic large cell lymphoma (ALCL), B-cell acute lymphoblastic leukemia (BALL), B-cell non-Hodgkin lymphoma (BNHL), Burkitt' s lymphoma, chronic myelogenous lymphoma (CML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, or T-cell acute lymphoblastic leukemia (TALL). In some more specific aspects of the fourth embodiment, the subject is suffering from a hematological cancer selected from anaplastic large cell lymphoma (ALCL), B-cell acute lymphoblastic leukemia (BALL), B- cell non-Hodgkin lymphoma (BNHL), Burkitt' s lymphoma, chronic myelogenous lymphoma (CML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, or T-cell acute lymphoblastic leukemia (TALL).

[0019] In some aspects of the fourth embodiment, the retinoid is a RARA-specific agonist. In more specific aspects of the fourth embodiment, the retinoid is tamibarotene. In other aspects of the fourth embodiment, the anti-CD38 antibody is daratumumab.

[0020] In some aspects of the fourth embodiment, the subject is administered a retinoid (e.g., a RARA agonist, e.g., tamibarotene) for a period of time prior to administration of an anti- CD38 antibody (e.g., daratumumab); and is co-administered the anti-CD38 antibody only when the CD38 level in the subject (e.g., in the subject's hematological cancer cells) is determined to be CD38^hl following initial administration of the retinoid (e.g., tamibarotene). In some aspects of the fourth embodiment, the CD38 level in the subject is determined between 6 and 120 hours following the initial administration of the retinoid (e.g., tamibarotene).

[0021] In some aspects of the fourth embodiment, only subjects that have: i) a level of retinoic acid receptor alpha mRNA previously determined to be equal to or above a predetermined threshold in a sample of diseased cells from the subject; and/or ii) a level of IRF8 mRNA previously determined to be equal to or above a pre-determined threshold in a sample of diseased cells from the subject are administered the retinoid (e.g., tamibarotene) and the anti- CD38 antibody (e.g. daratumumab).

[0022] In some aspects of the fourth embodiment, a sample of a subject's diseased cells is tested for CD38 induction prior to any treatment. Typically, this would involve obtaining a sample of the subject's diseased cells; growing such cells ex vivo; optionally measuring a baseline level of CD38 induction in those cells; treating the ex vivo cells with a retinoid (e.g., tamibarotene); and determining the level of CD38 induction following such treatment. If the CD38 level in this sample of the subject's diseased cells is determined to be CD38^HI after treatment with the retinoid, then the subject is determined to be a candidate for the retinoid (e.g., tamibarotene)/anti-CD38 antibody (e.g., daratumumab) combination treatment.

[0023] In some aspects of the fourth embodiment, the anti-CD38 antibody (e.g., daratumumab) is administered prior to or after administration of the retinoid (e.g., a RARA agonist, e.g., tamibarotene). In some aspects of the fourth embodiment, when the CD38 level in the diseased cells of a subject is determined to be inducible by a retinoid using the ex vivo technique described above, the treatment regimen does not require any specific order of administering the retinoid and the anti-CD38 antibody. Thus, it may be advantageous to administer the anti-CD38 antibody prior to the retinoid so as to limit or eliminate any side effects of the antibody and/or to optimize the pharmacokinetics of each agent. In some aspects of the fourth embodiment, the anti-CD38 antibody (e.g., daratumumab) is administered between 6 and 120 hours prior to administration of the retinoid (e.g., a RARA agonist, e.g., tamibarotene).

[0024] In a fifth embodiment, the invention provides a method for treating a subject having a hematological cancer that is characterized as CD38^", CD38^lD or CD38^dim, wherein the method comprises co-administering to the subject a retinoid and an anti-CD38 antibody. In some aspects of the fifth embodiment, the subject is suffering from a hematological cancer selected from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), anaplastic large cell lymphoma (ALCL), B-cell acute lymphoblastic leukemia (BALL), B-cell non-

Hodgkin lymphoma (BNHL), Burkitt's lymphoma, chronic myelogenous lymphoma (CML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, or T-cell acute lymphoblastic leukemia (TALL). In some more specific aspects of the fifth embodiment, the subject is suffering from a hematological cancer selected from anaplastic large cell lymphoma (ALCL), B- cell acute lymphoblastic leukemia (BALL), B-cell non-Hodgkin lymphoma (BNHL), Burkitt's lymphoma, chronic myelogenous lymphoma (CML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, or T-cell acute lymphoblastic leukemia (TALL).

[0025] In some aspects of the fifth embodiment, the retinoid is a RARA-specific agonist. In more specific aspects of the fifth embodiment, the retinoid is tamibarotene, ATRA, or a derivative thereof (e.g., fenretinide). In other aspects of the fifth embodiment, the anti- CD38 antibody is daratumumab.

[0026] In still other aspects of the fifth embodiment, the CD38 level in the subject

(e.g., in the subject's hematological cancer cells) is determined prior to administration of the therapy.

[0027] In some aspects of the fifth embodiment, a sample of a subject's diseased cells is tested for CD38 induction prior to any treatment as described for the fourth embodiment. If the CD38 level in this sample of the subject's diseased cells is determined to be CD38^HI after treatment with the retinoid, then the subject is determined to be a candidate for the retinoid (e.g., tamibarotene)/anti-CD38 antibody (e.g., daratumumab) combination treatment.

[0028] In some aspects of the fifth embodiment, only subjects that have: i) a level of retinoic acid receptor alpha mRNA previously determined to be equal to or above a predetermined threshold in a sample of diseased cells from the subject; and/or ii) a level of IRF8 mRNA previously determined to be equal to or above a pre-determined threshold in a sample of diseased cells from the subject are administered the retinoid (e.g., tamibarotene) and the anti- CD38 antibody (e.g. daratumumab).

[0029] In yet other aspects of the fifth embodiment, the subject is administered a retinoid (e.g., a RARA agonist, e.g., tamibarotene) for a period of time prior to administration of the anti-CD38 antibody (e.g., daratumumab); and is co-administered the anti-CD38 antibody only when the CD38 level in the subject (e.g., in the subject's hematological cancer cells) is determined to be CD38^hl following initial administration of the retinoid (e.g., tamibarotene). In some aspects of the fifth embodiment, the CD38 level in the subject is determined between 6 and 120 hours following the initial administration of the retinoid (e.g., tamibarotene).

[0030] In some aspects of the fifth embodiment, the anti-CD38 antibody (e.g., daratumumab) is administered prior to or after administration of the retinoid (e.g., a RARA agonist, e.g., tamibarotene). In some aspects of the fifth embodiment, when the CD38 level in the diseased cells of a subject is determined to be inducible by a retinoid using the ex vivo technique described above, the treatment regimen does not require any specific order of administering the retinoid and the anti-CD38 antibody. Thus, it may be advantageous to administer the anti-CD38 antibody prior to the retinoid so as to limit or eliminate any side effects of the antibody and/or to optimize the pharmacokinetics of each agent. In some aspects of the fifth embodiment, the anti-CD38 antibody (e.g., daratumumab) is administered between 6 and 120 hours prior to administration of the retinoid (e.g., a RARA agonist, e.g., tamibarotene).

[0031] In any and all embodiments, in some aspects, the present invention features a method for treating a subject having cancer (e.g., non-APL AML, MDS or MM), wherein the composition comprises an agonist of RARA (e.g., tamibarotene) co-administered with an antibody specific for CD38 (e.g., daratumumab). In some aspects, tamibarotene is administered orally. In some aspects, the subject is administered tamibarotene at a dosage of between 6 mg/m²/day and 12 mg/m²/day, wherein said dosage is divided into two doses. In some aspects, the anti-CD38 antibody is administered no more than once a week at a dose of between 10-20 mg/kg body weight of the subject.

[0032] The details of one or more embodiments of the invention are set forth herein.

Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Fig. 1 shows the correlation between RARA mRNA expression (log₂(l+TPM)) and RARA SE strength (RARA/M AL AT 1 fold enrichment) for 48 different AML patient samples using RNA-Seq.

[0034] Fig. 2 depicts a logio rank-ordered graph of RARA super enhancer strength ordinal in 94 AML samples including four AML cell lines -- Sig-M5, MV411, HEL and Kasumi.

[0035] Fig. 3 depicts a logio rank-ordered graph of RARA super enhancer strength ordinal in 70 AML patient samples. The lighter colored bars represent samples whose RARA super enhancer strength ordinal was equal to or above the prevalence cutoff. Darker colored bars represent samples whose RARA super enhancer strength ordinal was below the prevalence cutoff.

[0036] Fig. 4 depicts the correlation between RARA enhancer strength and sensitivity to tamibarotene in 11 different AML cell lines.

[0037] Fig. 5A depicts RARA mRNA levels from 70 AML patient samples and binned according to whether their RARA super enhancer strength ordinal was above (or equal to) the prevalence cutoff ("High RARA) or below the prevalence cutoff ("Low RARA") as described in Example 2. Fig. 5B depicts the correlation between RARA mRNA level and sensitivity to tamibarotene in 11 different AML cell lines.

[0038] Fig. 6 depicts IRF8 mRNA levels in seven different AML cell lines. The four cell lines shown on the left side of the chart demonstrated substantial responsiveness to tamibarotene treatment. The three cell lines shown on the right side of the chart demonstrated little or no responsiveness to tamibarotene treatment.

[0039] Fig. 7 shows correlation of tamibarotene anti-proliferative potency (EC5₀ value, nM) with IRF8 mRNA levels as measured by RNA-seq. Note that the top-left point with IRF8 mRNA level = 1 (loglO) and tamibarotene EC5₀ value imputed as 50 μΜ (non-responsive) represents data from 2 AML cell lines with low IRF8 mRNA levels and no anti-proliferative response to tamibarotene. The correlation of tamibarotene sensitivity with IRF8 mRNA levels was highly significant (p = 0.0001, Spearman's correlation, two-tailed).

[0040] Fig. 8 depicts a rank order graph of IRF8 mRNA level in individual patient

AML samples and AML cell lines as measured by RNA-Seq. The AML cell lines PL21, which was the cell line responsive to tamibarotene that had the lowest IRF8 mRNA level, and Kasumi, which was the cell line unresponsive to tamibarotene that had the highest IRF8 mRNA level are indicated. In this population, a 25% prevalence cutoff is equal to a RNA-Seq TPM value of approximately log₂(7).

[0041] Fig. 9 depicts the correlation between IRF8 mRNA level and RARA mRNA level in non-APL AML cell lines tested for response to tamibarotene. [0042] Fig. 10 depicts the correlation between IRF8 mRNA level and RARA mRNA level in a population of AML patient samples. The dotted lines represent a 25% prevalence cutoff for each mRNA.

[0043] Figs. 1 lA-1 IF depict the effect of tamibarotene ("SY1425") on CD38 levels in various AML cell lines. Figs. 11 A-1 ID demonstrate the effect of tamibarotene on different cell lines as measured by FITC cell sorting. Fig. 1 IE is a graphical representation of the CD38 mRNA levels in various cell lines before and after treatment with tamibarotene. Fig. 1 IF is a graphical representation of the % of cells that are positive for CD38 as determined by FITC cell sorting.

[0044] Figs. 12A-12D depict the effect of tamibarotene and daratumumab ("Dara"), alone or in combination, on NK cell proliferation and tumor cell death in various NK cell/AML cell line co-cultures, as observed by phase contrast microscopy.

[0045] Figs. 13A-13C depict the effect of a control antibody or Dara alone or in combination with tamibarotene on the number of apoptotic cells in NK cell/AML co-culture for various AML cell lines.

[0046] Fig. 14 depicts the effect of a control antibody or Dara alone or in combination with tamibarotene on interferon-gamma secretion in a NK cell/AML co-culture for the various AML cell lines depicted in Figs. 13A-13C.

[0047] Figs. 15A-15B depict the effect of a control antibody or Dara alone or in combination with tamibarotene on CD38 levels in two different multiple myeloma cell line

(MM1S; Fig. 15A) (HUNS1 ; Fig. 15B) as measured by FITC cell sorting.

[0048] Fig. 16 depicts the effect of a control antibody or Dara alone or in combination with tamibarotene on the number of apoptotic cells in two different multiple myeloma cell line co-cultured with NK cells.

[0049] Fig. 17 depicts the effect of a control antibody or Dara alone or in combination with tamibarotene on interferon-gamma secretion in a NK cell/multiple myeloma co-culture for the various multiple cell lines depicted in Figs. 15A-15B and 16.

[0050] Fig. 18A depicts the effect of tamibarotene treatment on the expression of

CD38 in various AML and MDS patient samples after 24 or 48 hours. Fig. 18B depicts the effect of tamibarotene treatment on the expression of CD38 in various AML and MDS patient samples after 24 or 48 hours as measured by FACS, as well as the expression of RARA and IRF8 mRNA as measured by qPCR.

[0051] Figs. 19A-19D compares the effect of tamibarotene treatment on CD38 levels in an AML cell line, an AML patient sample, and a multiple myeloma cell line, as well as the baseline CD38 level in a CD38 multiple myeloma patient sample. [0052] Figs 20A-20D compare the effect of tamibarotene and all-trans retinoic acid

(ATRA) on CD38 levels in a mouse MV4-11 xenograft model after one week (Figs. 20A and 20B) and three weeks (Figs. 20C and 20D). Tumor cells were tested for the presence of CD38 by both FACS (Figs. 20A and 20C) and immunohistochemical (IHC) staining (Figs. 20B and 20D).

[0053] Fig. 21 compares the effect of tamibarotene and all- trans retinoic acid (ATRA) on CD38 levels in a mouse THP-1 xenograft model after one week as measured by FACS.

[0054] Fig. 22 compares the effect of tamibarotene and all-trans retinoic acid (ATRA) on CD38 levels in a mouse Kasumi-1 xenograft model after one week as measured by FACS.

[0055] Fig. 23 compares the effect of tamibarotene and all-trans retinoic acid (ATRA) on CD38 levels in mouse THP-1, Kasumi-1 and MV4-11 xenograft models after one week as measured by IHC.

DEFINITIONS

[0056] As used herein, the terms "administer," "administering," or "administration," as used herein refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing an inventive compound, or a pharmaceutical composition thereof.

[0057] As used herein, the term the term "agonist" may be used to refer to an agent, condition, or event whose presence, level, degree, type, or form correlates with increased level or activity of another agent (i.e. , the agonized agent). In general, an agonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant activating activity. In some embodiments, an agonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an agonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g. , by interacting with a regulator of the target, so that level or activity of the target is altered).

[0058] The term "biological sample" refers to any sample including tissue samples

(such as tissue sections and needle biopsies of a tissue); cell samples (e.g. , cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsied tissue (e.g. , obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. Biological samples also include those biological samples that are transgenic, such as transgenic oocyte, sperm cell, blastocyst, embryo, fetus, donor cell, or cell nucleus. In some aspects, a biological sample from a subject suffering from AML, MDS or MM is a bone marrow aspirate.

[0059] As used herein, the term "biomarker" refers to an entity whose presence, level, or form, correlates with a particular biological event or state of interest, so that it is considered to be a "marker" of that event or state. To give but a few examples, in some embodiments, a biomarker may be or comprises a marker for a particular disease state or stage, or for likelihood that a particular disease, disorder or condition may develop. In some embodiments, a biomarker may be or comprise a marker for a particular disease or therapeutic outcome, or likelihood thereof. Thus, in some embodiments, a biomarker is predictive, in some embodiments, a biomarker is prognostic, in some embodiments, a biomarker is diagnostic, of the relevant biological event or state of interest. A biomarker may be an entity of any chemical class. For example, in some embodiments, a biomarker may be or comprise a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (e.g. , a metal or ion), or a combination thereof. In some embodiments, a biomarker is a cell surface marker. In some embodiments, a biomarker is intracellular. In some embodiments, a biomarker is found outside of cells (e.g. , is secreted or is otherwise generated or present outside of cells, e.g. , in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc. In some embodiments, term refers to a gene expression product that is characteristic of a particular tumor, tumor subclass, stage of tumor, etc. Alternatively or additionally, in some embodiments, a presence or level of a particular marker correlates with activity (or activity level) of a particular signaling pathway, for example that may be characteristic of a particular class of tumors. The statistical significance of the presence or absence of a biomarker may vary depending upon the particular biomarker. In some embodiments, detection of a biomarker is highly specific in that it reflects a high probability that the tumor is of a particular subclass. Such specificity may come at the cost of sensitivity (e.g. , a negative result may occur even if the tumor is a tumor that would be expected to express the biomarker). In some embodiments, a biomarker comprises a RARA biomarker (e.g., one or more RARA biomarkers (e.g. , presence, level, form, and/or activity of one or more RARA gene components or products, including for example RARA super enhancer strength, ordinal rank, or prevalence rank and RARA mRNA level or prevalence rank). In some embodiments, a biomarker comprises an IRF8 biomarker (e.g., presence, level, form, and/or activity of one or more IRF8 gene components or products, including for example IRF8 super enhancer strength, ordinal rank, or prevalence rank and IRF8 mRNA level or prevalence rank). In some embodiments, a biomarker refers to a combination of one or more biomarkers, such as a RARA biomarker or an IRF8 biomarker.

[0060] The terms "co-administer" or "co-administering" as used herein in the context of the administration of therapies (e.g., a RARA agonist and an antibody specific for CD38), indicates that one therapy may be used in combination with another therapy or therapies during the course of the subject's affliction with the disorder. In some embodiments, the administration of therapies is simultaneous or concurrent, meaning that the delivery of one treatment is still occurring when the delivery of the second begins. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. Here, a RARA agonist and an antibody specific to CD38 can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the RARA agonist can be administered first, and the antibody specific to CD38 can be administered second, or the order of administration can be reversed.

[0061] As used herein, the terms "condition," "disease," and "disorder" are used interchangeably.

[0062] An "effective amount" of a compound described herein, such a RARA agonist and/or an antibody specific for CD38, refers to an amount sufficient to elicit the desired biological response, i.e. , treating the condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound described herein, such a RARA agonist and/or an antibody specific for CD38, may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In some embodiments, an effective amount encompasses therapeutic and prophylactic treatment. In other embodiments, an effective amount encompasses only therapeutic treatment. For example, in treating cancer, an effective amount of an inventive compound or composition may reduce the tumor burden or stop the growth or spread of a tumor. [0063] A "subject" to which administration is contemplated includes, but is not limited to, humans (i.e. , a male or female of any age group, e.g. , a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g. , young adult, middle-aged adult, or senior adult)) and/or other non-human animals, for example, mammals (e.g. , primates (e.g. , cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g. , commercially relevant birds such as chickens, ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal. In certain embodiments, the subject is a human.

[0064] A "therapeutically effective amount" of a compound described herein, such a

RARA agonist and/or an antibody specific for CD38, is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0065] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a "pathological condition" (e.g. , a disease, disorder, or condition, or one or more signs or symptoms thereof) described herein. In some embodiments, "treatment," "treat," and "treating" require that signs or symptoms of the disease disorder or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g. , in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0066] The term "RARA gene" refers to a genomic DNA sequence that encodes a functional retinoic acid receptor-a gene and specifically excludes gene fusions that comprise all or a portion of the RARA gene. In some embodiments, the RARA gene is located at

chrl7:38458152-38516681in genome build hgl9.

[0067] The term "IRF8 gene" refers to a genomic DNA sequence that encodes an interferon consensus sequence-binding protein or splice variant thereof and specifically excludes gene fusions that comprise all or a portion of the IRF8 gene. In some embodiments, the IRF8 gene is located at chrl6:85862582-85990086 in genome build hgl9.

[0068] The term "enhancer" refers to a region of genomic DNA acting to regulate genes up to 1 Mbp away. An enhancer may overlap, but is often not composed of, gene coding regions. An enhancer is often bound by transcription factors and designated by specific histone marks.

[0069] The term "super enhancer" refers to a subset of enhancers that contain a disproportionate share of histone marks and/or transcriptional proteins relative to other enhancers in a particular cell. Because of this, a gene regulated by a super enhancer is predicted to be of high importance to the function of that cell. Super enhancers are typically determined by rank ordering all of the enhancers in a cell based on strength and determining using available software such as ROSE (https://bitbucket.org/young_computation/rose), the subset of enhancers that have significantly higher strength than the median enhancer in the cell (see, e.g., United States patent 9,181,580, which is herein incorporated by reference).

[0070] The term "strength" when referring to a portion of an enhancer or a super enhancer, as used herein means the area under the curve of the number of H3K27Ac or other genomic marker reads plotted against the length of the genomic DNA segment analyzed. Thus, "strength" is an integration of the signal resulting from measuring the mark at a given base pair over the span of the base pairs defining the region being chosen to measure.

[0071] The term "prevalence rank" for a specified value (e.g., the strength of a super enhancer associated with a RARA gene) means the percentage of a population that are equal to or greater than that specific value. For example, a 35% prevalence rank for the strength of a super enhancer associated with a RARA gene in a test cell means that 35% of the population have a RARA gene enhancer with a strength equal to or greater than the test cell.

[0072] The term "prevalence cutoff for a specified value (e.g., the strength of a super enhancer associated with a RARA gene) means the prevalence rank that defines the dividing line between two subsets of a population (e.g., responders and non-responders). Thus, a prevalence rank that is equal to or higher (i.e., a lower percentage value) than the prevalence cutoff defines one subset of the population; and a prevalence rank that is lower (e.g., a higher percentage value) than the prevalence cutoff defines the other subset of the population.

[0073] The terms "cutoff and "cutoff value" mean a value measured in an assay that defines the dividing line between two subsets of a population (e.g., responders and non- responders). Thus, a value that is equal to or higher than the cutoff value defines one subset of the population; and a value that is lower than the cutoff value defines the other subset of the population.

[0074] The terms "threshold" and "threshold level" mean a level that defines the dividing line between two subsets of a population (e.g., responders and non-responders). A threshold level may be a prevalence cutoff or a cutoff value.

[0075] The term "population" or "population of samples" means a sufficient number

(e.g., at least 30, 40, 50 or more) of different samples that reasonably reflects the distribution of the value being measured in a larger group. Each sample in a population of samples may be a cell line, a biological sample obtained from a living being (e.g., a biopsy or bodily fluid sample), or a sample obtained from a xenograft (e.g., a tumor grown in a mouse by implanting a cell line or a patient sample), wherein each sample is from a living being suffering from or from a cell line or xenograft representing, the same disease, condition or disorder.

[0076] The term "ordinal rank" of a specified value means the rank order of that value as compared to a set of other values. For example, an ordinal rank of 100 in terms of the strength of a super enhancer associated with a RARA gene in a test cell as compared to other super enhancers in the test cell means that 99 other super enhancers in the test cell had greater strength than the super enhancer associated with a RARA gene.

[0077] The term "rank ordering" means the ordering of values from highest to lowest or from lowest to highest. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

RARA and IRF8

[0078] The present invention features methods for treating a subject having cancer (e.g., non- APL AML, MDS or MM) with a RARA agonist in combination with an antibody specific for CD38, for example, by determining the prevalence of a RARA biomarker and/or an IRF8 biomarker (e.g., determining the level, strength, ordinal rank, prevalence rank and/or activity of one or more RARA or IRF8 gene components or products, including for example RARA or IRF8 super enhancer strength, ordinal rank, or prevalence rank and RARA or IRF8 mRNA level or prevalence rank) and co-administering a RARA agonist and an antibody specific for CD38 accordingly.

[0079] The retinoic acid receptor subtype alpha (RARA) is a nuclear hormone receptor that acts as a transcriptional repressor when unbound or bound by an antagonist, and as a gene activator in the agonist-bound state. The natural ligand of RARA is retinoic acid, also known as all-trans retinoic acid (ATRA), which is produced from vitamin A.

[0080] Super-enhancers (SEs) are large, highly-active chromatin regions that regulate key cell identity genes, including oncogenes in malignant cells. Using a gene control platform, SEs were identified genome- wide in 60 primary AML patient samples that enabled the discovery of novel tumor vulnerabilities. One of the SEs that exhibited a differential presence among patient samples was associated with the RARA gene encoding RARA.

[0081] Using various AML cell lines and patient samples previously analyzed for strength and ordinal of RARA enhancers, interferon response factor 8 (IRF8) mRNA levels were found to be upregulated in similar patient populations as RARA. IRF8 is an interferon responsive transcription factor known to be critical to hematopoiesis and whose signaling loss causes aberrant expansion of immature myeloid cells. In AML, IRF8 overexpression is observed and may correlate with poor clinical outcome. Despite this upregulation, IRF8 signaling is actually impaired by repressive transcriptional cofactors and potentially RARA when it is in a SE-driven repressive state. Furthermore, interferon-oc itself, the upstream signaling ligand for IRFs, exhibits pro-differentiation effects in AML and signaling cross-talk with the RARA pathway.

[0082] The present disclosure describes recent findings that a RARA agonist (e.g., tamibarotene) may induce CD38 upregulation in non-APL AML, MDS or MM in a RARA or IRF8 SE-dependent manner. In addition, it provides various compositions and methods useful in, among other things, characterizing, identifying, selecting, or stratifying patients based on likely responsiveness to co-administration of tamibarotene and an antibody specific for CD38.

RARA and IRF8 Super-Enhancer Identification and Determination of Threshold Levels

[0083] Described herein are methods to treat a cancer by administering a RARA agonist (e.g., tamibarotene) and an antibody specific for CD38 upon determination of the presence of a RARA biomarker and/or an IRF8 biomarker (e.g., the determination of the prevalence, level, form, and/or activity of one or more RARA or IRF8 gene components or products, including for example RARA or IRF8 super enhancer strength, ordinal rank, or prevalence rank and RARA or IRF8 mRNA level or prevalence rank).

[0084] The identification of an enhancer or super enhancer may be achieved by various methods known in the art, for example as described in Cell 2013, 155, 934-947 and PCT/US2013/066957, both of which are incorporated herein by reference. In some embodiments, the identification of a super enhancer is achieved by obtaining cellular material and DNA from a cancer sample in a patient (e.g., from a biopsy). The important metrics for enhancer measurement occur in two dimensions— the length of the DNA over which genomic markers (e.g., H3K27Ac) are contiguously detected— and the compiled incidence of genomic marker at each base pair along that span of DNA constituting the magnitude. The measurement of the area under the curve ("AUC") resulting from integration of length and magnitude analysis determines the strength of the enhancer. It is the strength of the IRF8 or RARA super enhancer relative to a control that is used in one aspect of the present invention to determine whether or not a subject will be responsive a RARA agonist (e.g., tamibarotene) and an antibody specific for CD38. It will be readily apparent to those of skill in the art that if the length of DNA over which the genomic markers is detected is the same for both RARA or IRF8 and the control, then the ratio of the magnitude of the RARA or IRF8 super enhancer relative to the control will be equivalent to the strength and may also be used to determine whether or not a subject will be responsive to a RARA agonist and an antibody specific for CD38. In some embodiments, the strength of the RARA or IRF8 enhancer in a cell is normalized before comparing to other samples.

Normalization is achieved by comparison to a region in the same cell known to comprise a ubiquitous super-enhancer or enhancer that is present at similar levels in all cells. One example of such a ubiquitous super-enhancer region is the MALAT1 super-enhancer locus

(chrl 1 :65263724-65266724) (genome build hgl9).

[0085] It has been determined through H3K27Ac ChlP-seq methods that there is a super-enhancer locus associated with the RARA gene at chrl7:38458152-38516681 (genome build hgl9). ChlP-sequencing, also known as ChlP-seq, is used to analyze protein interactions with DNA. ChlP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChlP-on-chip was the most common technique utilized to study these protein-DNA relations. Successful ChlP-seq is dependent on many factors including sonication strength and method, buffer compositions, antibody quality, and cell number.; see, e.g., T. Furey, Nature Reviews Genetics 13, 840-852 (December 2012); M.L. Metzker, Nature Reviews Genetics 11, 31-46 (January 2010); and P.J Park, Nature Reviews Genetics 10, 669-680 (October 2009)) . Genomic markers other that H3K27Ac that can be used to identify super enhancers using ChlP-seq include, P300, CBP, BRD2, BRD3, BRD4, components of the mediator complex (J Loven, et al., Cell, 153(2):320- 334, 2013), histone 3 lysine 4 monomethylated (H3K4mel), or other tissue specific enhancer tied transcription factors (E Smith & A Shilatifard, Nat Struct Mol Biol, 21(3):210-219, 2014) (S Pott & Jason Lieb, Nature Genetics, 47(1):8-12, 2015).

[0086] In some embodiments, H3K27ac or other marker ChlP-seq data super-enhancer maps of the entire genome of a cell line or a patient sample already exist. In these embodiments, one would simply determine whether the strength, or ordinal rank of the enhancer or super- enhancer in such maps at the chrl7:38458152-38516681 (genome build hgl9) locus was equal to or above the pre-determined threshold level.

[0087] It should be understood that the specific chromosomal location of RARA, IRF8, and MALAT1 may differ for different genome builds and/or for different cell types. However, one of skill in the art, in view of the instant specification, can determine such different locations by locating in such other genome builds specific sequences corresponding to the RARA and/or MALAT1 loci in genome build hg 19.

[0088] Other methods for identifying super enhancers include chromatin

immunoprecipitation (JE Delmore, et al., Cell, 146(6)904-917, 2011) and chip array (ChlP-chip), and chromatin immunoprecipitation followed by qPCR (ChlP-qPCR) using the same

immunoprecipitated genomic markers and oligonucleotide sequences that hybridize to the chrl7:38458152-38516681 (genome build hgl9) RARA locus or chrl6:85862582-85990086 (genome build hgl9) IRF8 locus. In the case of ChlP-chip, the signal is typically detected by intensity fluorescence resulting from hybridization of a probe and input assay sample as with other array based technologies. For ChlP-qPCR, a dye that becomes fluorescent only after intercalating the double stranded DNA generated in the PCR reaction is used to measure amplification of the template.

[0089] In some embodiments, determination of whether a cell has a RARA or IRF8 super enhancer above a requisite threshold level is achieved by comparing RARA or IRF8 enhancer strength in a test cell to the corresponding RARA or IRF8 strength in a cell known to not respond to RARA or IRF8 (a "control cell"). Preferably the control cell is the same cell type as the test cell. In one aspect of these embodiments, the control cell is such cell in a HCC1143.

[0090] In some embodiments, determination of whether a cell has a RARA or IRF8 super enhancer strength above a requisite threshold level is achieved by comparing RARA or IRF8 enhancer strength in a test cell to the corresponding RARA or IRF8 strength in a population of cell samples, wherein each of the cell samples is obtained from a different source (i.e., a different subject, a different cell line, a different xenograft). In some aspects of these embodiments, only primary tumor cell samples from subjects are used to determine the threshold level. In some aspects of these embodiments, at least some of the samples in the population will have been tested for responsiveness to a specific RARA agonist and an antibody specific for

CD38 in order to establish: a) the lowest RARA enhancer strength of a sample in the population that responds to that specific RARA agonist and an antibody specific for CD38 ("lowest responder"); and/or b) the lowest IRF8 enhancer strength of a sample in the population that responds to that specific RARA agonist and an antibody specific for CD38 ("lowest responder"); and optionally, c) the highest RARA enhancer strength of a sample in the population that does not respond to that specific RARA agonist and an antibody specific for CD38 ("highest non- responder") and/or d) the highest IRF8 enhancer strength of a sample in the population that does not respond to that specific RARA agonist and an antibody specific for CD38 ("highest non- responder"). In these embodiments, a cutoff of RARA or IRF8 enhancer strength above which a test cell would be considered responsive to that specific RARA agonist and an antibody specific for CD38 is set: i) equal to or up to 5% above the RARA or IRF8 enhancer strength in the lowest responder in the population; or ii) equal to or up to 5% above the RARA or IRF8 enhancer strength in the highest non-responder in the population; or iii) a value in between the RARA or IRF8 enhancer strength of the lowest responder and the highest non-responder in the population.

[0091] It should be understood that in the above embodiments typically not all of the samples in a population need to be tested for responsiveness to the RARA agonist and an antibody specific for CD38, but all samples are measured for RARA or IRF8 enhancer strength. In some embodiments, the samples are rank ordered based on RARA or IRF8 enhancer strength. The choice of which of the three methods set forth above to use to establish the cutoff will depend upon the difference in RARA or IRF8 enhancer strength between the lowest responder and the highest non-responder in the population and whether the goal is to minimize the number of false positives or to minimize the chance of missing a potentially responsive sample or subject. When the difference between the lowest responder and highest non-responder is large (e.g., when there are many samples not tested for responsiveness that fall between the lowest responder and the highest non-responder in a rank ordering of RARA or IRF8 enhancer strength), the cutoff is typically set equal to or up to 5% above the RARA or IRF8 enhancer strength in the lowest responder in the population. This cutoff maximizes the number of potential responders. When this difference is small (e.g., when there are few or no samples untested for

responsiveness that fall between the lowest responder and the highest non-responder in a rank ordering of RARA or IRF8 enhancer strength), the cutoff is typically set to a value in between the RARA or IRF8 enhancer strength of the lowest responder and the highest non-responder. This cutoff minimizes the number of false positives. When the highest non-responder has a RARA or IRF8 enhancer strength that is greater than the lowest responder, the cutoff is typically set to a value equal to or up to 5% above the RARA or IRF8 enhancer strength in the highest non- responder in the population. This method also minimizes the number of false positives.

[0092] In some embodiments, determination of whether a cell has a RARA or IRF8 super enhancer above a requisite threshold level is achieved by comparing the ordinal of RARA enhancer strength in a test cell to the ordinal of RARA or IRF8 enhancer strength in a population of cell samples, wherein each of the cell samples is obtained from a different source (i.e., a different subject, a different cell line, a different xenograft). In these embodiments, at least some of the samples in the population will have been tested for responsiveness to a specific RARA agonist in order to establish: a) the lowest RARA enhancer strength ordinal of a sample in the population that responds to that specific RARA agonist and an antibody specific for CD38 ("lowest ordinal responder"); b) the lowest IRF8 enhancer strength ordinal of a sample in the population that responds to that specific RARA agonist and an antibody specific for CD38

("lowest ordinal responder"); and, optionally, c) the highest RARA enhancer strength ordinal of a sample in the population that does not respond to that specific RARA agonist and an antibody specific for CD38 ("highest ordinal non-responder") and/or d) the highest RARA enhancer strength ordinal of a sample in the population that does not respond to that specific RARA agonist and an antibody specific for CD38 ("highest ordinal non-responder"). In these embodiments, a cutoff of RARA or IRF8 enhancer strength ordinal above which a test cell would be considered responsive to that specific RARA agonist and an antibody specific for CD38 is set: i) equal to or up to 5% above the RARA or IRF8 enhancer strength ordinal in the lowest ordinal responder in the population; or ii) equal to or up to 5% above the RARA or IRF8 enhancer strength ordinal in the highest ordinal non-responder in the population; or iii) a value in between the RARA or IRF8 enhancer strength ordinal of the lowest ordinal responder and the highest ordinal non-responder in the population.

[0093] It should be understood in the above embodiments, that typically not all of the samples in a population need to be tested for responsiveness to the RARA agonist and an antibody specific for CD38, but all samples are measured for RARA or IRF8 enhancer strength and the ordinal of RARA or IRF8 enhancer strength compared to other enhancers in the same sample is established. The ordinal is typically obtained by measuring the strength of all other enhancers in the cell and determining what rank (i.e., the ordinal) in terms of strength the RARA or IRF8 enhancer has as compared to the other enhancers.

[0094] In some embodiments, the samples are rank ordered based on the ordinal of

RARA enhancer strength. In some embodiments, the samples are rank ordered based on the ordinal of IRF8 enhancer strength. The choice of which of the methods set forth above to use to establish the cutoff will depend upon the difference in ordinal of RARA or IRF8 enhancer strength between the lowest ordinal responder and the highest ordinal non-responder in the population and whether the cutoff is designed to minimize false positives or maximize the number of responders. When this difference is large (e.g., when there are many samples not tested for responsiveness that fall between the lowest ordinal responder and the highest ordinal non-responder in a rank ordering of ordinals of RARA or IRF8 enhancer strength), the cutoff is typically set equal to or up to 5% above the ordinal of RARA or IRF8 enhancer strength in the lowest ordinal responder in the population. When this difference is small (e.g., when there are few or no samples untested for responsiveness that fall between the lowest ordinal responder and the highest ordinal non-responder in a rank ordering of ordinal of RARA or IRF8 enhancer strength), the cutoff is typically set to a value in between the ordinal of RARA or IRF8 enhancer strength of the lowest ordinal responder and the highest ordinal non-responder. When the highest ordinal non-responder has an ordinal of RARA or IRF8 enhancer strength that is greater than that of the lowest responder, the cutoff is typically set to a value equal to or up to 5% above the ordinal of RARA or IRF8 enhancer strength in the highest ordinal non-responder in the population.

[0095] In some aspects of embodiments where a test cell or sample is compared to a population, the cutoff value(s) obtained for the population (e.g., RARA enhancer strength or

RARA enhancer ordinal and/or IRF8 enhancer strength or IRF8 enhancer ordinal) is converted to a prevalence rank and the cutoff is expressed as a percent of the population having the cutoff value or higher, i.e., a prevalence cutoff. Without being bound by theory, applicants believe that the prevalence rank of a test sample will be similar regardless of the methodology used to determine RARA or IRF8 enhancer strength. Thus, a prevalence cutoff determined for one parameter (e.g., RARA enhancer strength ordinal or IRF8 enhancer strength ordinal) is portable and can be applied to another parameter (e.g., RARA mRNA level or IRF8 mRNA level) to determine the cutoff value for that other parameter. This allows the determination of a cutoff value for any parameter without having to experimentally determine the correlation between levels of such parameter and responsiveness to the RARA agonist and an antibody specific for CD38.

RARA and IRF8 mRNA Level Determination

[0096] The present invention features methods for treating a cancer with a RARA agonist (e.g., tamibarotene) and an antibody specific for CD38 when a RARA biomarker and/or an IRF8 biomarker are present. It has been determined that the identification of the RARA and/or IRF8 super enhancer loci allows one to use a RNA transcripts to determine sensitivity instead of super-enhancer level to determine sensitivity to a RARA agonist and an antibody specific for CD38. RNA transcripts from the super-enhancer locus itself may be quantified and correlate very well with super-enhancer levels at that locus. It has also been shown that mRNA transcripts encoding RARA also correlate with sensitivity to RARA agonists alone or in combination with an antibody specific for CD38, and thus mRNA levels can be used to identify cells that will respond to this combination of therapies. Thus, in some embodiments, RARA or IFR8 mRNA levels may be used instead of super-enhancer strength or ordinal rank to determine sensitivity to a RARA agonist and an antibody specific for CD38. [0097] In some embodiments, the RNA transcript level from the super-enhancer locus is quantified using quantitative techniques that compare RARA or IRF8 enhancer RNA transcript levels in a sample with corresponding RARA or IRF8 enhancer RNA transcript levels in a cell or cell line known to be non-responsive to a RARA agonist and an antibody specific for CD38. Such methods include RNA array or sequencing based methods for reading the eRNA associated with enhancer read through (N Hah et al., PNAS, 112(3):E297-302, 2015), as well as RNA qPCR.

[0098] In alternate embodiments, the RARA or IRF8 mRNA levels in a subject (i.e., in a tumor sample, in a cancer cell sample, in a blood sample, etc.) are compared, using RNA-Seq or RNA-qPCR techniques, to the RARA or IRF8 mRNA levels in a population of subjects having the same disease or condition to identify responders to a RARA agonist and an antibody specific for CD38. In these embodiments, at least some of the samples in the population will have been tested for responsiveness to a specific RARA agonist and an antibody specific for CD38 in order to establish: a) the lowest RARA mRNA level of a sample in the population that responds to that specific RARA agonist and an antibody specific for CD38 ("lowest mRNA responder"); and/or b) the lowest IRF8 mRNA level of a sample in the population that responds to that specific RARA agonist and an antibody specific for CD38 ("lowest mRNA responder"); and, optionally, c) the highest RARA mRNA level of a sample in the population that does not respond to that specific RARA agonist and an antibody specific for CD38 ("highest mRNA non- responder") and/or d) the highest IRF8 mRNA level of a sample in the population that does not respond to that specific RARA agonist and an antibody specific for CD38 ("highest mRNA non- responder"). In these embodiments, a cutoff of RARA or IRF8 mRNA level above which a test cell would be considered responsive to that specific RARA agonist and an antibody specific for CD38 is set: i) equal to or up to 5% above the RARA or IRF8 mRNA level in the lowest mRNA responder in the population; or ii) equal to or up to 5% above the RARA or IRF8 mRNA level in the highest mRNA non-responder in the population; or iii) a value in between the RARA or IRF8 mRNA level of the lowest mRNA responder and the highest mRNA non-responder in the population.

[0099] In some embodiments, not all of the samples in a population need to be tested for responsiveness to a RARA agonist and an antibody specific for CD38, but all samples are measured for RARA or IRF8 mRNA levels. In some embodiments, the samples are rank ordered based on RARA mRNA levels. In some embodiments, the samples are rank ordered based on IRF8 mRNA levels. The choice of which of the three methods set forth above to use to establish the cutoff will depend upon the difference in RARA or IRF8 mRNA levels between the lowest mRNA responder and the highest mRNA non-responder in the population and whether the cutoff is designed to minimize false positives or maximize the potential number of responders. When this difference is large (e.g., when there are many samples not tested for responsiveness that fall between the lowest mRNA responder and the highest mRNA non- responder in a rank ordering of RARA or IRF8 mRNA levels), the cutoff is typically set equal to or up to 5% above the RARA or IRF8 mRNA level in the lowest mRNA responder in the population. When this difference is small (e.g., when there are few or no samples untested for responsiveness that fall between the lowest mRNA responder and the highest mRNA non- responder in a rank ordering of RARA or IRF8 mRNA levels), the cutoff is typically set to a value in between the RARA or IRF8 mRNA levels of the lowest mRNA responder and the highest mRNA non-responder. When the highest mRNA non-responder has RARA or IRF8 mRNA levels that are greater than the lowest mRNA responder, the cutoff is typically set to a value equal to or up to 5% above the RARA or IRF8 mRNA levels in the highest mRNA non- responder in the population.

[00100] In some embodiments, the population is rank ordered based on RARA mRNA level. In some embodiments, the population is rank ordered based on IRF8 mRNA level. In these embodiments, the RARA or IRF8 mRNA level in each sample is measured and compared to the mRNA levels of all other mRNAs in the cell to obtain an ordinal ranking of the RARA or IRF8 mRNA level. A cutoff based on RARA or IRF8 mRNA ordinal ranking is then determined based on samples in the population tested for responsiveness to a RARA agonist in the same manner as described previously for determining a RARA or IFR8 super enhancer strength ordinal cutoff. The determined RARA or IRF8 mRNA ordinal cutoff is then used either directly or to determine a prevalence cutoff, either of which is then used to stratify additional samples for potential responsiveness to the RARA agonist and the antibody specific to CD38.

[00101] In some embodiments, the cutoff for RARA or IRF8 mRNA levels is determined using the prevalence cutoff established based on RARA enhancer strength or RARA enhancer strength ordinal, or IRF8 enhancer strength or IRF8 enhancer strength ordinal as described above. In some aspects of these embodiments, a population is measured for mRNA levels and the prior determined prevalence cutoff is applied to that population to determine an mRNA cutoff level. In some aspects of these embodiments a rank-order standard curve of RARA or IRF8 mRNA levels in a population is created, and the pre-determined prevalence cutoff is applied to that standard curve to determine the RARA or IRF8 mRNA cutoff level.

[00102] In some aspects of embodiments where a test cell or sample is compared to a population, the cutoff mRNA level value(s) obtained for the population is converted to a prevalence rank and the mRNA level cutoff is expressed as a percent of the population having the cutoff value or higher, i.e., a prevalence cutoff. [00103] Without being bound by theory, applicants believe that the prevalence rank of a test sample and the prevalence cutoff in a population will be similar regardless of the methodology used to determine or IRF8 RARA mRNA levels.

[00104] In some aspects of these embodiments, a subject is identified as a RARA agonist responder if its RARA or IRF8 mRNA level corresponds to a prevalence rank in a population of 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%, 42%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20% as determined by RARA or IRF8 mRNA levels in the population. In one aspect of these embodiments, the cutoff value is established based on the prevalence cutoff established for RARA or IRF8 enhancer strength. In an alternate aspect of these embodiments, the cutoff value is established based on the prevalence cutoff established for RARA or IRF8 enhancer strength ordinal. In another alternate aspect of these embodiments, the cutoff value is established based on RARA or IRF8 mRNA levels. In other more specific aspects of these embodiments, a cutoff value for AML patients is established based on the prevalence value determined for RARA or IRF8 enhancer strength ordinal, and that prevalence value is used to determine the cutoff value for RARA or IRF8 mRNA levels In even more specific aspects of these embodiments, the cutoff value for AML patients is determined using a prevalence cutoff of between 25-45%,e.g., between 25-30%, 25-35%, 25-40%, 30-35%, 30-40%, 35-45%, 35-40%, 31-35%, 32-35%, 33- 35%, 34-35%, 31-36%, 32-36%, 33-36%, 34-36%, or 35-36%. In other even more specific aspects of these embodiments, the cutoff value for AML patients is determined using a prevalence value of 36%. In yet other even more specific aspects of these embodiments, the cutoff value for AML patients is determined using a prevalence value of 25%.

[00105] In still other embodiments, a population may be divided into three groups— responders, partial responders and non-responders and two cutoff values or prevalence cutoffs are set. The partial responder group may include responders and non-responders, as well as those population members whose response to a RARA agonist and an antibody specific to CD38 was not as high as the responder group. In these embodiments, two cutoff values or prevalence cutoffs are determined. This type of stratification may be particularly useful when in a population the highest RARA or IRF8 mRNA non-responder has a RARA or IRF8 mRNA levels that are greater than the lowest RARA mRNA responder. In this scenario, the cutoff level or prevalence cutoff between responders and partial responders is set equal to or up to 5% above the RARA or IRF8 mRNA level of the highest RARA or IRF8 mRNA non-responder; and the cutoff level or prevalence cutoff between partial responders and non-responders is set equal to or up to 5% below the RARA or IRF8 mRNA level of the lowest RARA or IRF8 mRNA responder. The determination of whether partial responders should be administered the RARA agonist and an antibody specific to CD38 will depend upon the judgment of the treating physician and/or approval by a regulatory agency.

[00106] Methods of quantifying specific RNA sequences in a cell or biological sample are known in the art and include, but are not limited to, fluorescent hybridization such as utilized in services and products provided by NanoString Technologies, array based technology

(Affymetrix), reverse transcriptase qPCR as with SYBR® Green (Life Technologies) or TaqMan® technology (Life Technologies), RNA sequencing (e.g., RNA-seq), RNA

hybridization and signal amplification as utilized with RNAscope® (Advanced Cell

Diagnostics), or northern blot.

[00107] In some aspects of these embodiments, the level of RNA transcript (either mRNA or another RARA or IRF8 transcript) in both the test cell and the control cell or all members of the population are normalized before comparison. Normalization involves adjusting the determined level of a RARA or IRF8 RNA transcript by comparison to either another RNA transcript that is native to and present at equivalent levels in both of the cells (e.g., GADPH mRNA, 18S RNA), or to a fixed level of exogenous RNA that is "spiked" into samples of each of the cells prior to super-enhancer strength determination (J Loven et al., Cell, 151(3):476-82 (2012); J Kanno et al., BMC Genomics 7:64 (2006); J Van de Peppel et al., EMBO Rep 4:387- 93 (2003)).

RARA Agonists and Antibodies Targeting CD38

[00108] The present invention features methods of treating a subject having cancer (e.g., non-APL AML, MDS or MM) with a RARA agonist in combination with an antibody specific for CD38, for example, when a biomarker for RARA or IRF8 is identified

RARA Agonists

[00109] The choice of RARA agonist with which to treat a patient identified as having a super enhancer associated with a RARA gene may be made from any RARA agonist known in the art. It is preferable that the RARA agonist utilized in the methods of the invention be specific for RARA and have significantly less (at least 10X less, at least 100X less, at least

1,000X less, at least 10,000X less, at least 100,000X less) agonistic activity against other forms of RaR, e.g., RaR-β and RaR-γ.

[00110] In some embodiments, the RARA agonist is selected from a compound disclosed in or any compound falling within the genera set forth in any one of the following United States patents: US 4,703,110, US 5,081,271, US 5,089,509, US 5,455,265, US 5,759,785, US 5,856,490, US 5,965,606, US 6,063,797, US 6,071,924, US 6,075,032, US 6, 187,950, US 6,355,669, US 6,358,995, and US 6,387,950, each of which is incorporated by reference.

[00111] In some embodiments, the RARA agonist is selected from any of the following known RARA agonists set forth in Table 1, or a pharmaceutically acceptable salt thereof, or a solvate or hydrate of the foregoing:

Table 1. Exemplary RARA Agonists useful in the invention.

Structure Code Name(s)

0

0

0

A-112

o ¹

BD-4; BJ-1

0 Tazarotene;

AGN-190168

embodiments, the RARA agonist is O F (AGN-195183). Antibodies Specific for CD38

[00113] CD38 is a transmembrane glycoprotein involved in a number of metabolic functions including the catabolism of extracellular nucleotides, receptor-mediated adhesion, regulation of migration, and a variety of signaling events. Antibodies targeting CD38 may modulate any one of these functions, thus contributing to the efficacy of a RARA agonist in the treatment of a cancer. In some embodiments, the antibody specific for CD38 may recognize and/or bind to any portion of fragment of CD38. The antibody specific for CD38 may comprise a monoclonal antibody, a humanized antibody, or a human antibody. Exemplary antibodies that are specific for CD38 include isatuximab, daratumumab, MOR202, Ab79, Abl9, and EPR4106. In some embodiments, the antibody specific for CD38 is daratumumab.

Therapeutic Regimens

[00114] The methods of the present invention are theoretically useful to treat a cancer that is characterized by the association of a RARA biomarker or an IRF8 biomarker (e.g., the presence, level, form, and/or activity of one or more RARA or IRF8 gene components or products, including for example RARA or IRF8 super enhancer strength, ordinal rank, or prevalence rank and RARA or IRF8 mRNA level or prevalence rank). Super enhancer-associated RARA or IRF8 genes may be more prevalent in certain types of cancers than others. The present invention in particular is directed to treatment of AML, e.g., non-APL AML and in other forms of AML that are not characterized by a chromosomal translocation involving a RARA gene, as well as MDS and MM. In some embodiments, the disease to be treated is non-APL AML, MDS or MM that is not characterized by a chromosomal translocation involving an RARA gene. In some embodiments, the disease to be treated is non-APL AML, MDS or MM that is not characterized by a chromosomal translocation involving an IRF8 gene.

[00115] In some embodiments, the subject to be treated with a RARA agonist (e.g. , tamibarotene) and an antibody specific for CD38 is suffering from relapsed or refractory non- APL AML. A subject is classified as having relapsed or refractory non-APL AML if they: a) do not demonstrate a partial response after a first cycle of induction chemotherapy; or b) do not demonstrate a complete response after a second cycle of induction chemotherapy; or c) relapse after conventional chemotherapy; or d) relapse are undergoing a single stem cell transplantation. In some embodiments, the subject to be treated with a RARA agonist (e.g. , tamibarotene) and an antibody specific for CD38 is suffering from refractory MM.

[00116] In other embodiments, the subject to be treated with a RARA agonist (e.g. , tamibarotene) is an elderly unfit subject. The term "elderly unfit" as used herein means the subject is a human at least 60 years of age and who is determined by a physician to not be a candidate for standard induction therapy.

[00117] In some embodiments, the subject is co-administered a RARA agonist (e.g., tamibarotene) and an antibody specific to CD38 (e.g., daratumumab). In some embodiments, the RARA agonist (e.g., tamibarotene) is administered simultaneously with the antibody specific to CD38 (e.g., daratumumab). In some embodiments, the RARA agonist (e.g., tamibarotene) and the antibody specific to CD38 (e.g., daratumumab) are administered within about 1 hour to about 48 hours of one another. In some embodiments, the RARA agonist (e.g., tamibarotene) and an antibody specific to CD38 (e.g., daratumumab) are administered within about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours, or about 48 hours of one another.

[00118] In some embodiments, a patient population includes one or more subjects (e.g. , comprises or consists of subjects) who received previous therapy for treatment of cancer (e.g. , non-APL AML, MDS or MM). In some embodiments, a patient population includes one or more subjects (e.g. , comprises or consists of subjects) who have not received previous therapy for treatment of cancer (e.g. , non-APL AML, MDS or MM). In some embodiments, a patient population comprises or consists of patients who have not received previous therapy for treatment of non-APL AML, MDS or MM.

[00119] In some embodiments, a patient who received previous therapy may have received previous therapy selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, palliative care, surgery, and combinations thereof. In some embodiments, a patient has received a transplant. In some embodiments, a patient has received standard cytotoxic chemotherapy. In some embodiments, standard cytotoxic chemotherapy includes cytarabine and/or an anthracycline. In some embodiments, standard cytotoxic chemotherapy may include additional chemotherapy and/or hematopoietic stem cell transplantation (HSTC). In some embodiments, a patient has received hypomethylating agents. In some embodiments, a patient has received lenalidomide.

[00120] In some embodiments, a patient population includes one or more subjects (e.g., comprises or consists of subjects) who have received and/or are receiving other therapy, e.g. , so that a RARA agonist therapy (e.g., tamibarotene) composition is administered in combination with the other therapy (e.g. chemotherapy agents), in addition to an antibody specific for CD38. In some embodiments, such other therapy may comprise or consist of therapy for cancer (e.g., as described herein), pain, nausea, constipation, for treatment of one or more side effects (e.g., pruritus, hair loss, sleeplessness, etc.) associated with cancer therapy, etc., or any combination thereof. The present invention provides a method of treating non-APL AML, MDS or MM, which comprises treating a patient identified as having non-APL AML, MDS or MM, with a therapeutically effective amount of RARA agonist therapy (e.g., tamibarotene) or a

pharmaceutically acceptable salt thereof and an antibody specific for CD38.

[00121] In some embodiments, the invention provides a method for treating a patient for non-APL AML, MDS or MM previously treated with a treatment regimen comprising chemotherapy by administering to such a patient a therapeutically effective amount of a RARA agonist (e.g., tamibarotene) and an antibody specific CD38. In some embodiments, the present disclosure provides a method for treating a patient for non-APL AML, MDS or MM where no standard therapies exist. In some embodiments, the present disclosure provides a method for treating a patient that is not suited for standard therapy.

[00122] In some embodiments, a patient or patient population may not be (e.g. , may exclude) a patient who has a previous history of hypersensitivity to an ingredient of

tamibarotene. In some embodiments, a patient or patient population may not be (e.g. , may exclude) a patient who is receiving vitamin A formulations. In some embodiments, a patient or patient population may not be (e.g. , may exclude) a patient who has hypervitaminosis A.

[00123] In some embodiments, a patient or patient population may not be (e.g. , may exclude) an elderly patient. In some embodiments, a patient or patient population may be or include one or more elderly patients. In some embodiments, an elderly patient may be monitored more frequently to detect potential adverse events (including for example, low levels of serum albumin and/or elevated concentrations of free drug in plasma, etc.) as compared with one or more younger patients. In some embodiments, the administration of the RARA agonist and/or the antibody specific CD38 may be reduced, suspended, and/or terminated for an elderly patient determined to display one or more signs of such an adverse event.

Dose Forms and Dosing Regimens

[00124] In general, each active agent (e.g., a RARA agonist or an antibody specific for

CD38) for use in accordance with the present invention is formulated, dosed, and administered in a therapeutically effective amount using pharmaceutical compositions and dosing regimens that are consistently with good medical practice and appropriate for the relevant agent(s) and subject. In principle, therapeutic compositions can be administered by any appropriate method known in the art, including, without limitation, oral, mucosal, by-inhalation, topical, buccal, nasal, rectal, or parenteral (e.g., intravenous, infusion, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other kinds of administration). In some embodiments, a RARA agonist (e.g., tamibarotene) will be administered orally. In some embodiments, an antibody specific for CD38 will be administered intravenously.

[00125] In some embodiments, a dosing regimen for a particular active agent may involve intermittent or continuous administration, for example to achieve a particular desired pharmacokinetic profile or other pattern of exposure in one or more tissues or fluids of interest in the subject receiving therapy.

[00126] In some embodiments, different agents administered in combination may be administered via different routes of delivery and/or according to different schedules.

Alternatively or additionally, in some embodiments, one or more doses of a first active agent is administered substantially simultaneously with, and in some embodiments via a common route and/or as part of a single composition with, one or more other active agents.

[00127] Factors to be considered when optimizing routes and/or dosing schedule for a given therapeutic regimen may include, for example, the particular indication being treated, the clinical condition of a subject (e.g., age, overall health, prior therapy received and/or response thereto, etc.) the site of delivery of the agent, the nature of the agent, the mode and/or route of administration of the agent, the presence or absence of combination therapy, and other factors known to medical practitioners. For example, in the treatment of cancer, relevant features of the indication being treated may include, among other things, one or more of cancer type, stage, location, etc.

[00128] In some embodiments, one or more features of a particular pharmaceutical composition and/or of a utilized dosing regimen may be modified over time (e.g., increasing or decreasing amount of active in any individual dose, increasing or decreasing time intervals between doses, etc.), for example in order to optimize a desired therapeutic effect or response. [00129] In general, type, amount, and frequency of dosing of active agents in accordance with the present invention are governed by safety and efficacy requirements that apply when relevant agent(s) is/are administered to a mammal, preferably a human. In general, such features of dosing are selected to provide a particular, and typically detectable, therapeutic response as compared with what is observed absent therapy.

[00130] In context of the present invention, an exemplary desirable therapeutic response may involve, but is not limited to, inhibition of and/or decreased tumor growth, tumor size, metastasis, one or more of the symptoms and side effects that are associated with a tumor, as well as increased apoptosis of tumor cells, therapeutically relevant decrease or increase of one or more cell marker or circulating markers and the like. Such criteria can be readily assessed by any of a variety of immunological, cytological, and other methods that are disclosed in the literature.

[00131] In some embodiments, it may be desirable to tailor dosing regimens, and particularly to design sequential dosing regimens, based on timing and/or threshold expression levels of inducible markers, whether for particular types of tumors, particular tumors, particular patient populations (e.g., carrying genetic markers), and/or particular patients. In some such embodiments, therapeutic dosing regimens may be combined with or adjusted in light of detection methods that assess expression of one or more inducible markers prior to and/or during therapy.

[00132] In some embodiments, a RARA agonist (e.g. , tamibarotene) therapy regimen comprises at least one (or includes or consists of exactly one) dose of about 1 mg/m², 2 mg/m², 3 mg/m², 4 mg/m², 5 mg/m², 6 mg/m², 7 mg/m², 8 mg/m², 9 mg/m², 10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15 mg/m², 16 mg/m², or a dose between any two of these values of a RARA agonist (e.g. , tamibarotene). In some embodiments, a RARA agonist (e.g. , tamibarotene) therapy regimen comprises a dose of between 1 mg/m² and 50 mg/m². In some embodiments, a RARA agonist (e.g. , tamibarotene) therapy regimen comprises a dose of between 5 mg/m² and 25 mg/m². In some embodiments, a RARA agonist (e.g. , tamibarotene) therapy regimen comprises a dose of between 5 mg/m² and 15 mg/m². In some embodiments, a RARA agonist (e.g. , tamibarotene) therapy regimen comprises a dose of 12 mg/m². In some embodiments, a RARA agonist (e.g. , tamibarotene) therapy regimen comprises a dose of 6 mg/m².

[00133] In some embodiments, a RARA agonist (e.g., tamibarotene) therapy regimen comprises a plurality of doses of a tamibarotene composition. In some such embodiments, a tamibarotene therapy regimen comprises, for example 2, 5, 10, 20, 30, 60, 90, 180, 365 doses or a number of doses between any two of these values and/or comprises a repeated pattern of doses (e.g. , at least one cycle of two daily doses, which cycle may be repeated, optionally with a period of alternative administration, or optionally no administration, separating different cycles). In some embodiments, a tamibarotene therapy regimen is administered twice a day. In some embodiments, a tamibarotene therapy regimen is administered once a day. In some

embodiments, a tamibarotene therapy regimen comprises a total dose of 6 mg/m² to 12 mg/m², divided as twice daily oral dosing.

[00134] In some embodiments, an antibody specific for CD38 (e.g. , daratumumab) therapy regimen comprises at least one (or includes or consists of exactly one) dose of about 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, or a dose between any two of these values of a an antibody specific for CD38 (e.g. , daratumumab). In some embodiments, an antibody specific for CD38 (e.g. , daratumumab) therapy regimen comprises a dose of between 1 mg/kg and 100 mg/kg. In some embodiments, an antibody specific for CD38 (e.g. , daratumumab) therapy regimen comprises a dose of between 5 mg/kg and 50 mg/kg. In some embodiments, an antibody specific for CD38 (e.g. , daratumumab) therapy regimen comprises a dose of between 10 mg/kg and 20 mg/kg.

[00135] In some embodiments, an antibody specific for CD38 (e.g. , daratumumab) therapy regimen comprises a plurality of doses of a daratumumab composition. In some such embodiments, a daratumumab therapy regimen comprises, for example 2, 5, 10, 20, 30, 60, 90, 180, 365 doses or a number of doses between any two of these values and/or comprises a repeated pattern of doses (e.g. , at least one cycle of two daily doses, which cycle may be repeated, optionally with a period of alternative administration, or optionally no administration, separating different cycles). In some embodiments, a daratumumab therapy regimen is administered once a week. In some embodiments, a daratumumab therapy regimen is administered no more than once a week. In some embodiments, a daratumumab therapy regimen is administered once every two weeks. In some embodiments, a daratumumab therapy regimen comprises a total dose of 10 mg/kg to 20 mg/kg no more than once a week. Formulations

[00136] A pharmaceutical composition, as used herein, refers to a mixture of a compound, such as tamibarotene or an antibody specific for CD38, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical compositions containing a compound may be administered in therapeutically effective amounts by any conventional form and route known in the art including, but not limited to: intravenous, oral, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, otic, nasal, and topical administration.

[00137] For oral administration, a compound can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers or excipients well known in the art. Such carriers permit the compounds described herein to be formulated as tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Generally, excipients such as fillers, disintegrants, glidants, surfactants, recrystallization inhibitors, lubricants, pigments, binders, flavoring agents, and so forth can be used for customary purposes and in typical amounts without affecting the properties of the compositions. In some embodiments, the excipient is one or more of lactose hydrate, corn starch, hydroxypropyl cellulose and/or magnesium stearate. In some embodiments, tamibarotene may be formulated with one or more of lactose hydrate, corn starch, hydroxypropyl cellulose and/or magnesium stearate.

[00138] The identification of acceptable formulations of tamibarotene can be achieved by various methods known in the art, for example as described in US 20100048708, which is incorporated herein by reference.

Packaged Pharmaceutical Compositions

[00139] The packaged pharmaceutical compositions of the present invention comprise a written insert or label comprising instructions to use the RARA agonist and the antibody targeting CD38 in a subject suffering from a cancer and who has been determined to have a super enhancer associated with a RARA gene having a strength, or ordinal rank equal to or above a threshold level, or a RARA mRNA level equal to or above a threshold level. As described in detail above, the threshold level is determined in a population of samples from either subjects diagnosed as suffering from the same disease or cell lines or xenograft models of the same disease as that for which the pharmaceutical composition is indicated for treatment. The instructions may be adhered or otherwise attached to a vessel comprising the RARA agonist and the antibody targeting CD38. Alternatively, the instructions and the vessel comprising the RARA agonist will be separate from one another, but present together in a single package, box or other type of container.

[00140] The instructions in the packaged pharmaceutical composition will typically be mandated or recommended by a governmental agency approving the therapeutic use of the RARA agonist and the antibody targeting CD38. The instructions may comprise specific methods of determining whether a super enhancer is associated with a RARA or IRF8 gene, as well as quantification methods to determine whether an enhancer associated with a RARA or IRF8 gene is a super enhancer, quantification methods to determine RARA or IRF8 mRNA levels; and/or threshold levels of super enhancers or RARA or IRF8 mRNA at which treatment with the packaged RARA agonist and the antibody targeting CD38 are recommended and/or assumed therapeutically effective. In some aspects, the instructions direct that the composition be administered to a subject whose RARA or IRF8 mRNA level falls in at least the 30^th percentile of a population whose RARA or IRF8 mRNA levels have been measured. In some aspects of these embodiments, a subject is identified as a RARA agonist responder if its RARA or IRF8 mRNA level prevalence rank is 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%, 42%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20% in a population whose RARA or IRF8 mRNA levels have been measured. In some aspects, the instructions direct that the composition be administered to a subject whose RARA or IRF8 mRNA level as measured by a specific assay

[00141] The instructions may optionally comprise dosing information, the types of cancer for which treatment with the RARA agonist and/or and the antibody targeting CD38 were approved, physicochemical information about the RARA agonist and/or and the antibody targeting CD38; pharmacokinetic information about the RARA agonist and/or and the antibody targeting CD38; or drug-drug interaction information. In some aspects, the instructions direct that the composition be administered to a subject diagnosed as suffering from non-APL AML. In some aspects, the instructions direct that the composition be administered to a subject diagnosed as suffering from non-APL MM. In some aspects, the pharmaceutical composition comprises tamibarotene. In some aspects, the pharmaceutical composition comprises AGN- 195183. In some aspects, the pharmaceutical composition comprises daratumumab. In some embodiments, the pharmaceutical composition comprises both tamibarotene and daratumumab.

EXAMPLES

[00142] In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope. Example 1. Measurement of RARA RNA and Protein Expression Levels

[00143] Expression level measurements are employed to ascertain the level of mRNA for the RARA gene tagged. mRNA levels correlate well with enhancer levels and therefore are also predictive sensitivity to RARA agonists. We use various means of measuring RNA as set forth below.

[00144] /. Array-Based Technology. Expression levels in HCC 1143 and AU565 are assessed with triplicate batches of lxlO⁶ cells. RNA is extracted from cells using Trizol® and purified using the mirVana™ RNA purification kit (both from Life Technologies), following the manufacturer's protocol. RNA levels are read out on Affymetrix Prime View™ arrays at the Dana Farber Cancer Institute Microarray Core (http://mbcf.dfci.harvard.edu/).

[00145] Fig. 1 shows the levels of mRNA expression of various RAR subtypes in a tamibarotene responsive (Au565) and non-responsive (HCC1143) cell line measured using the above protocol. Expression of RARA mRNA is 8-fold higher in the responsive cell line versus the non-responsive cell line, while expression of RaR-β and RaR-γ is not significantly different between the cell lines. This confirms that RARA mRNA expression analysis correlates with

RARA super enhancer strength and sensitivity to an RARA agonist, as well as demonstrating that RARA mRNA level can be used to predict sensitivity to such agonist.

[00146] //. RNA-Seq. RARA expression levels are quantified by RNA-Seq. Poly-A

RNA-Seq is performed and reads are aligned to the HG19 transcriptome using rsem vl.2.21 software (rsem-calculate-expression; parameters = -p 4 -samtools-sort-mem 3G -ci-memory 3072— bowtie-chunkmbs 1024 -quiet -output-genome-bam -bowtie2 -bowtie2-path

/data/devtools/bowtie2-2.0.5 -strand- specific) and then mRNA quantification was done using the same rsem suite (rsem-parse- alignments, rsem-build-read-index, rsem-run-em) and reported in transcripts per million (TPM), which represents an estimate of the number of transcripts for a given gene for every million transcripts detected. All protein coding genes were then extracted for each sample and their scores were quantile normalized together. We processed and normalized all AML patient samples from PDX models, primary AML patient samples, and AML cell lines together to create a universal mRNA score. The plotted values show the log2(TPM+l) levels for RARA (y-axis) versus the super-enhancer strength (RARA/MALAT1) for 48 primary AML patients (Fig. 1).

Example 2. RARA Super Enhancer Strength Ordinal Rank Cutoff in AML

[00147] The total enhancer/super enhancer profile of 95 AML samples (both patient samples and AML cell lines, including SigM5, MV411, HEL and Kasumil) are analyzed using H3K27Ac and ChlP-Seq. In each of the samples, the ordinal rank of the RARA-associated enhancer in terms of strength (as measured by H3K27Ac) is determined as compared to other enhancers and super-enhancers in the same cell and the determined ordinal ranks are plotted on a rank-order bar graph (Fig. 2). In MV411, it was determined that the RARA-associated enhancer was the 133 strongest enhancer. MV411 is the confirmed tamibarotene-responsive cell line having the lowest super enhancer strength ordinal. In HEL, it was determined that the RARA- associated enhancer was the 155^th strongest enhancer. HEL is the confirmed tamibarotene non- responsive cell line having the highest super enhancer strength ordinal. Based upon these values, we set the RARA enhancer strength ordinal cutoff at 150, a value in between the HEL ordinal and the MV411 ordinal.

[00148] As determined from our analysis of 70 primary AML cell samples from human subjects, 36% of those samples had a RARA super enhancer that was at least the 150^th strongest in those cells (Fig. 3). Therefore, the prevalence cutoff was set at 36%. That same prevalence cutoff is also used as the RARA mRNA prevalence cutoff when identifying potential AML responders to tamibarotene based on RARA or IRF8 mRNA measurements.

[00149] We also quantified the enhancers for an expanded panel of AML cell lines by ratio of RARA enhancer to MALAT1 enhancer ("Normalized Enhancer Strength). Plotting this normalized enhancer strength value against sensitivity to tamibarotene, we confirmed that 5 out of 6 cell lines bearing RARA enhancer strength ratios above 1 are responsive, while only 4 out of 7 cell lines bearing enhancers below this level are responsive (Fig. 5). When the cutoff is moved to normalized enhancer strength of 1.4 or higher, all of the cell lines (4 out of 4) are responsive.

Example 3. RARA Super Enhancer Strength Ordinal Rank Cutoff in AML Correlates with RARA mRNA levels

[00150] The AML patient samples used to determine the 36% RARA super enhancer strength ordinal prevalence cutoff, are binned into two groups - those having a prevalence rank of 36% or higher (i.e., a lower % value) and those having a prevalence rank lower than 36% (i.e., a higher % value) - and assayed for RARA mRNA level using RNA-seq as described in Example 1. The results are shown in Fig. 5A. The group at or higher than the 36% prevalence rank in RARA super enhancer strength ordinal has a statistically significant higher level of RARA mRNA than the group below the prevalence rank (p<0.001). This again confirmed that a prevalence cutoff determined at the super-enhancer level can also be used as the prevalence cutoff at the mRNA level.

[00151] We also determined the RARA mRNA levels in 11 different AML cell lines using RNA-seq and compared the mRNA levels to sensitivity to tamibarotene. The tested AML cell lines partitioned into two distinct groups based on their sensitivity or insensitivity to tamibarotene. Tamibarotene-sensitive cell lines all had RARA mRNA measured by RNAseq >10 TPM, while three insensitive cell lines had levels below this cut-off level (Fig. 5B).

Example 4: IRF8 mRNA Levels in Non-APL AML Cell Lines Correlate with

Responsiveness to a RARA Agonist

[00152] We used an Affymetrix GeneChip^® Prime View^™ Human Gene Expression

Array to examine seven of these AML cell lines (four sensitive to tamibarotene - NOMO-1, AML3, MV-4-11, and Sig-M5; and three insensitive - KG la, OCI-M1 and Kasumi-1) for other mRNAs that might be specifically elevated in the tamibarotene sensitive cell lines and identified IRF8 mRNA as a potential candidate. We then quantified IRF8 mRNA levels in each of these seven AML cell lines, as well as several other AML cell lines tested for sensitivity to tamibarotene by performing RNA-seq analysis as set forth below. The results for the first seven cell lines are shown in Fig. 6. Interestingly, NOMO-1 did not have a high RARA mRNA level, but was responsive to tamibarotene. The fact that NOMO- 1 had elevated IRF8 mRNA levels helped clarify this seeming inconsistency and further validated the use of IRF8 mRNA levels to predict responsiveness to tamibarotene. RNA isolation, preparation, and RNA-seq data processing was carried out in a similar manner to that described in Example 1. We then compared sensitivity to tamibarotene to IRF8 mRNA levels as shown in Fig. 6 and Table 2. Table 2: AML cell line IRF8 mRNA levels and tamibarotene anti-proliferative potency

*HL60 is an APL cell line.

[00153] As can be seen from the above table, all tamibarotene-responsive cell lines, except for HL60, had an IRF8 mRNA level of greater than 190 TPM (log₂(7.57)) in the assay, while non-responsive cell lines all had an IRF8 mRNA level of less than 16.5 TPM (log₂(4.03)). The responsiveness of HL60 to tamibarotene without a concomitant high level of IRF8 mRNA (6.73 TPM) suggests that correlation between IRF8 mRNA level and tamibarotene sensitivity may not hold for APL and thus may be better suited to stratify subjects suffering from non-APL AML. Figure 7 removes the data point for HL60.

[00154] Table 3 shows similar data to Table 2, but with the IRF8 mRNA values set forth as log₂ values and shows additional data for the Sig-M5 and THP-1 cell lines.

Table 3. AML cell line mRNA level expressed as log₂ values and tamibarotene anti-proliferative potency

*HL60 is an APL cell line.

Example 5: Determination of IRF8 mRNA Threshold Values for RARA Agonist

Treatment

[00155] The AML cell line results suggest a cutoff value of between 15.5 and 190 TPM

(i.e. , between log₂(4.03) and log₂(7.57) in the RNA-Seq assay. We chose a population of AML patient samples (kindly provided by Stanford University) in order to examine the distribution of IRF mRNA levels and to determine prevalence cutoffs based on the cutoff values. We added to that population AML cell lines and then generated a rank-ordered graph. Fig. 8 shows that rank- ordered distribution of IRF8 mRNA levels in the combined patient sample/ AML cell line population. We determined that a prevalence cutoff of 25% corresponded to an IRF8 mRNA value of approximately log₂(7).

Example 6: Correlation of IRF8 mRNA and RARA mRNA Levels

[00156] We next compared IRF8 and RARA mRNA levels in AML cell lines and patient population to determine correlation. Fig. 9 shows that some cell lines that responded to tamibarotene have relatively low RARA mRNA, but a high level of IRF8 mRNA. Fig. 10 shows that a subset of patients, too, demonstrates high IRF8 mRNA levels, but relatively low RARA mRNA levels and vice versa. This supports the idea that measuring both IRF8 and RARA mRNA in a patient and selecting that patient for treatment with a RARA agonist, such as tamibarotene, if either mRNA level is above a threshold value may optimize the treatable patient population.

Example 7: RARA mRNA -dependent CD38 induction in AML cell lines by tamibarotene

[00157] We monitored the induction of cell surface CD38 expression following tamibarotene treatment by measuring cell surface mean fluorescent intensity (MFI) staining by CD38-FITC antibody. Fig. 11A shows that 72h tamibarotene treatment at a concentration of 50 nmol/L does not induce CD38 expression in a RARA mRNA low cell line, Kasumi (CD38 ). Fig. 1 IB demonstrates that following 72h tamibarotene treatment of RARA mRNA high cell line, MV411, the entire cell population expresses high levels of CD38 (CD38 ). Furthermore, in Fig. llC, OCI-AML3 (RARA mRNA high), at baseline, has low CD38 MFI (CD38^DM). Upon tamibarotene treatment, CD38 expression is further induced and the cell population shifts to CD38 HI . Fig. 1 ID shows 72h tamibarotene treatment does not induce CD38 expression in another RARA mRNA low cell line, OCI-M1 (CD38 ). These data indicate that CD38^HI induction by tamibarotene can be predicted by RARA mRNA level. These results are depicted as a bar graph in Fig. HE, which shows the level of CD38 mRNA expression detected in each cell line before and after treatment. Fig. 11F shows the percentage of CD38^HI cells based on FACS are shown before and after tamibarotene treatment. In Figs. HE and 11F the APL cell line, NB4, is also shown. Example 8: RARA mRNA predicts NK cell mediated cytotoxicity of AML cell line, MV411, following tamibarotene and daratumumab combination therapy

[00158] To functionally evaluate the efficacy of a tamibarotene and daratumumab combination, AML cell lines with differential RARA mRNA levels were treated with tamibarotene for 72 hours then co-cultured with human NK cells with daratumumab or control antibody. NK cell proliferation and tumor cell death were then imaged in phase contrast during a 38h co-culture time course. Figs. 12A-12D are representative images of phase contrast images of RARA mRNA high MV411 cell line in co-culture assay with the following treatment conditions: Fig. 12A) 72h DMSO MV411 cell line pretreatment and 38h co-culture control antibody treatment. Fig. 12B) 72h SY1425 (50nM) MV411 cell line pretreatment and 38h co- culture control antibody treatment. Fig. 12C) 72h DMSO MV411 cell line pretreatment and 38h co-culture daratumumab treatment. Fig. 12D) 72h SY1425 (50nM) MV411 cell line pretreatment and 38h co-culture daratumumab treatment. Example 9: RARA mRNA level predicts NK cell mediated cytotoxicity of AML cell linesfollowing tamibarotene and daratumumab combination therapy

[00159] Kinetic measurement of NK cell-mediated tumor cell death by annexin V staining of tumor cells supports the finding that tamibarotene and daratumumab treatment combination result in increased tumor cell death in only some AML cell lines (Figs. 13A-13C) in comparison to single agent treatment. These data in combination with the increased induction of CD38 expression in RARA mRNA high AML cell lines (i.e. OCI-AML3 and MV411), but not in RARA mRNA low AML cell lines (i.e., OCI-M1) (Figs. 11B-11D) support the conclusion that tamibarotene induces CD38 phenotype in a RARA mRNA-dependent manner, which is required for efficient NK cell-mediated tumor cell death following daratumumab treatment.

Example 10: NK cell activation in co-culture assay only occurs post tamibarotene and daratumumab combination treatment as determined by NK cell IFN gamma secretion.

[00160] Interferon gamma (IFNy) secretion is an indication of NK cell activation. IFNy secretion was quantitated 38h post AML cell line and NK cell co-culture following the indicated treatment conditions (Fig. 14). Significantly increased levels of IFNy are observed following combination treatment with tamibarotene and daratumumab only in the RARA mRNA high AML cell lines (MV411 and OCI-AML3) and NK cell co-culture assay in comparison to single agent treatment conditions. In addition, these are the only cell lines that demonstrate the CD38 HI phenotype following tamibarotene treatment. These data further support the Annexin V quantitation (Figs. 12A-12D and 13) indicating efficient NK cell activation requires the combination treatment.

Example 11: Tamibarotene increases the intensity of CD38 expression in multiple myeloma cells.

[00161] Daratumumab is clinically approved for the treatment of multiple myeloma

(MM). However, only a fraction of these patients respond due to low levels of CD38 expression.

We demonstrate here that tamibarotene increases the CD38 phenotype in an already CD38 multiple myeloma cell line (MM IS) (Fig. 15 A) and can increase a CD38^" multiple myeloma cell line to a dim state (HUNS1) (Fig. 15B). Based on the previous, AML experiments, we predict that this will may increase the response of CD38 high subjects to CD38 therapeutic antibody directed immune-mediated death.

Example 12: Tamibarotene increase in CD38^ffl phenotype further sensitizes MM cell line to daratumumab dependent NK cell-mediated cytotoxicity.

[00162] To investigate if treatment of MM cell lines with tamibarotene will increase sensitivity to CD38 antibody treatment, we repeated the NK cell co-culture assay described previously with HUNS1 and MM1S MM cell lines. Fig. 16 demonstrates that following tamibarotene and daratumumab combination treatment, MM IS tumor cell death is increased in comparison to single agent anti-CD38 antibody treatment as quantitated by Annexin V staining. We further confirm this by directly monitoring NK cell activation by IFNy secretion after 38h treatment in this assay (Fig. 17). The multiple myeloma cell line that achieved high CD38 intensity, MM1S, showed strong cell killing in response to combination treatment. Example 13: Tamibarotene induces CD38^ffl phenotype in primary non-APL AML and MDS patient samples.

[00163] To confirm tamibarotene induction of CD38^HI levels in AML cell lines can be extrapolated to AML and MDS patient samples, we cultured PBMCs obtained from 15 different AML or MDS patients with 50nM tamibarotene or with DMSO as a negative control. We tested viability and CD38 induction 24h and 48h post-treatment as measured by flow cytometry.

Samples that had at least 10% viability were analyzed for CD38 levels. The results are shown in Fig. 18A. The majority of patient samples (11/15) showed an increase in CD38^HI cells 24 hours after treatment. One patient sample showed no response (Patient 4) and three patient samples did not meet viability criteria after 24h (Patients 2, 6 and 10). After 48h, four additional patient samples that had shown CD38 induction after 48h no longer met viability criteria and were excluded (patients 3, 7, 9 and 14). All of the remaining patient samples except for non- responder Patient 4 showed further induction of CD38 level after 48 h.

[00164] We then measured both RARA and IRF8 mRNA levels in a subset of these patients (Patients 1, 5, 11, 12 and 13), which represented patients that showed some response, were still viable after 48 h, and represented both AML and MDS. As shown in Fig. 18B, those patient samples that showed higher levels of either RARA mRNA and/or IRF8 mRNA as measured by RNA qPCR (the lower the dCq value, the higher the level of mRNA) demonstrated a greater percentage of CD38 upon tamibarotene treatment as compared to samples with lower RARA mRNA and/or IRF8 mRNA.

[00165] The CD38 induction levels observed for AML patient samples correlated well with results obtained from high RARA RNA AML cell lines (e.g., compare tamibarotene- induced CD38 MFI in patient sample AML_1 with AML cell line MV411 in Fig. 19C-19D). For both, tamibarotene caused an increase in the CD38^HI phenotype similar to that observed in daratumumab-sensitive multiple myeloma cell lines and multiple myeloma patients (e.g., compare patient sample MM_1 with multiple myeloma cell line MM1S in Fig. 19A-19B).

Based on this correlation, the inventors believe that high RARA mRNA AML patients will benefit from a combination treatment of a RARA-specific agonist, such as tamibarotene, and an anti-CD38 antibody, such as daratumumab. Example 15: Tamibarotene Causes a Greater Increase in CD38 Induction than ATRA in AML cell line xenografts having high RARA or IRF8 Levels.

[00166] It has been reported that ATRA causes a greater induction in CD38 levels than tamibarotene in HL-60 cells, an AML cell lines typically characterized as APL (A Uruno et al., 2011, J Leuk Biol, 90, pp 235-247). We wanted to compare the CD38 induction effects of ATRA versus tamibarotene in non-APL AML cell lines having varying levels of either RARA or IRF8 mRNA. In this experiment, we used mouse xenografts of MV411, THP-1, or Kasumi-l. MV411 has a high level of RARA mRNA and IRF8 mRNA (i.e., above a threshold), while THP- 1 has a high level of IRF-8 mRNA. Kasumi-l cells are considered to have levels of IRF8 and RARA that are below the threshold.

[00167] Each of the cell lines were maintained in vitro as suspension cultures in the appropriate medium (THP-1 cells: RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum and 0.05mM β-mercaptoethanol; MV4-11 cells: IMDM medium

supplemented with 10% heat inactivated fetal bovine serum; Kasumi-l cells: RPMI1640 medium supplemented with 20% heat inactivated fetal bovine serum), at 37°C in an atmosphere of 5% CO₂ in air. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

[00168] All mice were γ-irradiated (200 rads) 24 h before tumor cell inoculation. Each mouse was inoculated subcutaneously at the right flank region with the appropriate tumor cell line (THP-1 cells: lx 10⁷ cells in 0.1 ml of PBS (1: 1 Matrigel); MV4-11 cells: 5 x 10⁶ cells in 0.1 ml of PBS (1 : 1 Matrigel); Kasumi-1 cells: 1 x 10⁷ cells in 0.1 ml of PBS (1: 1 Matrigel)) for tumor development. Treatment with tamibarotene, ATRA or vehicle was started when the mean tumor size reached approximately 100-200 mm³. The mice were divided into 3 groups of nine and each was orally administered drug (ATRA 4 mg/kg; tamibarotene 3 mg/kg) or vehicle alone BID for up to 28 days. The date of tumor cell inoculation was denoted as day 0. Tumor volumes were measured twice per week in two dimensions using a caliper, and the volume expressed in mm³ using the formula: V = 0.5 a x b² where a and b are the length and width of the tumor, respectively. Three mice per group were sacrificed on day 7, 14, and 21 after grouping (4-week study total). Half of the tumor was collected for embedding into a FFPE block for immunohistochemistry staining (IHC), and the other half of tumor was collected for CD38 FACS analysis of the tumor cells. IHC staining was performed utilizing a BOND RX autostainer using an anti-CD38 antibody directed against the C-terminus of CD38 (clone SP149; Abeam; Cat. No. abl 83326). Antibody was diluted 1 : 100 in pH 9.0 EDTA buffer and then incubated with tumor slices for 20 minutes. Signal was developed using DAB and the manufacturer's Bond Polymer Refine Detection kit.

[00169] As seen in FIG. 20 A, after 7 days, tamibarotene-treated MV4-11 xenograft mice showed a mean fluorescence intensity (MFI) of over 200 with over 80% of the tumor cells being CD38^HI by FACS analysis. ATRA-treated MV4-11 xenograft mice showed a MFI of slightly over 100 with approximately 70% of the tumor cells being CD38 . Immunohistochemical staining of tumor sections with an anti-CD38 antibody confirmed the surprising and unexpected superiority of tamibarotene over ATRA in this xenograft model (FIG. 20B). This trend became even more pronounced at 3 weeks. As shown in FIG. 20C, at three weeks, in tamibarotene- treated MV4-11 xenografts, over 60% of the tumor cells remained CD38^HI, with a MFI of around 110, while in ATRA-treated xenografts less than 20% of the tumor cells remained CD38^HI with a MFI of around 50. FIG 20D shows immunohistochemical staining of tumor cells after three weeks, which confirms the superiority of tamibarotene over ATRA.

[00170] As shown in FIG. 21, the superiority of tamibarotene in inducing CD38 after 7 days was also observed in THP-1 cells. Although ATRA-treated xenografts showed a similar

HI

percentage of CD38 cells as compared to tamibarotene, tamibarotene treatment resulted in a higher MFI (over 200) as compared to ATRA (-175). In contrast, Kasumi-1 cells showed very little CD38 induction with either treatment (FIG. 22). An immunohistochemical comparison of each type of xenograft and each type of treatment after one week is shown in FIG. 23.

Equivalents and Scope

[00171] In the embodiments articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[00172] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed embodiments is introduced into another embodiment. For example, any embodiment that is dependent on another embodiment can be modified to include one or more limitations found in any other embodiment that is dependent on the same base embodiment. Where elements are presented as lists, e.g. , in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms "comprising" and "containing" are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included.

Furthermore, unless otherwise indicated or otherwise evident from the context and

understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[00173] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.

[00174] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following embodiments.

Claims

Claims We claim:

1. A method of diagnosing and treating a human subject suffering from a disease selected from non-APL AML, multiple myeloma, and MDS comprising:

a. diagnosing whether the subject has a tamibarotene- sensitive form of the disease based on:

i. a level of retinoic acid receptor alpha mRNA previously determined to be equal to or above a pre-determined threshold in a sample of diseased cells from the subject; and/or ii. a level of IRF8 mRNA previously determined to be equal to or above a pre-determined threshold in a sample of diseased cells from the subject

b. administering to the subject an amount of tamibarotene effective to treat the disease; and c. co-administering to the subject an antibody specific for CD38.

2. The method of claim 1, wherein the antibody specific for CD38 is daratumumab.

3. The method of any of claims 1-2, wherein the subject is administered tamibarotene for a period of time prior to administration of the antibody specific for CD38; and is co-administered the antibody specific for CD38 only when the CD38 level in the subject has been determined to be CD38^hl following initial administration of tamibarotene.

4. The method of claim 3, wherein the CD38 level in the subject was determined between 6 and 72 hours following the initial administration of tamibarotene.

5. A method of treating a human subject suffering from a disease selected from non-APL AML, multiple myeloma, or MDS, wherein the level of retinoic acid receptor alpha mRNA and/or the level of IRF8 mRNA in diseased cells has been determined to have a level of retinoic acid receptor alpha mRNA equal to or above a pre-determined threshold, or a level of IRF-8 mRNA equal to or above a pre-determined threshold, the method comprising a step of administering to a subject an amount of tamibarotene effective to treat the disease; and coadministering to the subject an antibody specific for CD38.

6. The method of claim 5, wherein the antibody specific for CD38 is daratumumab.

7. The method of claim 6, wherein the subject is administered tamibarotene for a period of time prior to administration of the antibody specific for CD38; and is co-administered the antibody specific for CD38 only when the CD38 level in the subject is determined to be CD38^hl following initial administration of tamibarotene.

8. The method of claim 7, wherein the CD38 level in the subject is determined between 6 and 72 hours following the initial administration of tamibarotene.

9. A method of diagnosing and treating a human subject suffering from non-APL AML, multiple myeloma or MDS comprising:

a. diagnosing whether the subject has a tamibarotene- sensitive form of the disease based on a level of retinoic acid receptor alpha mRNA and/or a level of IRF8 mRNA, either or both which were previously determined to be present in a sample of diseased cells from the subject; and b. administering therapy to the subject, wherein:

i. the therapy comprises administering an amount of tamibarotene effective to treat the disease and co-administering an antibody specific for CD38 if the level indicates that the disease is tamibarotene- sensitive; and

ii. the therapy comprises administering an agent other than tamibarotene if the level indicates that the disease is not tamibarotene-sensitive.

10. The method of claim 9, wherein the retinoic acid receptor alpha mRNA and/or the IRF8 mRNA level indicates that the disease is tamibarotene-sensitive if it is above a pre-determined threshold and that the disease is not tamibarotene-sensitive if it is below the pre-determined threshold.

11. The method of claim 9 or 10, wherein the antibody specific for CD38 is daratumumab.

12. The method of any one of claims 9-11, wherein if the level of retinoic acid receptor alpha mRNA or IRF8 mRNA indicates that the disease is tamibarotene-sensitive the subject is administered tamibarotene for a period of time prior to administration of the antibody specific for CD38; and is co- administered the antibody specific for CD38 only when the CD38 level in the subject is determined to be CD38^hl following initial administration of tamibarotene.

13. The method of claim 12, wherein the CD38 level in the subject is determined between 6 and 72 hours following the initial administration of tamibarotene.

14. The method of any one of claims 1-13, wherein tamibarotene is administered orally.

15. The method of any one of claims 1-14, wherein the subject is administered tamibarotene at a dosage of between 6 mg/m²/day and 12 mg/m²/day, wherein said dosage is divided into two doses.

16. The method of any one of claims 1-15, wherein the anti-CD38 antibody is administered no more than once a week at a dose of between 10-20 mg/kg body weight of the subject.