RU2799789C2 - Rara agonists for the treatment of aml and mds - Google Patents

Abstract

FIELD: medicine, oncology.

SUBSTANCE: invention can be used in the treatment of non-APL (acute promyelocytic leukemia) acute myelocytic leukemia (non-APL AML). The method includes administering a combination of tamibarotene and a second therapeutic agent to a subject having non-ATL AML, wherein the cancer cells contained in a biological sample from the said subject contain an IRF8 biomarker and/or a RARA biomarker, the said second therapeutic agent being: azacitidine, midostaurin, cytarabine, daunorubicin, methotrexate, idarubicin, sorafenib, decitabine, quisartinib, ABT199, JQ1, prednisone, SAHA, GSKJ4, or EPZ6438.

EFFECT: use of the invention makes it possible to increase the effectiveness of the treatment of patients with non-APL AML, in which cancer cells express the IRF8 biomarker, due to the synergistic effect of the combination.

29 cl, 21 dwg, 3 tbl, 8 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 62/320,352, filed April 8, 2016, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Retinoids are a class of compounds structurally related to vitamin A, including natural and synthetic compounds. Several groups of retinoids have been found to be clinically useful in the treatment of dermatological and oncological diseases. Retinoic acid and its other naturally occurring retinoid analogues (9-cis-retinoic acid, all-trans-3,4-didehydroretinoic acid, 4-oxoretinoic 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 proliferation, differentiation, and morphogenesis of epithelial cells in the lungs. Retinoids exert their biological action through a series of hormonal nuclear receptors, which are ligand-inducible transcription factors belonging to the steroid/thyroid receptor superfamily.

[0003] Retinoid receptors are classified into two families - retinoic acid receptors (RAR) and retinoid X receptors (RXR), each of which consists of three different subtypes (α, β and γ). Each subtype of the RAR gene family encodes a variable number of isoforms resulting from the differential splicing of two primary RNA transcripts. All-trans retinoic acid is a physiological hormone for retinoic acid receptors and binds with approximately equal affinity to all three RAR subtypes, but does not bind to RXR receptors, for which 9-cis retinoic acid is the natural ligand. Retinoids have anti-inflammatory effects, alter the course of epithelial cell differentiation, and inhibit the production of stromal cell matrix. These properties have led to the development of topical and systemic retinoid-based therapeutics for dermatological disorders such as psoriasis, acne, and hypertrophic skin scars. Other uses include the control of acute promyelocytic leukemia, adeno- and squamous cell carcinoma, and hepatic fibrosis.

[0004] The limitation of the therapeutic use of retinoids is due to the relative toxicity observed for naturally occurring retinoids, all-trans retinoic acid and 9-cis-retinoic acid. These natural ligands are non-selective in the context of the RAR subtype and therefore have a whole body pleiotropic effect that is often toxic.

[0005] Various retinoids have been described that interact selectively or specifically with RAR or RXR receptors or with specific subtypes (α, β, γ) within a class. RARA-specific agonists have shown promise in the treatment of cancers and many have been taken into 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 clinical trials in the US and Europe. The discrepancy between the theoretical efficacy of RARA agonists in cancer and the lack of regulatory approval of such agents raises the question of why such agonists are not effective and safe in humans. Therefore, there is a need for a better understanding of why RARA agonists do not fulfill their therapeutic potential.

[0006] Recent advances in genomic technology and understanding of gene regulatory circuits have led to the discovery of super enhancers. While many genes in a given tissue or cancer type can be regulated by the presence of enhancers near the coding region of a gene, a small fraction of them present a highly asymmetric and disproportionately large accumulation of transcriptional marks and mechanisms compared to all other active genes. Recent discoveries suggest that such enhancers are associated with genes that are directly related to the function and survival of the cell in which they are found. Therefore, the association of a super enhancer with a gene indicates the relative importance of said gene for the survival of that cell.

SUMMARY OF THE INVENTION

[0007] The present invention provides technologies for detecting one or more IRF8 biomarkers (e.g., the presence, level, shape, and/or activity of one or more components or products of the IRF8 gene, including, for example, potency, ordinal rank or abundance of an IRF8 super enhancer and level or abundance of IRF8 mRNA). In the unstoping invention, it was demonstrated that the cells {for example, cancer cells or cells obtained from a subject suffering from non-folla OML or MDS) containing one or more IRF8 biomarkers, and the IRF8 biomarker represents or includes the expression of one or more of the increased levels of the IRF8 MRNC or the super-enhanser associated with the IRF8, associated with the IRF8. are more susceptible to the action of the Rara agonist, such as Tamibaroten.

[0008] In various embodiments, aspects, and alternatives, this invention addresses the problem of determining which cell populations are responsive to retinoic acid receptor alpha ("RARA") agonists, identifying patient populations that benefit from RARA agonist treatment (e.g., by stratifying patients for treatment with a RARA agonist; separating RARA agonist responsive from non-responsive patients), and providing treatment options. therapies targeting such patient populations. The decision is based at least in part on the discovery that increased expression of one or more IRF8 biomarkers in certain cancer cells indicates that such cells will be significantly more susceptible than similar cells lacking increased IRF8 biomarkers to treatment with an RARA agonist (e.g., tamibarotene).

[0009] In some embodiments, the invention relates to a method of treating cancer (e.g., non-ATL AML or MDS) in a subject (e.g., human) based on the level of IRF8 mRNA in the subject's cancer cells, comprising the step of administering to the subject an amount of a RARA agonist (e.g., tamibarotene) effective to treat the disease. In some aspects of these embodiments, the level of IRF8 mRNA in the subject's cancer cells is equal to or greater than a predetermined threshold level.

[0010] In some embodiments, the present invention relates to a method of treating cancer, comprising the step of administering tamibarotene to a subject having cancer, wherein the cancer is determined to be characterized by the presence of an IRF8 biomarker, wherein the IRF8 biomarker is or includes the expression of one or more of increased levels of IRF8 mRNA or a super enhancer associated with the IRF8 gene.

[0011] In some embodiments, the present invention relates to a method comprising the step of applying therapy to a subject that is determined not to express one or more elevated levels of RARA mRNA or a super enhancer associated with the RARA gene; and lack of expression of one or more of the increased levels of IRF8 mRNA or super enhancer associated with the IRF8 gene, and the therapy does not include the administration of tamibarotene.

[0012] In some embodiments, the invention relates to a method of treating cancer, comprising the step of administering therapy to a subject determined to (a) lack expression of one or more elevated levels of RARA mRNA or lack a super enhancer associated with the RARA gene whose potency and/or ordinal rank exceeds a predetermined threshold; and (b) the presence of expression of one or more of increased levels of IRF8 mRNA or super enhancer associated with the IRF8 gene, and the therapy is the use of tamibarotene.

[0013] In some embodiments, the present invention relates to a method of treating cancer, comprising the step of applying therapy to a subject that is determined to lack the expression of one or more elevated levels of RARA mRNA or a super enhancer associated with the RARA gene; and the presence of expression of one or more of increased levels of IRF8 mRNA or super enhancer associated with the IRF8 gene, and the therapy is the use of tamibarotene.

[0014] In some embodiments, the present invention relates to a method of treating cancer, comprising the step of obtaining information related to the level of IRF8 mRNA in a subject suffering from cancer; and administering tamibarotene to the subject if the information indicates that the IRF8 mRNA level or the super enhancer level is equal to or greater than the reference level. In some aspects, the reference level is a predetermined threshold value. In some aspects, the predetermined threshold is a cut-off or prevalence limit.

[0015] In some embodiments, the present invention relates to a method of treating cancer, comprising the steps of obtaining information related to the presence of a super enhancer associated with the IRF8 gene in a subject suffering from cancer; and administering tamibarotene to the subject if the information indicates that the super enhancer is associated with the IRF8 gene.

[0016] In some embodiments, the present invention relates to a method for predicting the effectiveness of a RARA agonist in treating cancer, comprising the steps of determining the presence of cancer cells having an IRF8 mRNA level equal to or greater than a reference level, wherein an IRF8 mRNA level equal to or greater than the reference level is predictive of the effectiveness of the RARA agonist in treatment. In some aspects, the reference level is a predetermined threshold value. In some aspects, the predetermined threshold is a cut-off or prevalence limit.

[0017] In some embodiments, the present invention relates to a method for predicting the efficacy of a RARA agonist in treating cancer, comprising the steps of determining, in a subject suffering from cancer, the presence of cancer cells characterized by the presence of an IRF8 gene-related super enhancer, wherein the presence of the IRF8 gene-related super enhancer is indicative of the efficacy of a RARA agonist in treating cancer.

[0018] In some embodiments, the present invention relates to a method comprising the steps of obtaining a biological sample containing cancer cells from a subject suffering from cancer; and detecting in the biological sample one or more of the IRF8 mRNA level equal to or greater than the reference level; or a super enhancer associated with the IRF8 gene. In some aspects, the reference level is a predetermined threshold value. In some aspects, the predetermined threshold is a cut-off or prevalence limit.

[0019] In some embodiments, the present invention relates to a method for diagnosing, predicting, or treating a subject suffering from cancer, comprising the steps of obtaining a cancer sample from the subject; and determining in the sample one or more of the level of IRF8 mRNA or the presence of a super enhancer associated with the IRF8 gene in the subject.

[0020] In some embodiments, the present invention relates to a method for diagnosing, predicting, or treating a subject suffering from cancer, comprising the steps of obtaining a cancer sample from the subject; determining the level of IRF8 mRNA in the sample or the presence of a super enhancer associated with the IRF8 gene in the subject; and administering a therapeutic composition comprising an RARA agonist if one or more of (a) the presence of an IRF8 mRNA level equal to or greater than a reference level; or (b) the presence of a super enhancer associated with the IRF8 gene. In some aspects, the reference level is a predetermined threshold value. In some aspects, the predetermined threshold is a cut-off or prevalence limit.

[0021] In some embodiments, the present invention relates to a method comprising detecting one or more of the level of RARA mRNA or the potency or ordinal rank of a super enhancer associated with the RARA gene in a biological sample obtained from a subject with cancer; and detecting one or more of an IRF8 mRNA level or an IRF8 gene-associated super enhancer in the biological sample, if the biological sample does not express one or more of an elevated RARA mRNA level equal to or greater than a reference level, or a RARA gene-associated super enhancer that has an potency or ordinal rank equal to or greater than a predetermined threshold. In some aspects, the reference level is a predetermined threshold value. In some aspects, the predetermined threshold is a cut-off or prevalence limit.

[0022] In some embodiments, the present invention relates to a method comprising detecting one or more of the level of RARA mRNA or the potency or rank of a super enhancer associated with the RARA gene in a biological sample obtained from a subject with cancer; and detecting one or more of an IRF8 mRNA level or an IRF8 gene-associated super enhancer in the biological sample, if the biological sample does not express one or more of an elevated RARA mRNA level equal to or greater than the reference level, or an potency or rank of the RARA gene-associated super enhancer equal to or greater than a predetermined threshold.

[0023] In some embodiments, the invention relates to a method comprising detecting one or more of an IRF8 mRNA level or an IRF8 gene-associated super enhancer in a biological sample obtained from a subject with cancer; and detecting one or more of the RARA mRNA level or the potency or rank of the super enhancer associated with the RARA gene in the biological sample, if the biological sample does not express one or more of an increased level of IRF8 mRNA equal to or greater than the reference level, or a super enhancer associated with the IRF8 gene.

[0024] In some embodiments, the invention relates to a method comprising detecting one or more of an IRF8 mRNA level or an IRF8 gene-associated super enhancer in a biological sample obtained from a subject with cancer; and detecting one or more of the level of RARA mRNA or the potency or rank of the super enhancer associated with the RARA gene in the biological sample if the biological sample expresses one or more of an increased level of IRF8 mRNA equal to or greater than the reference level or a super enhancer associated with the IRF8 gene.

[0025] In some embodiments, the present invention relates to a method for diagnosing and treating a human subject suffering from a disease selected from non-APL AML and MDS, comprising:

a. diagnosing whether the subject has a tamibarotene-susceptible form of the disease based on the level of IRF8 mRNA, which according to the previous determination is present in a sample of diseased cells from the subject; And

b. administering to the subject an amount of tamibarotene effective to treat the disease.

[0026] In some aspects of these embodiments, the level of IRF8 mRNA is equal to or greater than a predetermined threshold.

[0027] In some aspects of any of the above embodiments that involve treating a subject with tamibarotene, the subject is administered a combination of tamibarotene and a second therapeutic agent.

[0028] In some embodiments, the present invention relates to a method of treating a cancer selected from non-ATD and MDS in a subject based on the level of RARA mRNA or the level of IRF8 mRNA in the subject's cancer cells, the treatment comprising administering to the subject a combination of tamibarotene and a second therapeutic agent. In some aspects of these embodiments, the subject has a level of RARA mRNA equal to or greater than a threshold value. In some aspects of these embodiments, the subject has an IRF8 mRNA level equal to or greater than a threshold. In some aspects of these embodiments, the subject has a RARA mRNA level equal to or greater than a threshold and an IRF8 mRNA level equal to or greater than the threshold. In some aspects of these embodiments, the subject suffers from non-AML AML.

BRIEF DESCRIPTION OF GRAPHICS

[0029] FIG. 1 shows IRF8 mRNA levels in seven different AML cell lines. Cell lines indicated by red bars show significant susceptibility to tamibarotene treatment. Cell lines indicated by blue bars show little or no susceptibility to tamibarotene treatment.

[0030] FIG. 2 shows the antiproliferative efficacy of tamibarotene (EC ₅₀ value, nM) correlated with IRF8 mRNA levels determined by RNA sequencing. Of note, the upper left dot with IRF8 mRNA level=1 (log10) and tamibarotene _EC50 value set to 50 (not sensitive) represents data from 2 AML cell lines with low IRF8 mRNA levels and no antiproliferative response to tamibarotene. The correlation of susceptibility to tamibarotene with IRF8 mRNA levels was highly significant (p=0.0001, Spearman correlation, two-tailed).

[0031] In FIG. 3 is a rank plot of IRP8 mRNA levels in individual patient AML samples and in AML cell lines obtained by RNA sequencing. The PL21 AML cell line, which represented the cell line with the lowest level of IRP8 mRNA among all susceptible lines, and Kasumi, which represented the cell line with the highest level of IRF8 mRNA among all tamibarotene-resistant lines, are indicated. In this population, the prevalence cutoff of 25% is equal to an RNA sequencing value for TNM of approximately log ₂ (7).

[0032] FIG. 4 shows the correlation between IRF8 mRNA level and RARA mRNA level in non-APL AML cell lines that were tested for response to tamibarotene.

[0033] FIG. 5 shows the correlation between IRF8 mRNA level and RARA mRNA level in AML patient population samples. The dotted lines represent the 25% prevalence limit for each mRNA.

[0034] FIG. 6 shows the correlation between the antiproliferative efficacy of tamibarotene and the efficacy of the IRF8 enhancer in AML cell lines. Plot of AML cell line sensitivity to tamibarotene (EC ₅₀ value, nM) as a function of the RECOMB score for the IRF8 enhancer. It should be noted that the leftmost dot with IRF8 enhancer score=0 and tamibarotene EC50 value taken as ₅₀ pM (refractory) represents the results of 3 AML cell lines with no detectable IRF8 enhancer peak and no antiproliferative response to tamibarotene.

[0035] FIG. 7 depicts the efficacy of the IRF8 enhancer in AML patient samples. Ranking plot of IRF8 enhancer efficacy according to the RECOMB scoring method for samples from 66 AML patients. Each bar represents the potency of the IRF8 enhancer in one patient with AML. The y-axis plots the potency of a single IRF8 enhancer as a multiple of the cut-off (defined as 1.0, shown in dotted line) between super enhancers (>1.0) and typical enhancers (≤1.0). Patient samples above this threshold are displayed in white, while samples below this threshold are displayed in black. 14 of 66 patient samples (21% of the population) exceeded the threshold of 1.0 and were considered to contain IRF8 SE.

[0036] FIG. 8 shows the correlation of IRF8 mRNA levels with IRF8 enhancer potency in AML patient samples. Plot of IRF8 mRNA transcript representation from quantile normalized RNA sequencing (log ₂ THM; y-axis) as a function of IRF8 RECOMB enhancer efficiency (x-axis) in 49 primary patient samples (those with reported values for enhancer and expression). The estimated Spearman coefficient is ~0.81 with a p-value of 2.2×10 ^-12 .

[0037] FIG. 9 shows the distribution of IRF8 enhancer potency in AML cell lines. Plot of IRF8 enhancer efficacy according to the RECOMB scoring method for 26 AML cell lines. Each bar represents the potency of the IRF8 enhancer for one AML cell line. The y-axis plots the potency of a single IRF8 enhancer as a multiple of the cut-off (defined as 1.0, shown in dotted line) between super enhancers (>1.0) and typical enhancers (≤1.0). Cell lines above this threshold are shown in white, while cell lines below this threshold are shown in black. Nine of 26 AML cell lines (34% of the population) exceeded the threshold of 1.0 and were considered to contain IRF8 SE.

[0038] FIG. 10 shows the correlation of IRF8 mRNA levels with IRF8 enhancer potency in AML cell lines. Plot of IRF8 mRNA transcript representation from TNM RNA sequencing with quantile normalization (Y-axis) as a function of IRF8 RECOMB enhancer efficiency (X-axis) in all non-ATL AML cell lines for which both RNA sequencing and ChIP sequencing data were available. The estimated Spearman coefficient is ~0.82 with a p-value of ~2×10 ^-6 .

[0039] FIG. 11 depicts the response, expressed as % CD45 ⁺ cells, to daily dosing with tamibarotene in two patient-derived AML xenograft mouse models. In FIG. 11 also depicts the % of CD45 ⁺ cells in different organs and body fluids, as well as the survival time of mouse models.

[0040] In FIG. 12 depicts the response, expressed as % CD45 ⁺ cells, to daily dosing with tamibarotene in two additional patient-derived AML xenograft mouse models. In FIG. 12 also depicts the % of CD45 ⁺ cells in different organs and body fluids in these models, as well as the survival time in these models.

[0041] In FIG. 13 depicts the level of IRF8 mRNA and RARA mRNA in each of the four AML patient samples used in the xenograft experiments depicted in FIG. 11 and 12. Only tamibarotene-responsive AM8096, which produced a tamibarotene-responsive xenograft, showed an IRF8 mRNA level above the 100 THM threshold. AM8096 and AM5512 (which showed some susceptibility to tamibarotene) showed RARA mRNA levels above the 10 THM threshold.

[0042] FIG. 14 shows the rank ordering of IRF8 mRNA levels detected in different AML cell lines, primary samples of AML patients, normal blood cells, and AML AUC.

[0043] FIG. 15 shows isobolograms for combinations of tamibarotene and azacitidine in various AML cell lines. Asterisks indicate data beyond the isobologram maximum.

[0044] FIG. 16 shows isobolograms for combinations of tamibarotene and arsenic trioxide in various AML cell lines. Asterisks indicate data beyond the isobologram maximum.

[0045] FIG. 17 shows isobolograms for combinations of tamibarotene and ara-C in various AML cell lines. Asterisks indicate data beyond the isobologram maximum.

[0046] FIG. 18 shows isobolograms for combinations of tamibarotene and daunorubicin in various AML cell lines. Asterisks indicate data beyond the isobologram maximum.

[0047] FIG. 19 shows isobolograms for combinations of tamibarotene and methotrexate in various AML cell lines. Asterisks indicate data beyond the isobologram maximum.

[0048] FIG. 20 shows isobolograms for combinations of tamibarotene and idarubicin in various AML cell lines. Asterisks indicate data beyond the isobologram maximum.

[0049] FIG. 21 shows isobolograms for tamibarotene and sorafenib combinations in various AML cell lines. Asterisks indicate data beyond the isobologram maximum.

DEFINITIONS

[0050] In this application, unless the context clearly indicates otherwise, (i) the term "one" may mean "at least one"; (ii) the term "or" may mean "and/or"; (iii) the terms "comprising" and "comprising" can be understood as including the listed components or steps, presented alone or together with one or more additional components or steps; and (iv) the terms "about" and "approximately" can be understood as allowing for a standard deviation that is understood by those skilled in the art; and (v) when ranges are cast, endpoints are included.

[0051] Specialists in this field it is obvious that one or more chemical compounds, whose structure is given in this document, may have one or more isomeric (for example, enantiomeric, diastereomeric and geometric (or conformational)) and/or tautomeric forms; for example, R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. In some embodiments, the principles included herein may apply to and/or cover any and all such forms. Therefore, unless otherwise indicated, single stereochemical isomers as well as enantiomeric, diastereomeric and geometric (or conformational) mixtures of the present compounds may all be included within the scope of the invention. Likewise, unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention. In addition, those skilled in the art will appreciate that, in some embodiments, the chemical structures provided herein may include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds whose structure is identical to those shown, except for the replacement of hydrogen with deuterium or tritium, or the replacement of carbon with ¹³ C- or ¹⁴ C-rich carbon, are within the scope of this invention. In some embodiments, such compounds can be used, for example, as analytical tools, as probes in biological assay methods, and/or as therapeutic agents in accordance with the present invention.

[0052] Agonist: As used herein, the term "agonist" may be used to refer to an agent, condition, or phenomenon whose presence, level, extent, type, or form is correlated with an increase in the level or activity of another agent (i.e., the agent being agonized). 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 component that exhibits a relevant activating activity. In some embodiments, the agonist may be direct (in which case it acts directly on its target); in some embodiments, the agonist may be indirect (in which case it exerts an effect in a manner other than binding to its target; for example, by interacting with a target regulator such that a change in the level or activity of the target occurs).

[0053] Agonist therapy: As used herein, the term "agonist therapy" refers to the administration of an agonist that agonizes a particular target of interest in order to achieve the desired therapeutic effect. In some embodiments, agonist therapy comprises administering a single dose of an agonist. In some embodiments, agonist therapy comprises the administration of multiple doses of the agonist. In some embodiments, agonist therapy comprises administering an agonist according to a dosing schedule that is known or expected to achieve a therapeutic effect, e.g., because such a result has been proven with some degree of statistical certainty, e.g., by administration of a relevant population.

[0054] Antagonist: As used herein, the term "antagonist" may be used to refer to an agent, condition, or phenomenon whose presence, level, extent, type, or form is correlated with a reduction in the level or activity of another agent (i.e., the agent or target being inhibited). In general, the antagonist 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 components that exhibit relevant inhibitory activity. In some embodiments, the antagonist may be direct (in which case it affects its target directly); in some embodiments, the agonist may be indirect (in which case it exerts an effect in a manner other than binding to its target; for example, by interacting with a target regulator such that a change in the level or activity of the target occurs).

[0055] Approximately: In the context of this document, the term "approximately" or "about" applied to one or more values of interest refers to a value similar to a specified reference value. In some embodiments, the term "about" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction (more or less) from the established reference value, unless otherwise indicated or otherwise obvious from the context (except when such a number would exceed 100% of the possible value).

[0056] Acute promyelocytic leukemia: As used herein, the term "acute promyelocytic leukemia" or "APL" refers to a subtype of acute myelogenous leukemia ("AML") characterized by a genetic translocation between human chromosomes 15 and 17. Accordingly, the term "non-ATL AML" refers to any subtype of AML that is not characterized by such a genetic translocation.

[0057] Biological sample: in the context of this document, the term "biological sample" refers to any sample obtained from an individual suffering from a disease to be treated by the methods according to this invention, including tissue samples (such as tissue sections and tissue punch biopsy); cell samples (for example, cytological smears (such as Pap smears or blood smears) or cell samples obtained by microdissection; bone marrow samples (both fractions of whole, complete cells and subpopulations of cells within them); or cell fractions, fragments, or organelles (for example, obtained by cell lysis and separation of components by centrifugation or in some other way). Other examples of biological samples include blood, serum, urine, semen , feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsied tissue (e.g., obtained by surgical biopsy or punch biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that are obtained from a first biological sample.In some aspects, a biological sample is from a subject, suffering from non-ALD AML or MDS, is a bone marrow aspirate. In other aspects, the biological sample from a subject suffering from non-ATL AML or MDS is a fractionated whole blood sample. In more specific aspects, a biological sample from a subject suffering from non-ATL AML or MDS is a PBMC fraction from the subject's whole blood ("PBMC sample"). In more specific aspects, a PBMC sample from a subject suffering from non-ATL AML or MDS is further enriched for specific blasts using various enrichment methods, such as antibody-bound bead enrichment protocols, cell sorting based on fluorescent labels, or other methods known in the art ("enriched PBMC sample"). In some embodiments, as the context implies, the term "sample" refers to a preparation obtained by processing (for example, by removing one or more components and/or adding one or more agents) of the original sample. Such a "processed sample" may contain, for example, nucleic acids or proteins isolated from the sample or obtained by applying methods such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc. to the original sample.

[0058] Biomarker: As used herein, the term "biomarker" refers to a compound whose presence, level, or form correlates with a particular biological event or condition of interest such that it is considered a "marker" of that event or condition. To give some examples, in some embodiments, the biomarker may be or contain a marker of a particular condition or stage of the disease or the likelihood of developing a particular disease, disorder or pathological condition. In some embodiments, the biomarker may be or contain a marker for a particular disease or therapeutic outcome or likelihood thereof. Thus, in some embodiments the biomarker is predictive, in some embodiments the biomarker is predictive, in some embodiments the biomarker is diagnostic for a relevant biological event or condition of interest. The biomarker may be a compound of any chemical class. For example, in some embodiments, the biomarker may be or contain a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (eg, a metal or an ion), or a combination thereof. In some embodiments, the biomarker is a cell surface marker. In some embodiments, the biomarker is intracellular. In some embodiments, the biomarker is outside the cells (e.g., secreted or otherwise generated or found outside the cells, for example, in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc. In some embodiments, the term refers to a gene expression product that is characteristic of a particular tumor, tumor subclass, tumor stage, etc. In alternative or additional embodiments, the presence of a level of a particular marker correlates with the activity (or level of activity) of a particular signaling path, for example, characteristic of a particular class or subclass of tumors.The statistical significance of the presence or absence of a biomarker may vary depending on the particular biomarker.In some embodiments, the detection of a biomarker is highly specific in the sense that it reflects a high probability that the tumor belongs to a particular subclass.Such specificity may come at the expense of sensitivity (for example, a negative result may occur even if the tumor is a tumor for which the biomarker is expected to be expressed). In some embodiments, the biomarker may be or include an IRF8 biomarker (e.g., the presence, level, shape, and/or activity of one or more components or products of the IRF8 gene, including, for example, potency, ordinal rank or abundance of an IRF8 super enhancer and level or abundance of IRF8 mRNA). In some embodiments, the biomarker may be or contain an RARA biomarker (e.g., one or more RARA biomarkers (e.g., the presence, level, shape, and/or activity of one or more components or products of the RARA gene, including, for example, the potency, ordinal rank or prevalence rate of a RARA super enhancer and the level or prevalence of RARA mRNA). In some embodiments, a biomarker refers to a combination of one or more biomarkers, such as IRF8 and RARA.

[0059] Cancer: As used herein, the term "cancer" refers to a malignant neoplasm or tumor (Stedman's Medical Dictionary, 25th ed.; Hensly ed.; Williams & Wilkins: Philadelphia, 1990). The terms "neoplasm" and "tumor" are used interchangeably herein and refer to an abnormal mass of tissue for which mass growth exceeds and is inconsistent with normal tissue growth. A "malignant neoplasm" is generally poorly differentiated (anaplasia) and is characterized by rapid growth accompanied by progressive infiltration, invasion, and destruction of surrounding tissue. In addition, a malignant neoplasm can generally metastasize to distant sites. In some embodiments, the cancer is any cancer or tumor for which the IRF8 biomarker correlates with sensitivity to a RARA agonist, such as tamibarotene. In some embodiments, the cancer is acute myelocytic leukemia (AML). In some embodiments, the cancer is non-ATL AML.

[0060] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously treated with two or more therapeutic regimens (eg, two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered according to overlapping dosing schedules. In some embodiments, "administering" a combination therapy may include administering one or more agents to a subject receiving other agents in combination. To be clear, combination therapy does not require separate agents to be administered together in the same composition (or even necessarily at the same time), although in some embodiments, two or more active agents, compounds, or components may be administered together in a combined composition or even in a combined compound (e.g., as part of the same chemical complex or covalent compound).

[0061] Limit value: in the context of this document, the terms "limit" and "limit value" means the value measured in the analysis, which defines the dividing line between two subgroups of the population (for example, susceptible and non-susceptible). Thus, a value equal to or greater than the limit value defines one subgroup of the population; and a value less than the limit value defines another subgroup of the population.

[0062] Diagnostic information: in the context of this document, "diagnostic information" or "information for use in making a diagnosis" is information used in determining whether a patient has a disease, disorder, or condition, and/or in classifying a disease, disorder, or condition into a phenotypic category or any category of significance in connection with the prognosis of the disease, disorder, or condition, or the likelihood of response to treatment (treatment in general or any particular treatment) of the disease, disorder, or pathological condition. Similarly, "diagnosis" refers to the provision of any type of diagnostic information, including, but not limited to, information about the subject's likelihood of developing a disease, disorder, or condition, the condition, staging, or characteristics of the disease, disorder, or condition present in the subject, information regarding the nature or classification of the tumor, information regarding prognosis, and/or information used in selecting an appropriate treatment. The choice of treatment may include the choice of a particular therapeutic agent or other treatment procedure, such as surgery, radiation, etc., the choice of whether to stop or continue therapy, a choice regarding the dosing regimen (for example, the frequency of application or the level of one or more doses of a particular therapeutic agent or combination of therapeutic agents), etc.

[0063] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term "dosage form" can be used to refer to a physically discrete unit of an active agent (eg, therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such an amount is a single dosage amount (or an integer fraction thereof) suitable for administration in accordance with a dosing regimen that is predetermined to correlate with the desired or beneficial result when administered to the relevant population (i.e., the therapeutic dosing regimen). It will be apparent to those skilled in the art that the total amount of therapeutic composition or agent administered to a particular subject is determined by one or more physicians and may include the administration of multiple dosage forms.

[0064] Dosing schedule: Those skilled in the art will appreciate that the term "dosing schedule" can be used to refer to a set of unit doses (typically more than one) that are separately administered to a subject, typically at specific time intervals. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may include the use of one or more doses. In some embodiments, the dosing regimen includes the use of a number of doses, each of which is separated in time from other doses. In some embodiments, individual doses are separated from each other by a period of time of the same length; in some embodiments, the dosing regimen includes the use of a number of doses and at least two different periods of time separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dosage amount. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, the dosing regimen includes administering a first dose in a first dosage amount followed by administering one or more additional doses in a second dosage amount different from the first dosage amount. In some embodiments, the dosing regimen comprises administering a first dose in a first dosage amount followed by administering one or more additional doses in a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with the desired or beneficial result when administered within a relevant population (i.e., is a therapeutic dosing regimen).

[0065] Effective amount: As used herein, an "effective amount" of a compound described herein, such as a compound of formula (I), refers to an amount sufficient to elicit the desired biological response, i. for the treatment of a pathological condition. Those skilled in the art will appreciate that an effective amount of a compound described herein, such as a compound of formula (I), may vary depending on such factors as the desired biological end point, the pharmacokinetics of the compound, the condition being treated, the route of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment. For example, in the treatment of cancer, an effective amount of a compound of the invention may reduce tumor burden or stop tumor growth or spread.

[0066] Enhancer: As used herein, the term "enhancer" refers to a region of genomic DNA whose function is to regulate genes up to 1 mbp away from it. The enhancer may overlap, but is often not part of the gene coding regions. The enhancer is often bound by transcription factors and is labeled with specific histone marks.

[0067] Hydrate: As used herein, the term "hydrate" refers to a compound that is associated with water. Typically, the number of water molecules contained in the hydrate of a compound is in a certain ratio to the number of compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R⋅xH ₂ O, where R is a compound and x is a number greater than 0. A given compound may form more than one type of hydrate, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, such as hemihydrates (R⋅0.5H ₂ O)) and polyhydrates (x is a number greater than 1, such as di hydrates (R⋅2H ₂ O) and hexahydrates (R⋅6H ₂ O)).

[0068] IRF8 gene: As used herein, the term "IRF8 gene" refers to a genomic DNA sequence that encodes a protein that binds an interferon consensus sequence or a splice variant thereof, and specifically excludes gene fusions that contain all or part of the IRF8 gene. In some embodiments, the IRF8 gene is located on chromosome 16:85862582-85990086 in the hg19 genomic block.

[0069] "Improve", "increase" or "reduce": in the context of this document, these terms or their grammatical equivalents indicate meanings that are associated with a reference measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or a number of control individuals) in the absence of the treatment described herein. In some embodiments, a "control individual" is an individual afflicted with the same form of disease or injury as the individual being treated.

[0070] Messenger RNA transcript: As used herein, the term "messenger RNA transcript" or mRNA refers to an RNA transcription product from a DNA sequence that contains one or more gene coding regions.

[0071] Ordinal rank: In the context of this document, the term "ordinal rank" of a specified value refers to the rank order of that value compared to a set of other values. For example, an ordinal rank of 100 in the context of the effectiveness of the RARA gene-associated super enhancer in the cell under study compared to other super enhancers in the cell under study means that 99 other super enhancers in the cell under study are more effective than the RARA gene-associated super enhancer.

[0072] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a composition is administered or may be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the patient is a human. The concept of a person includes pre- and postnatal forms. In some embodiments, the patient suffers from or is predisposed to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions.

[0073] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to those salts that are medically suitable for use in contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, and the like, and a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describe in detail pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 1977, 66, 119, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by other methods known in the art such as ion exchange. . Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citran, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, format, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, hept anoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, beer lat, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Exemplary alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed with counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

[0074] Population: In the context of this document, the term "population" or "population of samples" means a sufficient number (for example, at least 30, 40, 50 or more) of different samples that objectively reflect the distribution of the measured value in a larger group. Each sample in a population of samples can be a cell line, a biological sample derived from a living being (e.g., a biopsy or body fluid sample), or a sample derived from a xenograft (e.g., when studying tumor growth in mice by implanting a cell line or a patient sample), where each sample is from a living being suffering from the same disease, condition, or disorder, or from a representative cell line or xenograft.

[0075] Prevalence cutoff: As used herein, the term "prevalence cutoff" for a particular value (e.g., potency of an IRF8 gene-associated super enhancer) refers to a prevalence that defines a dividing line between two subgroups of a population (e.g., susceptible and non-responder). Thus, a prevalence equal to or greater than (eg, a lower percentage) the prevalence cut-off defines one subset of the population; and a prevalence less (eg, a smaller percentage) than the cut-off prevalence defines a different subset of the population.

[0076] Prevalence score: As used herein, the term "prevalence score" for a particular value (eg, the potency of a super enhancer associated with the IRF8 gene) refers to the percentage of a population that equals or exceeds that particular value. For example, a 35% prevalence rank for the effectiveness of an IRF8 gene-associated super enhancer in a test cell means that 35% of the population has an IRF8 gene enhancer with an potency equal to or greater than the value for the cell under study.

[0077] Prognostic and predictive information: In the context of this document, the terms "prognostic information" and "predictive information" are used to refer to any information that can be used to indicate any aspect of the course of a disease or pathological condition in the absence or presence of treatment. Such information may include, but is not limited to, the patient's average life expectancy, the likelihood of the patient surviving over a given time period (e.g., 6 months, 1 year, 5 years, etc.), the likelihood of the patient recovering from the disease, the likelihood of the patient's disease susceptibility to a particular therapy option (susceptibility can be determined in any of a variety of ways). Prognostic and predictive information is included in the broad category of diagnostic information.

[0078] Rank ordering: in the context of this document, the term "rank ordering" means the ordering of values from largest to smallest or from smallest to largest.

[0079] RARA gene: As used herein, the term "RARA gene" refers to a genomic DNA sequence that encodes a functional retinoic acid receptor-α gene and specifically excludes gene fusions that contain all or part of the RARA gene. In some embodiments, the RARA gene is located on chromosome 17:38458152-38516681 in the hg19 genomic block.

[0080] Reference: in the context of this document, describes the standard or control against which comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the test and/or determination for the reference or control is performed substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a retrospective reference or control, optionally displayed on a tangible medium. Typically, as will be apparent to those skilled in the art, the determination or characterization of a reference or control is carried out under conditions or circumstances comparable to those for which the assessment is being made. It is obvious to those skilled in the art that there is sufficient similarity to validate and/or compare with a particular possible reference or control.

[0081] Susceptibility: In the context of this document, susceptibility to treatment can refer to any beneficial change in a subject's condition that occurs as a result of, or correlates with, treatment. Such a change may include stabilizing the condition (e.g., preventing worsening that might have occurred in the absence of treatment), reducing the intensity, slowing the onset and/or reducing the frequency of one or more symptoms of the condition, and/or improving the prospects for cure of the condition, etc. In some embodiments, the susceptibility may be the subject's susceptibility; in some cases, the response may be tumor susceptibility.

[0082] Solvate: As used herein, the term “solvate” refers to forms of a compound that are associated with a solvent, usually via a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. The compounds described herein, such as compounds of formula (I), may be prepared, for example, in crystalline form and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In some cases, it is possible for a solvate to be isolated, for example, when one or more solvent molecules are included in the crystal lattice of a crystalline solid. "Solvate" includes both solvates in the solution phase and isolated solvates. Exemplary solvates include hydrates, ethanolates, and methanolates.

[0083] Potency: As used herein, the term "potency" referring to the portion of an enhancer or super enhancer refers to the area under the curve of H3K27Ac counts or other genomic marker measures plotted against the length of the analyzed genomic DNA segment. Thus, "efficiency" is the integration of the signal obtained as a result of measuring the label in a given base pair over a number of base pairs that define the area selected for measurement.

[0084] Subject: As used herein, the term "subject" to whom administration is intended to be administered is a human (e.g., male or female of any group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or elderly adult)).

[0085] Super enhancer: In the context of this document, the term "super enhancer" refers to a subgroup of enhancers that contain a disproportionately large proportion of histone marks and/or transcriptional proteins compared to other enhancers in a particular cell. As a consequence, the superenhancer-regulated gene is predicted to be very important for the function of this cell. Super enhancers are generally determined by ranking all enhancers in a cell based on potency and by using available software such as ROSE (https://bitbucket.org/young_computation/rose) to identify subsets of enhancers that are substantially more potency than the median enhancer in the cell (see, for example, U.S. Patent 9,181,580, incorporated herein by reference.

[0086] Threshold: In the context of this document, the terms "threshold" and "threshold level" refer to a level that defines a dividing line between two subsets of a population (eg, susceptible and non-susceptible). The threshold or cut-off level may be a prevalence limit or threshold value.

[0087] Treatment: As used herein, the terms "treatment" and "treat" refer to reversing, alleviating, slowing the onset, or inhibiting the progression of the "pathological condition" (e.g., disease, disorder, or condition, or one or more symptoms thereof) described herein. In some embodiments, "treatment" and "treat" require that signs or symptoms of a disease, disorder, or pathological condition develop or be observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease or condition (eg, based on history of symptoms and/or genetic or other factors that influence susceptibility). Treatment may also be continued after symptoms have resolved, for example to slow or prevent recurrence.

[0088] In the context of this document, the terms "pathological condition", "disease" and "disorder" are used interchangeably.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

RARA and IRF8

[0089] The retinoic acid receptor alpha subtype (RARA) is a nuclear hormone receptor that acts as a transcriptional repressor in the unbound or antagonist bound state and as a gene activator in the agonist bound state. The natural ligand for RARA is retinoic acid, also known as all-trans retinoic acid (ATRA), which is produced from vitamin A.

[0090] Super enhancers (SEs) are large, highly active regions of chromatin that regulate key cell identity genes, including oncogenes in cancer cells. Using a gene-manipulation platform, we identified genome-wide SEs in 60 primary samples from AML patients so that new tumor vulnerabilities could be found. One of the SEs, which showed an uneven presence among patient samples, was associated with the RARA gene encoding RARA.

[0091] Studies have shown a good correlation between susceptibility to tamibarotene and either or both of super enhancer potency and RARA mRNA levels. However, for each of these potential RARA biomarkers, there was an average range within which susceptibility to tamibarotene was mixed. The present invention provides insights and techniques that help resolve such ambiguous susceptibility results and provides various compositions and methods useful, among other things, for characterizing, identifying, selecting, or stratifying patients based on likely susceptibility to tamibarotene therapy. For example, the present invention provides technologies that allow one or more IRF8 biomarkers (e.g., the presence, level, shape, and/or activity of one or more IRF8 gene components or products, including, e.g., potency, ordinal rank, IRF8 super enhancer prevalence rate, or IRF8 mRNA levels) to be realized, determined, and/or used, and demonstrate their applicability in anticancer therapy.

[0092] Using various AML cell lines and patient samples previously analyzed for potency and rank of RARA enhancers, RARA mRNA levels, and susceptibility to tamibarotene, we searched for additional biomarkers that would correlate with susceptibility to tamibarotene. Interferon response factor 8 (IRF8) mRNA levels have been found to be elevated in the same patient populations as RARA. IRF8 is an interferon-dependent transcription factor known to be critical for hematopoiesis and whose signaling cessation leads to aberrant proliferation of immature myeloid cells. In AML, overexpression of IRF8 is observed, which may correlate with an adverse clinical outcome. Despite this upregulation, in reality, IRF8 signaling is disrupted by repressive transcriptional cofactors and potentially by RARA when it is in a repressive state under the control of SE. In addition, interferon-α itself, an upstream IRF signaling ligand, has a differentiating effect in AML and interferes with the RARA pathway.

[0093] The present invention describes a genome-wide analysis of the expression and level of enhancers of a panel of AML patient samples and AML cell lines to investigate the correlation of IRF8 gene enhancer potency, IRF8 mRNA levels, and sensitivity to tamibarotene. A panel of AML cell lines has been investigated previously and showed a correlation between sensitivity to the antiproliferative effects of the RARA agonist tamibarotene and RARA enhancer potency and RARA mRNA levels. In this application, we demonstrate that IRF8 mRNA levels are also elevated in AML cell lines and AML patient samples that are characterized by elevated RARA mRNA levels, and that there is a correlation between IRF8 mRNA levels and susceptibility to an RARA agonist such as tamibarotene. We also demonstrate that there is a correlation between IRF8 enhancer potency (eg, the presence of an IRF8-associated superenhancer), IRF8 mRNA levels, RARA mRNA levels, and susceptibility to tamibarotene. Thus, IRF8 enhancer potency or IRF8 mRNA levels can be used alone or in combination with RARA enhancer potency and RARA mRNA levels to identify patients who would be responsive to treatment with an RARA agonist such as tamibarotene.

Identification of IRF8 and RARA super enhancers and determination of threshold levels

[0094] Identification of an enhancer or super enhancer can be accomplished by various methods known in the art, such as those described in Cell 2013, 155, 934-947 and PCT/US 2013/066957, both of which are incorporated herein by reference. In some embodiments, super enhancer identification is performed by obtaining cellular material and DNA from a patient's cancer sample (eg, from a biopsy sample). Important indicators for the measurement of enhancers lie in two directions - the length of DNA over which the continuous detection of genomic markers (for example, H3K27Ac) occurs - and the compiled frequency of occurrence of the genomic marker in each base pair along this DNA region, which is the amplitude. The measurement of the area under the curve ("AUC") resulting from the integration of the length and amplitude analysis determines the effectiveness of the enhancer. This is the potency of an IRF8 or RARA super enhancer compared to a control, which is used in one aspect of the present invention to determine whether a subject will be responsive to an RARA agonist (eg, tamibarotene) or not. Based on the present description, it will be apparent to those skilled in the art that if the length of DNA over which genomic markers are detected is the same for IRF8 or RARA and a control, then the amplitude ratio of the IRF8 or RARA super enhancer compared to the control will be equivalent to potency and can also be used to determine whether a subject will be responsive to a RARA agonist or not. In some embodiments, the effectiveness of the IRF8 or RARA enhancer in the cell is normalized before comparison with other samples. Normalization is performed by comparison with a region in the same cell that contains a universal super enhancer or enhancer that is present at the same levels in all cells. One example of such a universal super enhancer region is the MALAT1 super enhancer locus (xp11:65263724-65266724) (genomic block hg19).

[0095] Using H3K27Ac ChIP sequencing methods, it was determined that there is a super enhancer locus associated with the RARA gene at xp17:38458152-38516681 (genomic block hg19), and there is a super enhancer locus associated with the IRF8 gene at xp16:85862582-859900 86 (genomic block hg19).

[0096] ChIP sequencing, also known as ChIP-seq, is used to analyze protein interactions with DNA. ChIP sequencing combines chromatin immunoprecipitation (CHP) and massively parallel DNA sequencing to identify binding sites for DNA-associated proteins. This can be used to accurately map global binding sites for any protein of interest. Previously, on-chip ChIP was the most widely used technique used to investigate these protein-DNA interactions. Successful ChIP sequencing depends on many factors, including the efficiency and method of sonication, buffer compositions, antibody quality, and cell number; see, for example, 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 than H3K27Ac that can be used to detect super enhancers using ChIP sequencing include P300, CBP, BRD2, BRD3, BRD4, mediator complex components (J Loven, et al., Cell, 153(2):320-334, 2013), histone 3 lysine 4 monomethylated (H3K4mel) or other tissue specific enhancer-associated transcription factors (E Smith & A Shilatifard, Nat Struct Mol Biol, 2l(3):210-219, 2014) (S Pott & Jason Lieb, Nature Genetics, 47(1):8-12, 2015).

[0097] In some embodiments, super enhancer maps based on ChIP sequencing of H3K27Ac or another marker for the entire genome of the cell line or patient sample already exist. In some embodiments, one can simply determine whether the potency or ordinal rank of the enhancer or super enhancer on such maps at the xp17:38458152-38516681 locus (hg19 genomic block) was equal to or greater than a previously determined threshold level. In some embodiments, one can simply determine whether the potency or ordinal rank of an enhancer or super enhancer on such maps at the xp16:85862582-85990086 locus (hg19 genomic block) was equal to or greater than a previously determined threshold level.

[0098] It should be understood that the exact location of IRF8, RARA and MALAT1 on the chromosome may differ for different genomic blocks and/or different cell types. However, a person skilled in the art, given the present disclosure, can determine such a different position by positioning in such other genomic blocks specific sequences corresponding to the RARA and/or MALAT1 loci in the hg19 genomic block.

[0099] Other methods for identifying super enhancers include chromatin immunoprecipitation (JE Delmore, et al., Cell, 146(6)904-917, 2011) and the use of a template chip (ChIP chip) and chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) using the same immunoprecipitated genomic markers and oligonucleotide sequences, that hybridize to the xp17:38458152-38516681 locus (hg19 genomic block) of RARA or the xp16:85862582-85990086 locus (hg19 genomic block) of IRF8. In the case of the ChIP chip, the signal is usually recorded by the intensity of fluorescence resulting from the hybridization of the probe and the injected analytical sample, as in the case of other matrix-based techniques. In the case of ChIP-qPCR, a dye is used to measure template amplification, which becomes fluorescent only after intercalation of the double-stranded DNA created in the PCR reaction.

[0100] In some embodiments, the determination of whether a cell has an IRF8 super enhancer potency equal to or greater than a desired threshold level is performed by comparing the potency of the IRF8 enhancer in the cell under study with the corresponding potency of IRF8 in a population of cell samples, with each of the cell samples obtained from different sources (e.g., from different subjects, from different cell lines, from different xenografts) corresponding to the same disease being treated. In some aspects of these embodiments, only cell samples from primary tumors are used to determine the threshold level. In some aspects of these embodiments, at least some of the samples in the population will be tested for susceptibility to a specific RARA agonist to determine: a) the least effective IRF8 enhancer of the sample in the population that is susceptible to that specific RARA agonist (the "lowest score susceptible sample"); and, optionally, b) the highest IRF8 enhancer potency of the sample in the population that is not responsive to that specific RARA agonist (the "highest score non-responsive"). In these embodiments, the IRF8 enhancer potency threshold above which the test cell is considered susceptible to that specific RARA agonist is set to: i) equal to or greater by up to 5% of enhancer I potency RF8 in a susceptible sample with the lowest rate in the population; or ii) equal to or greater than up to 5% of the potency of the IRF8 enhancer in the highest rated non-responder sample in the population; or iii) as a value between the IRF8 enhancer potency of the lowest scoring susceptible sample and the highest scoring non-receptive sample in the population.

[0101] It should be understood that in the above embodiments, not all samples in a population are necessarily subject to RARA agonist susceptibility testing, but all samples are subject to measurement of IRF8 enhancer potency and/or IRF8 mRNA levels. In some embodiments, samples are ranked based on the potency of the IRF8 enhancer. The choice of which of the above three methods to use to set the cut-off will depend on the difference in IRF8 enhancer potency between the lowest scoring susceptible sample and the highest scoring non-susceptible sample in the population and whether the goal is to minimize the number of false positives or to minimize the possibility of missing a potentially susceptible sample or subject. When the difference between the lowest score susceptible sample and the highest score non-responsive sample is large (e.g., in the case of a large number of samples not tested for susceptibility that fall within the range between the lowest score susceptible sample and the highest score non-responsive sample in the ranking order of IRF8 enhancer efficacy), the limit value is generally set to or greater by up to 5% of the enhancer efficacy. sulfur IRF8 in a susceptible sample with the lowest index in the population. This limit value maximizes the number of potential susceptible samples. When the difference is small (for example, in the case of few or no samples that were not tested for susceptibility that fall between the lowest scoring receptive sample and the highest scoring non-susceptible sample in the ranking of IRF8 enhancer potency), the cut-off value is generally set to the value between the IRF8 enhancer potency of the lowest scoring susceptible sample and the highest scoring non-susceptible sample. This limit value minimizes the number of false positive samples. When the highest scoring non-responsive sample has an IRF8 enhancer potency greater than the lowest scoring receptive sample, the cut-off is typically set to a value equal to or greater than 5% of the IRF8 enhancer potency in the highest scoring non-responsive sample in the population. This method also minimizes the number of false positive samples.

[0102] In some embodiments, the determination of whether a cell has an IRF8 super enhancer potency equal to or greater than a desired threshold level is performed by comparing the ordinal value of the IRF8 enhancer potency in the cell under study with the ordinal value of the IRF8 enhancer potency in a population of cell samples, with each of the cell samples obtained from different sources (e.g., from different subjects, from different cell lines, from different xenografts). In these embodiments, at least some of the samples in the population will be tested for susceptibility to a specific RARA agonist to determine: a) the lowest rank of IRF8 enhancer potency of a sample in the population that is responsive to that specific RARA agonist ("lowest rank susceptible"); and, optionally, b) the highest sequencing IRF8 enhancer potency of a sample in a population that is unresponsive to that specific RARA agonist (the "highest scoring non-responsive sample"). In these embodiments, the IRF8 enhancer sequencing potency threshold above which a test cell is considered susceptible to that specific RARA agonist is set to: i) equal to or greater than up to 5% of the IRF8 enhancer potency sequencing value in the lowest sequencing sample in the population; or ii) equal to or greater than up to 5% of the IRF8 enhancer potency rank in the non-responsive sample with the highest rank in the population; or iii) as a value between the IRF8 enhancer potency score of the lowest score susceptible sample and the highest score non-receptive sample in the population.

[0103] It should be understood that in the above embodiments, as a rule, not all samples in the population need to be tested for susceptibility to the RARA agonist, but all samples are subject to measurement of the effectiveness of the IRF8 enhancer and the establishment of an ordinal value of the effectiveness of the IRF8 enhancer compared to other enhancers in the same sample. An ordinal value is generally obtained by measuring the potency of all other enhancers in a cell and determining what rank (eg, ordinal) the IRF8 enhancer has in terms of potency compared to other enhancers.

[0104] In some embodiments, samples are ranked based on the ordinal value of IRF8 enhancer potency. The choice of which of the above three methods to use to establish a cut-off will depend on the difference in IRF8 enhancer efficiency sequencing between the lowest sequencing susceptible sample and the highest sequencing non-responding sample in the population, and whether the cut-off serves to minimize the number of false positive samples or maximize the number of responding samples. When this difference is large (for example, in the case of a large number of samples not tested for susceptibility that fall between the lowest susceptible sample and the highest sequencing non-response sample in a ranking order of IRF8 enhancer potency ranks), the limit value is generally set to be equal to or greater by up to 5% of the IRF8 enhancer's susceptibility rank. imchivy sample with the lowest ordinal index in the population. When this difference is small (e.g., in the case of few or no samples that were not tested for susceptibility that fall between the lowest scoring receptive sample and the highest sequencing non-responsive sample in a ranking order of the IRF8 enhancer potency ranks), the cut-off is generally set to a value between the susceptible sample's IRF8 enhancer potency ranks with the lowest ordinal score and the non-receptive sample with the highest ordinal score. When the non-responsive sample with the highest ordinal value has an IRF8 enhancer efficacy ordinal greater than the lowest susceptible sample, the cut-off value is generally set to a value equal to or greater than up to 5% of the IRF8 enhancer efficacy ordinal in the highest ordinal non-responsive sample in the population.

[0105] In some embodiments in which the cell or sample under study is compared to a population, the cut-off value(s) obtained for the population (e.g., IRF8 enhancer potency or IRF8 enhancer ordinal value) is converted to a prevalence score and the cut-off value is expressed as a percentage of the population having the cut-off value or greater, e.g., the cut-off prevalence rate. Without being bound by theory, Applicants believe that the cut-off for the prevalence of a test sample will be the same regardless of the methodology used to determine the potency of the IRF8 enhancer. Thus, a prevalence cutoff defined for one parameter (eg, IRF8 enhancer potency ordinal value) can be transferred and applied to another parameter (eg, IRF8 mRNA level) to determine a cutoff for that other parameter. This allows the definition of a limit value for any parameter without experimental determination of the correlation between the levels of such a parameter and susceptibility to the RARA agonist. All that needs to be determined is what level of that other parameter corresponds to the previously determined population prevalence limit.

[0106] In some embodiments, the methods discussed above can be used simply to determine if a diseased cell from a subject has a super enhancer associated with the IRF8 gene. In these embodiments, the presence of an IRF8-bound superenhancer indicates that the subject will be responsive to the RARA agonist. In one aspect of these embodiments, a cell is determined to have an IRF8 gene-associated super enhancer when the IRF8-associated enhancer has a potency equal to or greater than the MALAT-1-associated enhancer. In alternative aspects of these embodiments, a cell is determined to have an IRF8 gene-associated super enhancer when the IRF8-associated enhancer has an potency that is at least 10 times greater than the median potency of all enhancers in the cell. In other alternative aspects of these embodiments, a cell is determined to have an IRF8 gene-associated super enhancer when the IRF8-associated enhancer has a potency that is above the point where the slope of the tangent is 1 on the potency rank plot of each of the enhancers in the cell.

[0107] In some embodiments, the methods discussed above can be used to further determine if a diseased cell from a subject expresses a super enhancer associated with the RARA gene that has potency, ordinal rank, or prevalence equal to or greater than a predetermined threshold level. In some aspects of these embodiments, determining that either: a) either the diseased cell has a super enhancer associated with the IRF8 gene (or that such super enhancer has a potency or ordinal rank equal to or greater than a predetermined threshold level); or b) the diseased cell has a super enhancer associated with the RARA gene that has a potency or ordinal rank equal to or greater than a predetermined threshold level, indicating that the subject will be responsive to the RARA agonist. In other aspects of these embodiments, determining that: a) the diseased cell has a super enhancer associated with the IRF8 gene (or that such super enhancer has a potency or ordinal rank equal to or greater than a predetermined threshold level); or b) the diseased cell has a super enhancer associated with the RARA gene that has a potency or ordinal rank equal to or greater than a predetermined threshold level, indicating that the subject will be responsive to the RARA agonist.

IFR8 mRNA level determination

[0108] In some embodiments, IFR8 mRNA levels can be used instead of super enhancer potency or ordinal rank to determine sensitivity to a RARA agonist. IFR8 mRNA can be quantified, which correlates very well with super enhancer potency at this locus (Figure 10). We have determined that mRNA transcripts encoding IRF8 correlate with sensitivity to RARA agonists (Figure 8) and that, in some embodiments, IFR8 mRNA levels can be used to identify cells that will be susceptible to RARA agonists.

[0109] In some embodiments, the sequences of one or more biomarkers (eg, epigenetic markers such as transcript level) are evaluated. In some embodiments, DNA sequencing can be used to sequence individual genes, larger genetic regions (eg, gene clusters or operons), entire chromosomes, or complete genomes. In some embodiments, RNA sequencing may be used. The person skilled in the art knows various methods for determining the sequences of individual genes, larger genetic regions (eg, gene clusters or operons), entire chromosomes, or complete genomes. In some embodiments, next generation sequencing may be used. In some embodiments, next generation whole genome sequencing may be used. In some embodiments, sequencing can be used to quantify the level of the transcript.

[0110] In some embodiments, IRF8 mRNA levels in a subject (e.g., in a tumor sample, in a cancer cell sample, in a blood sample, etc.) are compared using the same type of analysis with IRF8 mRNA levels in a population of subjects having the same disease or condition, to identify subjects susceptible to a RARA agonist. In these embodiments, at least some of the samples in the population will be tested for susceptibility to a specific RARA agonist to determine: a) the lowest IRF8 mRNA level of a sample in the population that is susceptible to that specific RARA agonist ("lowest mRNA susceptible sample"); and optionally b) the highest IRF8 mRNA level of a sample in the population that is immune to that specific RARA agonist (the "highest mRNA non-responsive sample"). In these embodiments, the IRF8 mRNA level threshold above which the test cell is considered susceptible to that specific RARA agonist is set to: i) equal to or greater than up to 5% of the IRF8 mRNA level in the susceptible sample with the lowest mRNA level in the population; or ii) equal to or greater by up to 5% of the level of IRF8 mRNA in the non-responder sample with the highest mRNA in the population; or iii) as a value between the IRF8 mRNA level of a susceptible sample with the lowest mRNA value and a non-receptive sample with the highest mRNA value in the population.

[0111] In some embodiments, not all samples in a population need to be tested for RARA agonist susceptibility, but all samples are to be measured for IRF8 mRNA levels. In some embodiments, samples are ranked based on IRF8 mRNA levels. The choice of which of the above three methods to use to establish a cutoff will depend on the difference in IRF8 mRNA levels between the lowest mRNA susceptible sample and the highest mRNA resistant nonresponder in the population, and whether the cutoff serves to minimize the number of false positives or maximize the potential number of responses. When this difference is large (for example, in the case of a large number of samples not tested for susceptibility that fall between a susceptible sample with the lowest mRNA score and a non-receptive sample with the highest mRNA score in a ranking of IRF8 mRNA levels), the cut-off value is generally set to be equal to or greater by up to 5% of the IRF8 mRNA level in the susceptible sample with the lowest mRNA score in the population. . When this difference is small (for example, in the case of few or no samples that were not tested for susceptibility that fall between a susceptible sample with the lowest mRNA score and a non-receptive sample with the highest mRNA score in a ranking of IRF8 mRNA levels), the cut-off value is generally set to the value between the IRF8 mRNA levels of the susceptible sample with the lowest mRNA score and the non-receptive sample with the highest mRNA. When the non-receptive sample with the highest mRNA has an IRF8 mRNA level greater than the susceptible sample with the lowest mRNA, the cut-off is generally set to equal or greater by up to 5% of the levels of IRF8 mRNA in the non-receptive sample with the highest mRNA in the population.

[0112] In some embodiments, the population is ranked based on the level of IRF8 mRNA. In these embodiments, the level of IRF8 mRNA in each sample is measured and compared to the mRNA levels of all other mRNAs in the cell to obtain an ordinal ranking of IRF8 mRNA levels. A cut-off value is then determined based on an ordinal ranking of IRF8 mRNA based on population samples that have been tested for susceptibility to a RARA agonist in the same manner as previously described for determining an ordinal cut-off for IRF8 super enhancer efficacy. The determined ordinal cut-off of IRF8 mRNA is then used directly or to determine a cut-off for prevalence, which is then used to stratify additional samples with respect to potential RARA agonist susceptibility.

[0113] In some embodiments, a cut-off for IRF8 mRNA levels is determined using a prevalence cut-off based on IRF8 enhancer potency or an IRF8 enhancer potency score as described above. In some aspects of these embodiments, a population is measured for mRNA levels and a predetermined prevalence cutoff is applied to that population to determine the mRNA cutoff. In some aspects of these embodiments, a standard curve is constructed to rank the levels of IRF8 mRNA in a population, and a predetermined prevalence cutoff is applied to this standard curve to determine the cutoff level of IRF8 mRNA.

[0114] In some aspects of the embodiments in which the cell or sample under study is compared to a population, the threshold(s) of mRNA levels obtained(s) for the population is converted to a prevalence rate, and the threshold value of the mRNA level is expressed as a percentage of the population having a threshold value or greater, for example, a prevalence threshold.

[0115] Without wishing to be bound by theory, Applicants believe that the prevalence rate of the test sample and the cut-off prevalence rate in the population will be the same regardless of the methodology used to determine IRF8 mRNA levels.

[0116] In some aspects of these embodiments, a subject is defined as susceptible to a RARA agonist if its IRF8 mRNA level corresponds to a population prevalence of about 80%, 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%, 4 2%, 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 IRF8 mRNA levels in the population . In some embodiments, the limit value is set based on the prevalence limit set for the effectiveness of the IRF8 enhancer. In some embodiments, the cutoff value is set based on the prevalence cutoff set for the ordinal value of the IRF8 enhancer's potency. In some embodiments, the cutoff is set based on IRF8 mRNA levels. In some embodiments, a cutoff for patients with AML, non-ATL AML, or MDS is set based on a prevalence value determined for an IRF8 enhancer potency ordinal value, and this prevalence value is used to determine a cutoff for IRF8 mRNA levels. In some embodiments, the cutoff for patients with AML, non-ATL AML, or MDS is defined using a cutoff prevalence of about 20-45% (e.g., about 20-25%, 25-30%, 25-35%, 2540%, 20-30%, 20-35%, 20-40%, 20-45%, 21-34%, 2 2-34%, 25-34%, 21-25%, 22-25%, 23-25%, 24-25% or 21-22%). In some embodiments, the cut-off for patients with AML, non-ATL AML, or MDS is determined using a prevalence value of 34%. In some embodiments, the cutoff for patients with AML, non-ATL AML, or MDS is determined using a prevalence value of 25%. In some embodiments, the cut-off for patients with AML, non-ATL AML, or MDS is determined using a prevalence value of 22%. In some embodiments, the cut-off value for patients with AML, non-ATL AML, or MDS is determined using a prevalence value of 21%.

[0117] In other embodiments, the population can be divided into three groups - susceptible, partially susceptible and non-susceptible, and two cut-offs or prevalence rates are set. A partially susceptible group may include susceptible and non-responders, as well as those members of the population whose response to the RARA agonist was not as high as in the susceptible group. In these embodiments, two limit values or prevalence limits are defined. This type of stratification may partially apply when, in a population, a non-receptive subject with the highest IRF8 mRNA score has IRF8 mRNA levels greater than a susceptible subject with the lowest RARA mRNA score. In this case, the cut-off value or cut-off rate between susceptible and partially susceptible subjects is set to be equal to or greater by up to 5% of the IRF8 mRNA level of the non-receptive subject with the highest IRF8 mRNA rate; and the cut-off or cut-off of prevalence between partially susceptible and non-responders is set to be equal to or less than 5% of the IRF8 mRNA level of the susceptible subject with the lowest IRF8 mRNA. Whether a RARA agonist should be administered to partially susceptible subjects will depend on the judgment of the treating physician and/or regulatory approval.

[0118] Methods for quantifying specific RNA sequences in a cell or biological sample are known in the art and include, but are not limited to, fluorescence hybridization such as used in services and products provided by NanoString Technologies, matrix technology (Affymetrix), reverse transcriptase qPCR, such as SYBR® Green (Life Technologies) or TaqMan® technology (Life Technologies), RNA sequencing (e.g., RNA-se q), RNA hybridization and signal amplification used in RNAscope® (Advanced Cell Diagnostics), or Northern blot.

[0119] In some aspects of these embodiments, the level of the RNA transcript (mRNA or other RARA or IRF8 transcript) in both the test cell and the control cell or all members of the population is normalized prior to comparison. Normalization involves adjusting a specific level of the IRF8 or RARA RNA transcript by comparison with another RNA transcript that is native and present at the same levels in both cells (eg, GADPH mRNA, 18S RNA), or with a fixed level of endogenous RNA that is "injected" into samples of each of the cells prior to determining the effectiveness of the super enhancer (J Loven et al., Cell, 151(3):476-82(201 2); J Kanno et al., BMC Genomics 7:64 (2006); J Van de Peppel et al., EMBO Rep 4:387-93 (2003)).

Cancer and other diseases

[0120] The methods of the present invention are applicable to the treatment of any cancer that is characterized by the association of a super enhancer with IRF8 or an IRF8 mRNA level equal to or greater than the threshold level for such cancer. Super enhancer-associated IRF8 genes may be more common in certain types of cancer than others. In some embodiments, super enhancer-associated IRF8 genes may be more prevalent in non-ATL AML and in MDS than in other types of cancer or precancerous conditions.

[0121] In some embodiments, the disease to be treated by the methods of the invention is cancer. In some embodiments, the disease being treated is selected from non-ALD AML and MDS. In some embodiments, the disease being treated is non-ALD AML and MDS that do not have a chromosomal translocation that includes the IRF8 gene.

[0122] In some embodiments, the subject to be treated with a RARA agonist (eg, tamibarotene) is suffering from relapsed or refractory non-ATL AML. Subjects are classified as having relapsed or refractory non-APL AML if they: a) do not demonstrate a partial response after the first cycle of induction chemotherapy; or b) do not show a complete response after the second cycle of induction chemotherapy; or c) demonstrate a relapse after conventional chemotherapy; or d) demonstrate relapse after undergoing a single stem cell transplant.

[0123] In some embodiments, the subject being treated with a RARA agonist (eg, tamibarotene) is suffering from refractory MDS. Subjects are classified as having refractory MDS if they are: a) defined as at high risk or intermediate stage-2 MDS (as defined by the International Predictive Staging System ("IPPS")) and have not achieved any hematologic improvement (as defined by the IWG 2006 criteria) after at least 4 cycles of induction therapy with hypomethylating agents (eg, azacitidine, decitabine) or demonstrated relapse after complete or partial response of any duration; or b) determined to be intermediate stage-1 or low risk for MDS by IPSS and dependent on transfusion or not responding to treatment with erythropoiesis-stimulating agents (ESAs).

[0124] In some embodiments, the subject to be treated with a RARA agonist (eg, tamibarotene) is an elderly, unfit subject. As used herein, the term "elderly unfit" refers to a subject who is a human at least 60 years of age and who, as determined by a physician, is not a candidate for standard induction therapy.

RARA agonists

[0125] The choice of RARA agonist to be treated in a patient having by definition some level of super enhancer can be made from any RARA agonist known in the art. Preferably, the RARA agonist used in the methods of the invention is specific for RARA and has substantially less (at least 10-fold less, at least 100-fold less, at least 1000-fold less, at least 10,000-fold less, at least 100,000-fold less) agonist activity against other forms of RaR, e.g., RaR -β and RaR-γ.

[0126] In some embodiments, the RARA agonist is selected from a compound described in any of the following US patents, or any compound corresponding to the class given them: 7, US 6071924, US 6075032, US 6187950, US 6355669, US 6358995 and US 6387950, each of which is incorporated by reference.

[0127] In some embodiments, the RARA agonist is selected from any of the following known RARA agonists listed in Table 1, or a pharmaceutically acceptable salt, or solvate, or hydrate thereof:

[0128] In some embodiments, the RARA agonist is tamibarotene.

Therapeutic regimens

Markers and stats

[0129] In some embodiments, the technologies proposed in the present invention include an assessment of the type of cancer from which the patient suffers. In some embodiments, the patient is suffering from non-APL acute myelocytic leukemia (AML). In some embodiments, the patient is suffering from myelodysplastic syndrome (MDS).

[0130] In General, the present invention provides technologies, in accordance with which carry out the analysis and/or evaluation of one or more markers or characteristics of the subject; in some embodiments, a decision regarding therapy is made based on such analysis and/or evaluation.

[0131] In some embodiments, a marker is an agent or compound whose presence, form, level, and/or activity correlates in a relevant population with a relevant trait (eg, type or stage of cancer). In some embodiments, the present invention contemplates the identification, classification and/or characterization of one or more biomarkers relevant to the treatment of non-AML AML with a RARA agonist. In some embodiments, the present invention involves the identification, classification and/or characterization of one or more biomarkers relevant to the treatment of MDS with a RARA agonist.

[0132] In some embodiments, classifying a patient as suffering from a particular type of cancer may include an assessment of the stage of the cancer. In some embodiments, classifying a patient as suffering from a particular type of cancer may include an assessment of the severity of the disease in the patient (eg, number of cancer cells, tumor size, and/or amount of cancer in the body).

[0133] In general, the type of cancer can be assessed in accordance with the present invention using a suitable type of analysis, as is obvious to those skilled in the art. Many assays are known in the art for evaluating the type of cancer, including, for example, those using histological assessment (e.g., biopsy sample), imaging (e.g., magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT)), ultrasound, endoscopy, x-rays (e.g., mammogram, barium swallow test, panoramic radiography), ductogram, or bone scintigraphy.

[0134] In some embodiments, RARA agonist therapy includes assessing the level of one or more biomarkers that characterize the stage or form of non-APL AML or MDS. In some embodiments, RARA agonist therapy includes assessing the level of IRF8 mRNA and, optionally, the level of RARA mRNA. In some embodiments, RARA agonist therapy includes the presence of a super enhancer associated with the IRF8 gene and optionally the potency or ordinal rank of the super enhancer associated with the RARA gene. In some embodiments, RARA agonist therapy includes assessing the level of IRF8 mRNA or the presence of a super enhancer associated with the IRF8 gene, and optionally, the level of RARA mRNA, potency, or ordinal rank of the super enhancer associated with the RARA gene.

[0135] Without being limited by theory, Applicants believe that PBMC subsets that have only one of the CD34 or CD117 markers can also be used to determine IRF8 or super enhancer mRNA levels. In addition, some IRF8 mRNA assays, such as RNAScope®, do not require enrichment of the PBMC sample prior to analysis, as these methods provide analytical enrichment of mRNA from the cells of interest using specific oligonucleotide hybridization/amplification procedures.

Patient populations

[0136] In some embodiments, RARA agonist therapy is administered in accordance with the present invention to one or more patients (eg, a population of patients) as described herein.

[0137] In some embodiments, the patient population includes one or more subjects (eg, includes or consists of subjects) suffering from cancer. In some embodiments, the patient population includes one or more subjects suffering from non-APL AML. In some embodiments, the patient population includes one or more subjects suffering from MDS.

[0138] In some embodiments, the patient population includes one or more subjects (eg, includes or consists of subjects) who have previously received therapy for the treatment of cancer (eg, non-APL AML or MDS). In some embodiments, the patient population includes one or more subjects (eg, includes or consists of subjects) who have not previously received therapy for the treatment of cancer (eg, non-AML AML or MDS). In some embodiments, the patient population includes or consists of patients who have not previously received therapy for the treatment of non-APL AML or MDS.

[0139] In some embodiments, a patient who has previously received therapy may have previously received therapy selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, palliative care, surgery, and combinations thereof. In some embodiments, the patient has undergone a transplant. In some embodiments, the patient received standard cytotoxic chemotherapy. In some embodiments, standard cytotoxic chemotherapy includes the use of cytarabine and/or anthracycline. In some embodiments, standard cytotoxic chemotherapy may include additional chemotherapy and/or hematopoietic stem cell transplantation (HSCT). In some embodiments, the patient received hypomethylating agents. In some embodiments, the patient received lenalidomide.

[0140] In some embodiments, the patient population includes one or more subjects (e.g., includes or consists of subjects) who have received and/or are receiving another therapy, for example, such that the RARA agonist therapeutic composition (e.g., tamibarotene) is administered in combination with another type of therapy (e.g., chemotherapeutic agents). In some embodiments, such other therapy may include or consist of cancer therapy (e.g., as described herein), pain, nausea, constipation, treatment of one or more side effects (e.g., itching, hair loss, insomnia, etc.) associated with cancer therapy, etc., or any combination thereof. The present invention provides a method for treating non-ATL AML or MDS, which comprises treating a patient defined as having non-ATL AML or MDS with a therapeutically effective amount of a RARA agonist therapeutic agent (e.g., tamibarotene) or a pharmaceutically acceptable salt thereof.

[0141] In some embodiments, the present invention provides a method of preventing or slowing the onset of non-ATL AML or MDS, comprising administering to a patient determined to be in need of preventing or slowing the onset of non-ATL AML or MDS, a prophylactically effective amount of a RARA agonist therapeutic agent (e.g., tamibarotene) or a pharmaceutically acceptable salt thereof.

[0142] In some embodiments, the present invention provides a method of treating a patient for non-APL AML or MDS who has previously been treated with a chemotherapy regimen by administering to such patient a therapeutically effective amount of a RARA agonist therapeutic agent (e.g., tamibarotene) or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating a patient for non-AKL AML or MDS in the absence of standard therapy options. In some embodiments, the present invention provides a method of treating a patient who is not eligible for standard therapy.

[0143] In some embodiments, the patient may also have diseases associated with MDS, such as bone marrow failure, peripheral blood cytopenia, and associated complications such as anemia, infection, or bleeding. In some embodiments, the patient may have MDS progressing to AML.

[0144] In some embodiments, the patient or patient population may not be (eg, may exclude) patients who are or may be pregnant. In some embodiments, a patient or patient population may be monitored for one or more signs of pregnancy, childbirth, and/or breastfeeding prior to and/or during RARA agonist therapy. In some embodiments, RARA agonist therapy may be reduced, temporarily interrupted, or discontinued for a patient who, by definition, exhibits one or more signs of pregnancy, labor, and/or breastfeeding.

[0145] In some embodiments, the patient or patient population may not be (eg, may exclude) patients who have a history of hypersensitivity to the tamibarotene ingredient. In some embodiments, the patient or patient population may not be (e.g., may exclude) patients who receive vitamin A formulations. In some embodiments, the patient or patient population may not be (e.g., may exclude) patients who have hypervitaminosis A.

[0146] In some embodiments, the patient or patient population may not be (eg, may exclude) elderly patients. In some embodiments, the patient or patient population may be or include one or more elderly patients. In some embodiments, elderly patients may be monitored more frequently for potential adverse events (including, for example, low serum albumin levels and/or elevated plasma free drug concentrations, etc.) compared to one or more younger patients. In some embodiments, RARA agonist therapy may be reduced, temporarily interrupted, and/or discontinued for an elderly patient who, by definition, exhibits one or more signs of such an adverse event.

[0147] In some embodiments, the patient or patient population may not be (eg, may exclude) pediatric patients. In some embodiments, the patient or patient population may be or include one or more pediatric patients. In some embodiments, pediatric patients may be monitored more frequently for potential adverse events (including, for example, increased intracranial pressure, etc.) compared to one or more older patients. In some embodiments, RARA agonist therapy may be reduced, temporarily interrupted, and/or discontinued for a pediatric patient who, by definition, exhibits one or more signs of such an adverse event.

[0148] In some embodiments, RARA agonist therapy according to the present invention is reduced, temporarily interrupted, or discontinued for a particular patient if and when the patient develops one or more adverse reactions, such as, for example, headache, rash, dry skin, eczema, exfoliative dermatitis, bone pain, joint pain, fever, increased white blood cell count, decreased hemoglobin, increased ACT, increased ALT, increased LDH, increased alkaline phosphatase, increased TG, increased TC, follicitis, folliculitis, increased CRP, and combinations thereof. In alternative or additional embodiments, RARA agonist therapy according to the present invention is reduced, temporarily interrupted, or discontinued for a particular patient if and when the patient develops one or more adverse reactions, such as, for example, thrombosis (e.g., cerebral infarction, pulmonary infarction, arterial thrombosis, venous thrombosis, etc.), vasculitis, delirium, toxic epidermal necrolysis (Lyell's syndrome), erythema multiforme, increased internal cranial pressure and their combinations.

[0149] In some embodiments, the present invention provides for the use of a compound (eg, tamibarotene) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for treating, preventing, or slowing the onset of non-ATL AML or MDS. In some embodiments, the patient is suffering from cancer (eg, non-ALD AML or MDS). In some embodiments, the patient is suffering from a cancer (eg, non-ALD AML or MDS) that is resistant to other therapies (eg, chemotherapy). In some embodiments, the cancer is characterized by the presence of an IRF8 biomarker, wherein the IRF8 biomarker is or includes the expression of one or more of increased levels of IRF8 mRNA or an IRF8 gene-related super enhancer. In some embodiments, the cancer is characterized by the expression of one or more of increased levels of RARA mRNA or a super enhancer associated with the RARA gene. In some embodiments, the cancer is characterized by a lack of expression of one or more of elevated levels of RARA mRNA or a super enhancer associated with the RARA gene.

Dosage Forms and Dosing Schedules

[0150] In general, each active agent (e.g., tamibarotene) for use in accordance with the present invention is formulated, dosed, and administered in a therapeutically effective amount using pharmaceutical compositions and dosage regimens consistent with good medical practice and appropriate for the relevant agent(s) and subject. In principle, therapeutic compositions may be administered by any suitable route known in the art, including, without limitation, oral, mucosal, inhalation, topical, buccal, nasal, rectal, or parenteral (e.g., intravenous, infusion, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other routes of administration). In some embodiments, the RARA agonist (eg, tamibarotene) will be administered orally.

[0151] In some embodiments, the dosage regimen for a particular active agent may include intermittent or continuous administration, for example, to obtain a particular desired pharmacokinetic profile or other mode of action in one or more tissues or body fluids of interest in the body of the subject receiving therapy.

[0152] In some embodiments, different agents administered in combination may be administered by different delivery routes and/or according to different schedules. In alternative or additional embodiments, one or more doses of the first active agent are administered substantially simultaneously, and in some embodiments, in the same way and/or as part of the same composition with one or more other active agents.

[0153] Factors to be considered when optimizing dosing routes and/or schedules for a given therapeutic regimen may include, for example, the specific indication to be treated, the subject's clinical condition (e.g., age, general health, previous therapy and/or responsiveness, etc.), site of agent delivery, nature of the agent, regimen and/or route of administration of the agent, presence or absence of combination therapy, and other factors known to medical practitioners. For example, in the treatment of cancer, relevant characteristics of the indication to be treated may include, but are not limited to, one or more of cancer type, stage, location, and so on.

[0154] In some embodiments, one or more characteristics of a particular pharmaceutical composition and/or dosage regimen used can be modified over time (e.g., increasing or decreasing the amount of active agent in any individual dose, increasing or decreasing time intervals between doses, etc.), for example, to optimize the desired therapeutic effect or response.

[0155] In general, the type, amount, and frequency of dosing of active agents in accordance with the present invention is adjusted according to the safety and efficacy requirements applicable when administering the relevant agent(s) to a mammal, preferably a human. In general, such dosing characteristics are chosen to provide a specific, and typically detectable, therapeutic response compared to that observed in the absence of therapy. In some embodiments, the RARA agonist (eg, tamibarotene) will be administered continuously.

[0156] In the context of the present invention, an example of a desired therapeutic response may include, but is not limited to, inhibition and/or reduction of tumor growth, tumor size, metastasis, one or more of the symptoms and side effects associated with the tumor, as well as an increase in tumor cell apoptosis, a therapeutically relevant decrease or increase in the number of one or more cellular markers or circulating markers, etc. Such criteria can be easily assessed by any or a variety of immunological, cytological and other methods described in the literature.

[0157] In some embodiments, there may be a need to formulate dosing regimens and, in particular, to develop sequential dosing regimens based on the time of appearance and / or threshold levels of expression of inducible markers for specific types of tumors, specific tumors, specific patient populations (for example, bearing genetic markers) and / or specific patients. In some such embodiments, therapeutic dosing regimens can be combined or adjusted for detection methods that assess the expression of one or more inducible markers before and/or during therapy.

[0158] 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/m ² , 2 mg/m ² , 3 mg/m ² , 4 mg/ ^{m 2} , 5 mg/m ² , 6 mg/m ² , 7 mg/m ² , 8 mg/m 2 , 9 mg/m ² , 10 mg/m ² , 11 mg/m ² , 12 mg/m ² , 13 mg/m 2 , 14 mg/m 2 , 15 mg/ ^{m 2 ,} 16 mg/m ^{2 ,} or a dose of tamibarotene intermediate between any two of these values. In some embodiments, the tamibarotene therapy regimen includes the use of a dose of 6 mg/m ² . In some embodiments, the tamibarotene therapy regimen includes the use of a dose of 4 mg/m ² . In some embodiments, the tamibarotene therapy regimen includes the use of a dose of 2 mg/m ² . In some embodiments, the tamibarotene therapy regimen includes the use of a dose of 1 mg/m ² .

[0159] In some embodiments, a RARA agonist (eg, tamibarotene) therapy regimen comprises the administration of a number of doses of the tamibarotene composition. In some such embodiments, a tamibarotene therapy regimen includes, for example, administering 2, 5, 10, 20, 30, 60, 90, 180, 365 doses, or a number of doses between any two of these values, and/or includes a repeatable dosing regimen (e.g., at least one cycle of two daily doses, which may be repeated, optionally, with an administration period of an alternative, or optionally, with a period without administration that separates different cycles). In some embodiments, the tamibarotene therapy regimen is administered twice daily. In some embodiments, the tamibarotene therapy regimen is administered once daily. In some embodiments, the tamibarotene therapy regimen comprises a total dose of 6 mg/m ² divided into two daily oral doses.

[0124] In some embodiments, a RARA agonist (eg, tamibarotene) therapy regimen may be applied to a subject or population of patients who have or have not taken some food before, during, or after administration. The terms "before administration" and "after administration" in relation to food intake refer to a period of time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 30, 42, or 72 hours or more before or after administration. In some embodiments, the term "introduction ... taking into account food intake" implies that the subject or population of patients take food prior to administration (eg, after a meal). In some embodiments, the term "introduction ... taking into account food intake" implies that the subject or population of patients eat after administration. In some embodiments, the term "introduction ... taking into account food intake" implies that the subject or patient population is eating at the time of administration. In some alternative implementations, the term "introduction ... taking into account food intake" means that the subject or population of patients are in a state of fasting at the time of administration.

[0160] In some embodiments, the meal includes a high fat meal or a high fat diet. In some embodiments, a RARA agonist (eg, tamibarotene) therapy regimen is administered to a subject in a fasted state. In some embodiments, a RARA agonist (eg, tamibarotene) therapy regimen is administered to the subject after a meal.

Lineups

[0161] As used herein, a pharmaceutical composition refers to a mixture of a compound, such as tamibarotene, with other chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickeners, and/or excipients. The pharmaceutical composition facilitates the administration of the compound into the body. Pharmaceutical compositions containing the compound may be administered in therapeutically effective amounts by any conventional route or route known in the art, including, but not limited to: intravenous, oral, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, otic, nasal, and topical administration.

[0162] The compound can be administered locally rather than systemically, for example, by injecting the compound directly into an organ, often as a depot or sustained release formulation. In addition, the pharmaceutical composition containing the compound can be administered in a targeted drug delivery system, for example, in a liposome coated with an organ-specific antibody. The liposomes will be selectively targeted and taken up by the organ. In addition, the pharmaceutical composition containing the compound may be provided in the form of an immediate release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. In some embodiments, the extended release formulation releases the compound for more than 1 hour, more than 2 hours, more than 3 hours, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, or more. In some embodiments, the extended release formulation releases the compound at a constant rate for more than 1 hour, more than 2 hours, more than 3 hours, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, or more.

[0163] For oral administration, the compound can easily be formulated by combining the active compounds with pharmaceutically acceptable carriers or excipients well known in the art. Such carriers allow the compounds described herein to be prepared in the form of tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like. for ingestion by the subject to be treated. In general, adjuvants such as fillers, disintegrants, glidants, surfactants, recrystallization inhibitors, lubricants, pigments, binders, fragrances, etc. can be used for conventional purposes and in typical amounts without affecting the properties of the compositions. In some embodiments, the excipient is one or more of lactose hydrate, cornstarch, hydroxypropyl cellulose, and/or magnesium stearate. In some embodiments, tamibarotene can be formulated with one or more of lactose hydrate, cornstarch, hydroxypropyl cellulose, and/or magnesium stearate.

[0164] The determination of acceptable formulations of tamibarotene can be carried out by various methods known in the art, for example, described in US 20100048708, which is incorporated herein by reference.

Combination Therapy

[0165] Those skilled in the art, reading the present disclosure, will readily appreciate that a RARA agonist described herein (e.g., tamibarotene) can, in some embodiments, be combined with other anti-cancer therapies, including, for example, the use of chemotherapeutic agents, other immunomodulatory agents, radiation therapy, high-frequency ultrasound therapy, surgery, FDA-approved therapies for cancer, etc.

[0166] In some embodiments, the RARA agonist is used in combination with one or more other therapeutic agents or modalities. In some embodiments, one or more other therapeutic agents or modalities are also anticancer agents or modalities; in some embodiments, the combination exhibits a synergistic effect in the treatment of cancer.

[0167] Known compounds or treatment options that demonstrate therapeutic efficacy in the treatment of cancer may include, for example, one or more alkylating agents, antimetabolites, microtubule-acting agents, topoisomerase inhibitors, cytotoxic antibiotics, angiogenesis inhibitors, immunomodulators, vaccines, cellular therapies, organ transplantation, radiation therapy, surgery, etc.

[0168] In some embodiments, the RARA agonist (and/or other therapy with which it is combined) can be combined with one or more types of palliative (e.g., pain relief, antinausea, antiemetic, etc.) therapy, in particular, when there is relief of one or more symptoms associated with a relevant type of cancer or with another disease, disorder, or pathological condition to which a particular cancer patient is susceptible or from which a particular cancer The new patient suffers.

[0169] In some embodiments, the agents used in combination are administered according to the dosage regimen for which they are approved for individual use. However, in some embodiments, the combination with a RARA agonist (e.g., tamibarotene) allows the administration of another agent according to a dosing regimen that includes the use of one or more smaller and/or less frequent doses and/or fewer cycles compared to using an agent without a RARA agonist (e.g., tamibarotene). In alternative or additional embodiments, a suitable dosing regimen includes the use of larger and/or more frequent doses and/or more cycles compared to the use of an agent without a RARA agonist (eg, tamibarotene).

[0170] In some embodiments, one or more doses of agents administered in combination are administered at the same time; in some such embodiments, the agents may be administered in a single composition. More often, however, the agents are administered in different formulations and/or at different times. In some embodiments, tamibarotene is administered sequentially and/or simultaneously with other therapeutic agents (eg, chemotherapy).

[0171] In some embodiments, the combination therapy options described herein are used only when the subject has a RARA mRNA level equal to or greater than a threshold value. In some embodiments, the combination therapy options described herein are used only when the subject has an IRF8 mRNA level equal to or greater than a threshold. In some embodiments, the combination therapy options described herein are used only when the subject has a RARA mRNA level equal to or greater than a threshold and an IRF8 mRNA level equal to or greater than a threshold. In some aspects of any of these embodiments, the subject is suffering from non-AML AML.

[0172] In some embodiments, the therapeutic agent for combination with a RARA agonist (e.g., tamibarotene) is selected from a DNA methyltransferase inhibitor, a DNA synthase inhibitor, a topoisomerase inhibitor, an FLT3 inhibitor, a folate inhibitor, a BRD4 inhibitor, a Zn-finger transcription factor inhibitor, a GCR inhibitor, a CDK7 inhibitor, an HDAC inhibitor, a J inhibitor. MJD3/JARID1B or an EZH2 inhibitor. In other specific aspects, the second agent is selected from an LSD1 inhibitor, a proteasome inhibitor, a DNA damage repair inhibitor, a PARP inhibitor, an mTOR inhibitor, a DOT1L inhibitor, a tubulin inhibitor, a PLK inhibitor, or an aurora kinase inhibitor.

[0173] In some embodiments, the RARA agonist (eg, tamibarotene) can be administered with decitabine, azacitidine, ara-C, daunorubicin, idarubicin, arsenic trioxide, and/or flt3 inhibitors. In some embodiments, a RARA agonist (eg, tamibarotene) can be administered with IDH inhibitors, BRD4 inhibitors (eg, JQ1), HDAC inhibitors (eg, SAHA and MC1568), HMT inhibitors (eg, EPZ6438, UNC0638, SGC707, EPZ5676, UNC037, and PFI-2), and/or KDM inhibitors (eg, G SKJ4, RN-1 and GSK-LSD1).

[0174] In some embodiments, the subject is suffering from AML and tamibarotene is administered in combination with a second agent selected from azacitidine, arsenic trioxide, midostaurin (only for those AML subjects who have high levels of FLT3 mRNA), cytarabine, daunorubicin, methotrexate, idarubicin, sorafenib (only for those AML subjects). with high FLT3 mRNA levels), decitabine, quisartinib (only for those AML subjects with high FLT3 mRNA levels), JQ1 (a BRD4 inhibitor), ATO, prednisone (only for those AML subjects with high GCR mRNA levels), SAHA, and GSKJ4 (only for those AML subjects with high JMJD3/JARID mRNA levels) 1B).

Sets

[0175] A kit containing one or more reagents for detecting one or more IRF8 biomarkers may be provided in a kit. In some cases, the kit contains packaged pharmaceutical compositions according to the present invention containing an insert or label with written instructions for the use of a RARA agonist (e.g., tamibarotene) in a subject suffering from cancer and which, by definition, has an IRF8 gene-related superenhancer having a potency or ordinal rank equal to or greater than a threshold level, or an IRF8 mRNA level equal to or greater than a reference (e.g., threshold level) or greater than it. As detailed above, the threshold level is determined in a population of samples from any subjects diagnosed with the same disease or cell lines or xenograft models of the same disease as the one for which the pharmaceutical composition is prescribed. The instructions may be glued or otherwise attached to the container containing the RARA agonist. Alternatively, the instructions and the container containing the RARA agonist will be separate from each other, but kept together in the same kit, package, box, or other type of container.

[0176] The instructions in the kit are generally authorized or recommended by the government agency that approves the therapeutic use of the RARA agonist. The instructions may include a description of specific methods for determining whether a super enhancer is associated with the IRF8 gene, as well as quantification methods for determining whether an enhancer associated with the IRF8 gene is a super enhancer, quantification methods for determining levels of IRF8 mRNA; and/or threshold levels of super enhancers or IRF8 mRNA at which treatment with the packaged RARA agonist is recommended and/or considered therapeutically effective. In some aspects, the instructions indicate that the composition should be administered to a subject whose IRF8 mRNA levels fall within at least the 30th percentile of the population for which IRF8 mRNA levels were measured. In some aspects of these embodiments, a subject is defined as susceptible to an RARA agonist if the IRF8 mRNA prevalence rate is about 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 6 4%, 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 the population for which IRF8 mRNA levels were measured. In some aspects, the instructions indicate that the composition is to be administered to a subject whose IRF8 mRNA levels are as measured by a particular assay method.

[0177] The instructions may optionally include dosing information, cancer types for which RARA agonist treatment has been approved, physicochemical information about the RARA agonist; pharmacokinetic information about the RARA agonist, information about the interaction between drugs. In some embodiments, the instructions indicate that the composition is to be administered to a subject diagnosed with AML. In some embodiments, the instructions indicate that the composition is to be administered to a subject diagnosed with non-APL AML. In some embodiments, the instructions indicate that the composition is to be administered to a subject diagnosed with MDS. In some aspects, the pharmaceutical composition contains tamibarotene.

EXAMPLES

[0178] In order to understand the invention described herein more fully, the following examples are given. The synthetic and biological examples described in this application are offered to illustrate the proposed compounds, pharmaceutical compositions and methods, and should not be taken as limiting their scope in any way.

Example 1: IRF8 mRNA Levels in Non-AML AML Cell Lines Correlate with RARA Agonist Sensitivity

[0179] Previously, we investigated several AML cell lines for susceptibility to tamibarotene and demonstrated that susceptibility correlated very well with each of RARA super enhancer potency, RARA super enhancer potency rank, and RARA mRNA level. This was done as described below.

[0180] On the day of the experiment, cells were homogenized using Accumax (EMD Millipore), counted, and adjusted to 60,000 cells/ml in a suitable growth medium. Using Biotek EL406, 50 μl of cells were dispensed into white (ATPlite) or black (CyQuant) 384-well plates (Thermo). The cells were returned to the 37° C. incubator for adhesion. After three hours, compounds were added to the plates using a 20 nl 384-well multichannel pipetting head on a Janus workstation. Stock was placed in four replicates to study 10-point dose-dependence in stock DMSO solution in 384-well compound plates. After the compound was added, the plates were incubated for five or ten days in a 37°C incubator.

[0181] Cell viability was read using ATPlite (Perkin Elmer) or CyQuant (Life Technologies). In the case of ATPlite, the plates were removed from the incubator and brought to room temperature before use. Lyophilized ATPlite reagent powder was resuspended in lysis buffer and diluted 1:2 with distilled water. 25 µl of this solution was added to each well using a Biotek fluid arm. The plates were incubated for 15 min at room temperature before reading the luminescence signal on an Envision plate reader (Perkin Elmer). In the case of CyQuant, the reagents were mixed according to the manufacturer's instructions in the FSB (Gibco). The reagent was added using a multichannel pipette and the plates were placed back in the incubator for 30 minutes before being read on an Envision plate reader (Perkin Elmer).

[0182] The data thus obtained was stored and grouped in Microsoft Excel and analyzed using the GraphPad Prism software. Curve fitting to calculate EC ₅₀ and E _max was performed in GraphPad Prism version 6.0 using four-parameter (Hill's slope was not taken as 1) non-linear regression with log 10 converted compound concentration data versus percent cell viability when normalized against wells treated with DMSO alone included in the plate. The outer holes were excluded.

[0183] We used the Affymetrix ^GeneChip® PrimeView™ Human Gene Expression Array to first examine seven of these AML cell lines (four tamibarotene-sensitive NOMO-1, OCI-AML3, MV-4-11 and Sig-M5; and three insensitive KG1a, OCI-M1 and Kasumi-1) for the presence of other mRNAs that can be specifically up-regulated in tamibarotene sensitive cell lines and identified IRF8 mRNA as a potential candidate. We previously quantified IRF8 mRNA levels in each of these seven AML cell lines, as well as several other AML cell lines being investigated for susceptibility to tamibarotene, using RNA sequencing assay as described below. The results for the first seven cell lines are shown in FIG. 1. Interestingly, NOMO-1 did not have a high level of RARA mRNA, but was sensitive to tamibarotene. The fact that NOMO-1 had elevated IRF8 mRNA levels helped to clarify this apparent discrepancy and further support the possibility of using IRF8 mRNA levels to predict susceptibility to tamibarotene.

[0184] Preparation of RNA: The cell suspension was transferred into microcentrifuge tubes and washed with 1 ml of PBS. The cell pellet was resuspended in 200 µl of TRIzol. 20 μl siRNA homogenate supplement from Ambion miKVana siRNA isolation kit (AM 1561) was added, mixed and incubated on ice for 10 minutes. Added 20 μl of bromochloropropane, mixed, incubated at room temperature for 5 minutes and centrifuged at 12000 × g for 10 minutes at 4°C. 62 μl of the aqueous phase was added to 78 μl of ethanol and transferred to a filter column. Isolation was performed according to the protocol for isolation of total RNA from Ambion. The sample was analyzed for quality control on a bioanalyzer and then sent to the Whitehead Sequencing Core (Cambridge, MA) for sequencing.

[0185] Processing of RNA sequencing data: Read results were aligned with 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-barn --bowtie2 --bow tie2 - path/data/devtools/bowtie2-2.0.5 --strand-specific) and then quantified mRNA using the same rsem package (rsem-parse-alignments, rsem-build-read-index, rsem-run-em) and presented in transcripts per million (TNM). Then, all protein-coding genes were extracted for each sample and quantile normalization of their parameters was performed.

[0186] We then compared susceptibility to tamibarotene with IRF8 mRNA levels as shown in FIG. 2 and in table 1.

*HL60 is an APL cell line.

[0187] As can be seen from the table above, all tamibarotene-responsive cell lines, with the exception of HL60, had an IRF8 mRNA level greater than 190 THM (log ₂ (7.57)) in this assay, while all non-responsive cell lines had an IRF8 mRNA level less than 16.5 THM (log ₂ (4.03)). HL60 susceptibility to tamibarotene without concomitant high levels of IRF8 mRNA (6.73 THM) suggests that the correlation between IRF8 mRNA level and susceptibility to tamibarotene may not be maintained in the case of ALI and therefore may be better suited to stratify subjects suffering from non-ALD AML. Figure 2 removed data for HL60.

[0188] IRF8 mRNA levels have been determined for a wide variety of sample types - normal blood cells, AML cell lines, primary AML patient samples, and AML APD. The data obtained were plotted in rank order and the results are plotted in Figure 14. As can be seen, Figure 14 does not show any correlation between IRF8 mRNA levels and the presence of disease; and it turned out that the levels of IRF8 are distributed in a fairly similar way in normal cells compared to diseased cells, cell lines and PPC.

Example 2 Determination of Thresholds for IRF8 mRNA for RARA Agonist Treatment

[0189] Results for AML cell lines suggest a cut-off of 15.5 to 190 TNM (i.e., log ₂ (4.03) to log ₂ (7.57) in RNA sequencing analysis. We selected a population of AML patient samples (courtesy of Stanford University) to investigate the distribution of IRF mRNA levels and determine prevalence cut-offs based on cut-offs. We added to this population AML cell lines and plotted in rank order.The rank distribution of IRF8 mRNA levels in the combined AML patient/cell line population is shown in Fig. 3. We determined that a prevalence cut-off of 25% corresponded to an IRF mRNA value of approximately log ₂ (7).

Example 3: Correlation of IRF8 mRNA levels and RARA mRNA levels

[0190] Next, we compared IRF8 and RARA mRNA levels in AML cell lines and patient populations to determine if there was a correlation. In FIG. 4 shows that some cell lines that were susceptible to tamibarotene have relatively low levels of RARA mRNA but high levels of IRF8 mRNA. In FIG. 5 shows that a subset of patients also show high levels of IRF8 mRNA but relatively low levels of RARA mRNA 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 the mRNA level is above a threshold could optimize the patient population to be treated.

Example 4: Super enhancer associated with IRF8. correlates with susceptibility to RARA agonist treatment

[0191] Next, we studied the effectiveness of the IRF8 enhancer in several cell lines and patient samples as follows.

[0192] Cell fixation: For cells in suspension, typically 1/10 volume of fresh 11% formaldehyde solution was added to the cell suspension, mixed, and allowed to stand at room temperature (RT) for 8 minutes. Then 1/20 volume 2.5 M glycine or 1/2 volume 1 M Tris, pH 7.5 was added to quench the formaldehyde and incubated for at least 1 min. Cells were washed 3 times with 20-50 ml of cold 1x phosphate-buffered saline (PBS), centrifuged for 5 min at 1250 x g to pellet the cells before and after each wash. The cells were then transferred into 15 ml conical tubes and centrifuged at 1250×g for 5 min at 4°C. The supernatant was removed, residual liquid was removed by blotting with Kimwipe wipes, and then the precipitated cells were quickly frozen in liquid nitrogen and stored at -80°C.

[0193] Bead preparation: Approximately 60 μl of Dynabeads® protein G beads per 2 ml immunoprecipitate (Invitrogen) were used. The beads were washed 3 times, 5 minutes each time, with 1.0 ml blocking buffer (0.5% BSA w/v in PBS) in a 1.5 ml Eppendorf tube. A magnet (Invitrogen) was used to collect the beads (and left to bind to the magnet for at least 1 full minute) after each wash and then the supernatant was aspirated. The washed beads were resuspended in 250 μl of blocking buffer, to which 6 μg of antibody was added and the mixture was left to incubate with mixing with inversion overnight (minimum 6 hours). The antibody-bound beads were washed 3 times, 5 minutes each time, with 1 ml blocking buffer and resuspended in blocking buffer (60 μl per IP). These last washes and resuspensions were performed after sonicating the cells (see 9.1.1.3) and just prior to overnight immunoprecipitation.

[0194] Cell lysis: protease inhibitors at 1x (full, Roche; prepared by dissolving one tablet in 1 ml H ₂ O for a 50x solution and stored in aliquots at -20°C) were added to all lysis buffers prior to use. Each tube of cells (approximately 5×10 ⁷ cells) was resuspended in 5-10 ml of lysis buffer 1 (LB1; 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and swirled at 4°C for 10 minutes. Cells were centrifuged at 1250 × g × 5 min in a tabletop centrifuge at 4°C and the supernatant was aspirated. Cells were resuspended in 5 ml lysis buffer 2 (LB2; 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris, pH 8) and incubated with inversion at 4° C. for 10 minutes. Cells were again pelleted at 1250×g for 5 min on a benchtop centrifuge at 4°C and washed in 2-5 ml Covaris sonication buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 0.1% SDS). The pellet was centrifuged at 1250×g for 5 min in a tabletop centrifuge at 4°C. Cells were pelleted at 1250×g for 5 min in a 4° C. benchtop centrifuge and resuspended at 20-50 million cells/1 ml Covaris sonication buffer.

[0195] Chromatin immunoprecipitation: Fifty µl of antibody-conjugated beads prepared as described above were added to a solution of purified cell extract (as described above in the cell lysis paragraph) in 1.5 ml tubes and swirled overnight at 4°C (minimum 8 hours) to immunoprecipitate DNA-protein complexes.

[0196] Washing, elution and cross-linking: all buffers used in these steps were ice cold. A magnetic stand was used to precipitate the magnetic beads, washed 3 times, 5 minutes each time, with stirring with gentle inversion, 1 ml wash buffer 1 (50 mM HEPES pH 7.5; 140 mM NaCl; 1 mM EDTA; 1 mM EGTA; 0.75% Triton-X; 0.1% SDS; 0.05% DOC); washed once for 5 minutes with 1 ml wash buffer 2 (50 mM HEPES pH 7.5; 500 mM NaCl; 1 mM EDTA; 1 mM EGTA; 0.75% Triton-X; 0.1% SDS; 0.05% DOC); and once for 5 minutes 1 ml wash buffer 3 (10 mM Tris, pH 8.0; 1 mM EDTA; 50 mM NaCl). Aspirate the residual wash buffer and gently centrifuge the beads at 1250×g for 1 min; the tubes were again placed on the magnet and all traces of buffer were removed. A volume of 210 µl of elution buffer (50 mM Tris, pH 8; 10 mM EDTA; 1% SDS) was added and eluted at 65°C for 60 minutes with brief vortexing to resuspend the beads every 15 minutes. The beads were separated from the supernatant with a magnet, and 200 μl of the supernatant was removed and placed in a clean tube to change cross-linking. Both IP and whole cell extract fractions were x-linked overnight at 65° C. (minimum 8 hours but maximum 18 hours). Heat was then used to separately cross-link the immunoprecipitation sample and whole cell extract fractions by overnight incubation at 65° C. (minimum 8 hours but maximum 18 hours). Heating facilitates the hydrolysis of formaldehyde cross-links.

[0197] Washing and purification of DNA: Tris-EDTA buffer (50 mM Tris, pH 8; 1 mM EDTA) and 2.7 μl of 30 mg/ml RNase A (to a final concentration of 0.2 mg/ml) in a volume of 200 μl were added to each sample, mixed and incubated at 37 ° C for 2 hours. Then, 5 µl of calcium chloride solution (300 mM CaCl ₂ in 10 mM Tris, pH 8.0) was added to each sample along with 4 µl of 20 mg/ml proteinase K (to a final concentration of 0.2 mg/ml), mixed and incubated at 55°C for 60 minutes. Then 400 µl of 25:24:1 phenol:chloroform:isoamyl alcohol (Sigma Aldrich #P3803) was added to each tube, vortexed at low settings (5/10), and each tube was inverted for additional mixing.

[0198] A PhaseLock Gel™ tube (Qiagen, 3 Prime) was prepared for each sample by centrifuging the tube at room temperature for 30 seconds at 10,000 rpm. Next, a DNA sample in phenol:chloroform:isoamyl alcohol was added to a PhaseLock Gel™ tube and centrifuged at 12,000-16,000 x g for 2 minutes at room temperature. The aqueous solution was then transferred to a new 1.6 ml tube (top fraction), 20 µl 5 M NaCl and 1.5 µl 20 µg/µl glycogen (total 30 µg) were added, then 1 ml EtOH was added and mixed by vortexing or inverting. The sample was then incubated at -20°C overnight (6-16 hours). The mixture was centrifuged at 20,000×g for 20 minutes at 4°C to precipitate the DNA, the supernatant was removed with 1 ml pipette tip, the pellet was washed with 800 μl of 80% EtOH, centrifuged at 20,000×g for 20 minutes at 4°C, and the supernatant was removed with 1 ml pipette tip. The sample was again centrifuged for 1 min at 20,000 x g, the supernatant was removed and the pellet was left to air dry for 5-20 minutes. The sediment should not have a halo of water around it and should be dried to a glossy or flaky appearance. The pellet was then dissolved in 60 µl of water, using 50 µl for sequencing.

[0199] ChIP sequencing data processing: IP and IN ChIP sequencing reads were aligned against the HG19 genome using Bowtie2 v.2.0.5 software (parameters = -p 4 -sensitive). As a result, BAM genome-wide files with generalized alignment were generated for IP and IN sequencing experiments.

[0200] Generating a Generic IRF8 Enhancer Score Dataset: A generic IRF8 enhancer metric dataset was created that can be used in all subsequent analyses. Peaks observed across the genome in MACS v1.4 aligned H3K27Ac read data using BAM IP aligned were defined as dynamic ChIP sequencing data and IN BAM aligned as control background data. A well-defined cut-off p-value of 10 ^-9 was used, but otherwise the default parameters were used. These peaks were then pooled if they contained ≥12500 base pairs between them in the reference human genome. This set of peaks is referred to as ROSE peaks, and the rank for the highest ROSE peak overlapping the IRF8 transcript for a given sample was recorded as the "ROSE rank GOP8" for that sample.

[0201] The set of ROSE peaks was then filtered against "blacklist regions" defined by ENCODE (https://sites.google.com/site/anshulkundaje/projects/blacklists) and ENCODE Project Consortium (2012) to remove CHP sequencing artifacts.

[0202] The filtered ROSE peaks from the primary patient samples were then combined into a universal H3K27Ac enrichment map by combining the coordinates of each peak from that sample with all peaks that overlap it from other samples. This made it possible to create a universal H3K27Ac enrichment map. Then, each enriched region within this universal map in each sample (including cell lines) was quantified by summing, for that region, the number of IP reads mapped within the region and dividing by the number of reads mapped in the entire experiment, multiplied by a million (“reads per million” or RPM (from English “reads per million”)). A similar RPM was calculated for IN reads. The IN RPM was subtracted from the IP RPM to give the total for that area within the universal map for that sample. The scores for this sample were fitted with a negative binomial distribution using the fitdistr function in the R MASS library, V7.3.45. The tail of the distribution was located at the point where the cumulative distribution function of this negative binomial distribution crossed 0.99 (equivalent to a p-value of 0.01). The total scores for all regions of the sample up to this point were divided such that any region of enrichment with a score corresponding to the bottom 99% of the approximate negative binomial distribution had a score less than 1 (considered a "typical enhancer"), and any region with a score above 99% had a score greater than 1 (considered a "typical super enhancer"). These indicators are called "RECOMB" indicators for each sample on the universal card. The RECOMB scores for each sample were then normalized relative to all other samples using quantile normalization with the lowest value set to 0.

[0203] Visualization of ChIP sequencing data: Genome-wide localization of H3K27Ac was visualized using the Integrative Genomics Viewer (IGV), version 2.3.60, after converting the BAM files to t-files of the IGV format using MACS2 to create an overlay (extsize 200) and the igvtools software, v2.3.9, for the TDF command. The y-axis of each track starts just above the noise level (0.25) and ends at about half the level needed to see the full height of the peak in the control region centered on the MALAT1 gene.

*HL60 is an APL cell line.

[0204] The above data demonstrates that the RECOMB score of the IRF8 RECOMB enhancer ≥1.0 (the RECOMB score ≥1.0 defines a super enhancer) correlates with susceptibility to tamibarotene. With the exception of the HL60 AML cell line, this cut-off resulted in 1 false positive (Kasumi-1) and one false negative (EOL-1) of the twelve non-AML AML cell lines tested. Increasing the RECOMB cutoff to ≥1.25 would remove a false positive, while lowering the RECOMB cutoff to ≥0.75 would remove a false negative. These data are also presented in graph form in Figure 6. A similar correlation was also observed between IRF8 mRNA level and susceptibility to tamibarotene, with cell lines having a THM(log ₂ ) value for IRF8 mRNA greater than 4.25 and all showing sensitivity to tamibarotene.

[0205] We then applied ChIP sequencing enhancer profiling to a subset of AML patient samples. The potency of the IRF8 locus enhancer was highly variable among 66 AML patient samples, with 21% (14/66) of patients having SE as indicated by a RECOMB score greater than 1.0 (Figure 7). The majority of patient samples showed minimal enhancer efficacy, including 14% (9/66) of patients with the lowest score who did not have quantifiable IRF8 enhancers.

Example 5: Correlation of IRF8 mRNA and IRF8 enhancer potency

[0206] IRF8 enhancer quantification and correlation of ChIP sequencing and RNA sequencing data: The RECOMB quantile normalized score was used for all patients for the region called enhancer on the universal map that overlapped IRF8: xp16:85862582-85990086. This correlated with the estimation of quantile-normalized TNM expression for the full IRF8 gene model by RSEM using Spearman's correlation. Only patients with both RNA sequencing and ChIP sequencing results were evaluated. The same analysis was performed in cell lines, but OPL cell lines were excluded.

[0207] To obtain a mediated assessment of IRF8 SE by measuring IRF8 mRNA, the correlation between these two parameters in one group of patients with AML was investigated. IRF8 mRNA levels measured by RNA sequencing were compared with measurements for the IRF8 locus enhancer by the RECOMB score for H3K27ac (Figure 8). IRF8 mRNA levels also varied greatly among samples in this group, and IRF8 mRNA levels strongly correlated with IRF8 enhancer potency (Spearman's correlation coefficient ~0.81, p-value 2.2×10 ^-12 ).

[0208] We also profiled the value of IRF8 enhancer potency and IRF8 mRNA levels in 26 AML cell lines. Some of these cell lines have been previously tested for antiproliferative susceptibility to tamibarotene (see Table 2). As observed in AML patient samples, the AML cell lines showed a broad distribution of IRF8 enhancer potency (Figure 9). IRF8 enhancer potency and IRF8 mRNA levels also varied greatly in 26 AML cell lines, 9 (34%) of which had RECOMB values for IRF8 ≥1.0.

[0209] As with AML patient samples, the AML cell lines showed a strong correlation of IRF8 mRNA levels with IRF8 enhancer potency (Figure 10; Spearman's correlation coefficient ~0.82, p-value 2×10 ^-6 ), thus confirming the use of IRF8 mRNA as an indirect measure of IRF8 enhancer potency.

Example 6: Susceptibility of AUC Models to Tamibarotene and Correlation with IRF8 mRNA Levels

[0210] Xenograft models derived from different AML patient samples (AM8096, AM5512, AM7577, and AM7440) using immunocompromised BALB/c nude mice were prepared by Crown Biosciences (Beijing, China) generally as follows.

[0211] Approximately 2×10 ⁶ cells from each patient sample were suspended in 100 μl of PBS and injected into individual mice (n=3 for each of the different patient samples and controls) by iv tail injection. For AM5512, AM7577, and AM7440 xenografts, the tumor burden was considered high enough to start treatment when the concentration of human CD45 ⁺ cells reached ~1-5% in the animal's peripheral blood. Human CD45 ⁺ cells are detected in mouse blood (obtained by ocular blood sampling) using a fluorescent activated cell sorter and anti-human CD45 FITC (Biolegend, Cat. # 304037). For AM8096 xenografts, treatment was started 40 days after cell injection.

[0212] Tamibarotene is administered orally in PBS adjusted to pH 8, 1% DMSO according to a daily schedule with a final dose of 6 mg per kg of body weight in a volume of 10 ml/kg. Mice in the stock solution treatment group are treated according to the same schedule, volume and formulation, but in the absence of tamibarotene. Levels of human CD45 ⁺ cells in the peripheral blood of treated animals and control animals are measured once a week.

[0213] Xenografts AM5512 and AM8096 show a significant reduction in total % CD45 ⁺ cells, as well as % CD45 ⁺ cells in blood, bone marrow and spleen when treated with tamibarotene compared to the control stock solution after 35 days of treatment (figure 11). On the other hand, AM7577 and AM7440 did not show a significant reduction in tumor volume among tamibarotene-treated and stock solution-treated animals, either in total or in blood, bone marrow, or spleen (FIG. 12).

[0214] We then measured both IRF8 and RARA mRNA levels in each of the four patient samples used in the xenograft study (FIG. 13). Two assay non-receptive sample xenografts, AM7577 and AM7440, have IRF8 mRNA levels that fall well below 100 THM in the assay. One of the susceptible samples, AM8096, has IRF8 mRNA levels that exceed 350 THM. Another susceptible sample, AM5512, has a very low level of IRF8 mRNA. Interestingly, these four samples showed a similar RARA mRNA level profile, with AM7577 and AM7440 having RARA mRNA levels below any defined prevalence cut-off {eg, 36% of the prevalence cut-off); AM8096 was well above the prevalence limit and AM5512 was also below the prevalence limit but had a significantly higher level of RARA mRNA than either of the non-responders AM7577 or AM7440.

Example 7: Obtaining and preparing patient samples for determination of IRF8 mRNA levels and ChIP sequencing

[0215] Blood (8 ml) was taken from patients with non-APL AML and collected in an 8 ml BD Vacutainer CPT tube with sodium citrate. After blood collection, the tube was gently inverted by hand 8-10 times to ensure proper mixing of the anticoagulant. The tube was kept upright at room temperature before centrifugation, which was carried out within two hours of collection. Then the blood sample was centrifuged at room temperature (18-25°C) for 20 minutes at 1500-1800 RCF (relative centrifugal force). After centrifugation, the blood was separated into layers. The lower layers under the gel plug were a red layer at the very bottom (red blood cells) and a thin gray layer above it (granulocytes and dense solution). Immediately above the gel plug was a clear layer of solid solution, then a white layer (mononuclear cells and platelets) and a yellowish layer (plasma) on top. The white layer (up to 1 ml by volume) containing PBMC was removed immediately after centrifugation using a Pasteur pipette. If necessary, PBMC can be stored in a cryotube containing 20% v/v. BloodStor® Freezing Medium (BioLife Solutions) added drop by drop followed by gentle hand-turning to mix.

[0216] Then, the PBMC fraction obtained in the previous step (thawed in the case of pre-freezing) was simultaneously treated with human CD117 microbeads (Miltenyi Biotec) and human CD34 microbeads (Miltenyi Biotec), following the manufacturer's instructions for magnetic labeling and magnetic separation of labeled cells. Then, messenger RNA was extracted from the isolated CD34 ⁺ /CD117 ⁺ cells and quantified by qPCR as described above.

Example 8: Synergistic effect between tamibarotene and other agents correlates with RARA mRNA levels.

[0217] Using Biotek EL406, 50 μl of cell medium containing 20-60,000 cells/ml was dispensed into 384-well Nunc plates (Thermo). The cells in suspension were then immediately supplemented with the compound, while the adherent cell lines were allowed one hour to reattach to the surface of the plate before adding the compound. The tamibarotene and second agents to be tested were dissolved in DMSO and dispensed into 384-well plates containing the compound (Greiner). Tamibarotene and one second agent were added to each compound tablet, each at 5 different doses centered on approximately the EC ₅₀ of the compound for a given cell line, giving a total of 25 different dose combinations of the two agents.

[0218] Compound arrays were dispensed into assay plates using a 20 nl 384-well multichannel pipetting head on a Janus MDT workstation (Perkin Elmer). Each plate contained 8 replicates of all 5 per 5 compound concentrations in addition to five doses of each compound alone in four replicates. After adding the compounds, the plates were incubated for 5 days in an incubator at 37°C. Cell viability was assessed using ATPlite (Perkin Elmer) following the manufacturer's protocols. Data was analyzed using commercially available CalcuSyn software and visualized using GraphPad Prism software. Isobolograms were obtained by plotting for each of the 25 dose combinations of tamibarotene and second agents and analyzed for synergistic effect. On the isobolograms, a straight line connecting the abscissa and ordinate values of 1.0 represents growth inhibition added to the combination of the two compounds. Graphs that are below the straight line represent synergistic growth inhibition, while graphs that are below this line and the line connecting the abscissa and ordinate values of 0.75 represent a weak synergistic effect. The graphs that lie between the line connecting the abscissa and ordinate values of 0.75 and the line connecting abscissa and ordinate values of 0.25 represent a moderate synergistic effect. The graphs that are below the line connecting the abscissa and ordinate values of 0.25 represent a strong synergistic effect. Data outside the maximum on each isobologram is indicated by a number of asterisks in the upper right corner of the isobologram and represents data with no synergistic effect.

[0219] We investigated azacitidine, arsenic trioxide, midostaurin, cytarabine, daunorubicin, methotrexate, idarubicin, sorafenib, decitabine, quisartinib, ABT199 (BCL2 inhibitor), JQ1 (BRD4 inhibitor), ATO, prednisone, SAHA, GSKJ4 (JMID3/JARID1B inhibitor), and EPZ 6438 (an EZH2 inhibitor) as second agents in this assay against various AML cell lines. Figures 15-21 depict isobolograms of various second agents in combination with tamibarotene in different cell lines.

[0220] For combinations of azacitidine and tamibarotene, a moderate to strong synergistic effect was observed for Sig-M5; a moderate synergistic effect was observed for KG-1a and NOMO-1; and a weak to moderate synergistic effect was observed for MV-4-11 (see figure 15). No synergistic effect was observed for Kasumi-1 or OCI-M1 (data not shown).

[0221] For combinations of arsenic trioxide and tamibarotene, a strong synergistic effect was observed for Sig-M5 and MV411; and a moderate synergistic effect was observed for NOMO-1 (see figure 16). No or little synergistic effect was observed for Kasumi-1, and no synergistic effect was observed for OCI-M1 (data not shown).

[0222] For combinations of cytarabine (ara-C) and tamibarotene, some moderate synergistic effect was observed for KG-1a and OCI-M1, but no synergistic effect was observed for HL-60 (see figure 17). For MV-411, the large number of data beyond the maximum (7 out of 25) and the large number that showed a strong synergistic effect make interpretation difficult.

[0223] For combinations of daunorubicin and tamibarotene, a strong synergistic effect was observed for Kasumi-1 and NOMO-1; and a moderate synergistic effect was observed for Sig-M5 and MV-4-11 (see figure 18). No synergistic effect was observed for OCI-M1 (data not shown).

[0224] For combinations of methotrexate and tamibarotene, a moderate synergistic effect was observed for NOMO-1, Sig-M5 and MV-4-11 (see figure 19). No or little synergistic effect was observed for Kasumi-1, and no synergistic effect was observed for OCI-M1 (data not shown).

[0225] For combinations of idarubicin and tamibarotene, a moderate synergistic effect was observed for NOMO-1, Sig-M5 and MV411 (see Figure 20). No synergistic effect was observed for Kasumi-1 or OCI-M1 (data not shown).

[0226] Inconclusive results were observed for the combination of sorafenib and tamibarotene in MV411, NOMO-1, KG-1a, and Sig-M5 due to the large amount of data beyond the maximum (see Figure 21), but no synergistic effect was observed for this combination in OCI-M1 (data not shown). Sorafenib is an inhibitor of FLT3 and we have also observed a synergistic effect for combinations of tamibarotene and other FLT3 inhibitors such as midostaurin and quisartinib in some of the cell lines. Without being limited by theory, we believe that high levels of RARA and/or IRF8 mRNA, as well as high levels of FLT3 mRNA, are required for a synergistic effect with FLT3 inhibitors. This "condition dependent" synergistic effect was also observed with the GCR inhibitor prednisone, which seems to require high levels of GCR mRNA as well as high levels of RARA and/or IRF8 mRNA. A similar effect was also observed with the JMJD3/JARID1B inhibitor GSKJ4, which seems to require high levels of JMJD3/JARID1B mRNA as well as high levels of RARA and/or IRF8 mRNA to exhibit a synergistic effect.

[0227] We did not observe a synergistic effect in any of the cell lines investigated for the combination of the BCL2 inhibitor ABT199 and tamibarotene. We also did not observe a synergistic effect with the combination of the EZH2 inhibitor EPZ6438 and tamibarotene.

[0228] However, we observed a synergistic effect for the combination of HDAC inhibitor SAHA and tamibarotene in AML cell lines with high levels of RARA and/or IRF8 mRNA.

[0229] In addition, a strong synergistic effect was observed for the combination of decitabine and tamibarotene in HL-60 and KG-1a cells (data not shown).

[0230] We also observed a synergistic effect for the combination of the Zn-finger transcription factor inhibitor ATO and tamibarotene.

[0231] While not wishing to be bound by any particular theory, non-ATL AML characterized by high levels of RARA, high levels of IRF8, or a combination thereof is likely to be synergistically responsive to combinations of tamibarotene and one or more of azacitidine, arsenic trioxide, midostaurin (in AML characterized by high levels of FLT3 mRNA), cytarabine, dauno rubicin, methotrexate, idarubicin, sorafenib (in AML characterized by high levels of FLT3 mRNA), decitabine, quisartinib (in AML characterized by high levels of FLT3 mRNA), JQ1 (BRD4 inhibitor), ATO, prednisone (in AML characterized by high levels of GCR mRNA), SAHA and GSKJ4 (in AML characterized by high levels of mRNA JMJD3/JARID1B).

LINKS

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3. Tamura, T., Kurotaki, D. & Koizumi, S. Regulation of myelopoiesis by the transcription factor IRF8. Int. J. Hematol. 101, 342-351 (2015).

4. Yang, J. et al. Cutting Edge: IRF8 Regulates Bax Transcription In Vivo in Primary Myeloid Cells. J Immunol. 187, 4426-4430 (2011).

5. Pogosova-Agadjanyan, E. L. et al. The Prognostic Significance of IRF8 Transcripts in Adult Patients with Acute Myeloid Leukemia. PLoS ONE 8, e70812 (2013).

6. Sharma, A. et al. Constitutive IRF8 expression inhibits AML by activation of repressed immune response signaling. Leukemia 29, 157-168 (2015).

7. Smits, E.L.J.M., Anguille, S. & Berneman, Z.N. Interferon a may be back on track to treat acute myeloid leukemia. Oncolmmunology 2, e23619 (2013).

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10. SY-1425-P003: Effects of tamibarotene (SY-1425) on proliferation of Acute Myeloid Leukemia (AML) cell lines in comparison to all-trans retinoic acid (ATRA).

11. SY-1425-P006: Characterization of the RARA enhancer and RARa mRNA in AML patient samples and cell lines.

Claims (37)

1. A method for the treatment of non-APL (acute promyelocytic leukemia) acute myelocytic leukemia (non-APL AML), including the step: administering a combination of tamibarotene and a second therapeutic agent to a subject having non-ATL AML, wherein the cancer cells contained in the biological sample from said subject contain an IRF8 biomarker and/or a RARA biomarker, wherein: the IRF8 biomarker is or includes an IRF8 RNA transcript level equal to or greater than a predetermined threshold, or a super enhancer associated with the IRF8 gene; the RARA biomarker is or includes a RARA RNA transcript level equal to or greater than a predetermined threshold, or a super enhancer associated with the RARA gene; And said second therapeutic agent is: azacitidine, midostaurin, cytarabine, daunorubicin, methotrexate, idarubicin, sorafenib, decitabine, quisartinib, ABT199, JQ1, prednisone, SAHA, GSKJ4, or EPZ6438, wherein: if said second therapeutic agent is midostaurin, sorafenib, or quisartinib, such midostaurin, sorafenib, or quisartinib is administered only if it is further determined that there is an elevated level of FLT3 mRNA in a biological sample containing cancer cells from said subject; if the second therapeutic agent is prednisone, then such prednisone is administered only if it is additionally determined that there is an increased level of GCR mRNA in a biological sample containing cancer cells from said subject; And if the second therapeutic agent is GSKJ4, then such GSKJ4 is administered only if it is additionally determined that there is an increased level of JMJD3 and/or JARID1B mRNA in a biological sample containing cancer cells from said subject. 2. The method of claim 1 wherein said second therapeutic agent is azacitidine. 3. The method of claim 1, wherein said second therapeutic agent is midostaurin. 4. The method according to p. 1, characterized in that the second therapeutic agent is cytarabine. 5. The method of claim 1 wherein said second therapeutic agent is daunorubicin. 6. The method of claim 1, wherein said second therapeutic agent is methotrexate. 7. The method of claim 1 wherein said second therapeutic agent is idarubicin. 8. The method of claim 1 wherein said second therapeutic agent is sorafenib. 9. The method of claim 1 wherein said second therapeutic agent is decitabine. 10. The method of claim 1 wherein said second therapeutic agent is quisartinib. 11. The method of claim 1 wherein said second therapeutic agent is ABT199. 12. The method of claim 1 wherein said second therapeutic agent is JQ1. 13. The method of claim 1 wherein said second therapeutic agent is prednisone. 14. The method of claim 1 wherein said second therapeutic agent is SAHA. 15. The method of claim 1 wherein said second therapeutic agent is GSKJ4. 16. The method of claim 1 wherein said second therapeutic agent is EPZ6438. 17. The method of claim 1, wherein said tamibarotene and the second therapeutic agent are administered simultaneously. 18. The method of claim 1, wherein said tamibarotene and the second therapeutic agent are administered sequentially. 19. The method according to claim 1, characterized in that an increased level of RNA transcript RARA and/or an increased level of RNA transcript IRF8 are independently determined using fluorescence hybridization, PCR, qPCR, qRT-PCR, RNA sequencing, RNA hybridization and signal amplification or Northern blotting. 20. The method of claim 1, wherein said biological sample obtained from said subject is a bone marrow aspirate or whole blood. 21. The method of claim. 20, characterized in that said bone marrow aspirate or whole blood is processed to remove one or more components from them. 22. The method of claim. 21, characterized in that said whole blood is processed to obtain a sample of peripheral blood mononuclear cells (PBMC) or a sample enriched in PBMC. 23. The method of claim 1, wherein said biological sample obtained from said subject is determined to have the IRF8 biomarker. 24. The method according to claim 23, characterized in that said IRF8 RNA transcript is transcribed from a genomic DNA sequence that encodes a protein that binds the interferon consensus sequence or its splice variant and specifically excludes gene fusions that include all or part of the IRF8 gene, while said IRF8 gene is located on chromosome 16: 85862582-85990086 in the hg19 genomic block. 25. The method of claim 1, wherein said biological sample obtained from said subject is determined to have an RARA biomarker. 26. The method according to claim 25, characterized in that said RARA RNA transcript is transcribed from a genomic DNA sequence that encodes a functional retinoic acid receptor-α gene and specifically excludes gene fusions that include all or part of the RARA gene, while said RARA gene is located on chromosome 17:38458152-38516681. 27. The method according to claim 1, wherein said IRF8 RNA transcript is IRF8 mRNA. 28. The method of claim 1, wherein said RARA RNA transcript is RARA mRNA. 29. The method according to any one of paragraphs. 1-28, characterized in that said subject has non-ATL AML. 30. The method of claim 1, wherein said method further comprises first detecting an IRF8 biomarker and/or an RARA biomarker in cancer cells of a biological sample from said subject.

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