Methods and compositions for the treatment of lymphoma

Abstract

The present disclosure provides, inter alia, methods and compositions for the treatment of a cancer such as, e.g., a lymphoma, in a subject in need thereof. Also provided are methods for treating or ameliorating the effects of hypoxia in the tumor microenvironment (TME) in a subject having a cancer.

Description

Docket No. 1035795.000884METHODS AND COMPOSITIONS FOR THE TREATMENT OF LYMPHOMACROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent Application Serial No. 63 / 715,112, filed on November 1 , 2024, the entire content of which is hereby incorporated by reference.FIELD OF THE DISCLOSURE

[0002] The present disclosure provides, inter alia, methods and compositions for the treatment of a cancer such as, e.g., a lymphoma, in a subject in need thereof. Also provided are methods for treating or ameliorating the effects of hypoxia in the tumor microenvironment (TME) in a subject having a cancer.GOVERNMENT FUNDING

[0003] This invention was made with government support under grant nos. CA203703, and CA222931 , awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE DISCLOSURE

[0004] Primary mediastinal B-cell lymphoma (PMBCL) exists on a clinicopathologic spectrum with Hodgkin lymphoma (HL), gray zone lymphoma (GZL), and diffuse large B-cell lymphoma (DLBCL), with an aggressive clinical course, predominantly affecting young women. Intensive infusional immunochemotherapy can offer substantial survival (OS) benefit in 97%1of cases; however, this comes at the cost of long-term toxicities including secondary malignancy, infertility, end organ damage, and poorer quality of life.2

[0005] There has been a lack of substantial improvement in frontline treatment and outcomes in the relapsed / refractory (R / R) remain poor with an OS of 15%.3PMBCL is frequently defined by amplification of chromosome 9p24.1 (9p) in up to 75% of patients,4leading to upregulation of PDL1 / 2 and JAK2.3 JAK2, in addition to its known role in the JAK / STAT pathway, has also been shown to act as an epigenetic modifier by phosphorylation of histone 3, tyrosine 41 (H3Y41 ), leading to euchromatin stateand expression of proliferative factors such as MYC.5 6When epigenetic modifications are coupled with upregulation of PDL1 / 2, the 9p amplicon leads to immune escape, unchecked cell proliferation, and tumorigenesis. Chromosome 9p amplification is also found in other lymphomas on a similar biologic spectrum as PMBCL.5 7Thus far, targeting 9p amplification depends largely on immunotherapy alone, and response rates correlate with the degree of 9p expression, with HL demonstrating the best overall response rate (ORR).8PDL-1 blockade is approved for R / R PMBCL; however, with an ORR of 41.5%, these effects are modest and inferior to what is observed in HL with ORR 71 .9%.9The addition of immunotherapy to chemotherapy is now being studied in the frontline setting in both PMBCL and HL (NCT03712202 and NCT03907488, respectively), the latter of which has recently demonstrated compelling evidence to introduce immunotherapy in frontline HL.10Given the modest effect of immunotherapy in PMBCL, it is critical to develop a deeper understanding of the other biologic drivers downstream of 9p amplification to build on and improve specific targeted therapy. Furthermore, amplification of chromosome 9 is frequently accompanied by rearrangements in chromosome 16p13.13, leading to downregulation of CIITA and MHCII; when coupled with upregulated PDL1 / 2, this reinforces immune evasion.11SUMMARY OF THE DISCLOSURE

[0006] PMBCL is a rare lymphoma that primarily effects a young population of women between median ages 32-39. And with a unique pathophysiology that has yet to be exploited. Though frontline treatment has evolved to improve outcomes, it continues to rely heavily on a chemotherapy backbone and comes at the cost of infertility, increased toxicity, risk of secondary malignancy, and cumbersome intensive intravenous infusions. The burden of treatment at a critical time point in life comes at a cost of career toxicity, family detriment, and psychological distress. Furthermore, outcomes in the R / R population are dismal with an OS of 15% at two years. Tailoring therapy in this young patient population to de-escalate frontline therapy or to improve OS in the R / R setting remains an urgent unmet clinical need. PMBCL exists within a clinicopathologic spectrum with HL, GZL, and DLBCL which all share a unique biology of 9p amplification. GZL is an especially challenging entity to treat and there is scant data for targeted therapy, most of which are case reports, highlighting the need for9p+ targeted therapy. The role of JAK2 as an epigenetic modifier by phosphorylation of H3Y41 has been appreciated in the last decade, but the biologic implications of this unique post-translational modification with respect to change in chromatin accessibility and gene expression have been poorly elucidated. Bridging these gaps in knowledge between JAK2 as an epigenetic modifier would introduce impactful information in the setting of a widely utilized drug such as ruxolitinib. To clearly delineate the epigenetic impact of ruxolitinib, the present dislcosure leveraged several advanced technologies including, e.g., top-down mass spectrometry (MS) to characterize the post- translational effects at the histone level with remarkable molecular detail which has never been performed using ruxolitinib, ATACseq and RNAseq to analyze changes within chromatin and gene expression. Organoids can also be used in PMBCL, which allows 3D recreation of lymphoma architecture and physiology to determine the effects of ruxolitinib more accurately. Humanized mouse models can also be used to more accurately display effects of combined immunotherapy with epigenetic modifier, bringing this novel concept closer to translation to patients. Defining 9p amplified lymphomagenesis at the epigenetic level is uncharted territory that is waiting to be explored, allowing for targeted therapy of complementary pathways driving disease. Data generated from this disclosure has the potential to define new therapies in the R / R setting or to help de-escalate chemotherapy in the frontline setting, which can be widely applicable to all 9p amplified lymphomas. The impact on this young patient population would be significant, potentially alleviating burdensome chemotherapy and changing the trajectory of these patients’ lives.

[0007] The present disclosure provides a method of treating or ameliorating the effect of a cancer in a subject. This method comprises administering to the subject an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor.

[0008] The present disclosure also provides a composition for treating or ameliorating the effect of a cancer in a subject, comprising an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor.

[0009] The present disclosure also provides a kit for treating or ameliorating the effect of a cancer in a subject, comprising an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor, packaged with instructions of use.

[0010] The present disclosure also provides a method for treating or ameliorating the effects of hypoxia in the tumor microenvironment (TME) in a subject having a cancer. This method comprises administering to the subject an effective amount of a Jumonji histone demethylase inhibitor.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] FIG. 1 shows that expression of PDL1 / 2 ligands is significantly reduced in 9p amplified cell lines. Cells treated at concentration of [500k] per flask using increasing doses of ruxolintib: 0, 100, 400, 1000, 2000, 5000nM. The L540 cell line required higher doses of ruxolitinib: 0, 1000, 2000, 5000, 8000, 1000nM. Data shown represents treatment for 72 hours.

[0013] FIG. 2 shows the RNA-sequencing performed on Karpas1106p (9p+) cells treated with ruxolintib 1000 nM for 48 hours. Vocano plot depicting differentially expressed genes (adjusted p value < 0.05), with down-regulated genes designated as having Log? fold change < -1 , and up-regulated genes with a Log? fold change > 1 .

[0014] FIG. 3 shows ICso (nM) of belinostat and YF2 for the cell lines HH (JAKWT), H9 (JAKGOF) and H9-belino-R (belinostat-resistant H9). Belinostat-treated cells were exposed for 3 days, and YF2-treated cells were exposed for 6 days. ICso was then measured using CellTiter-Glo Luminescent Cell Viabiliyt Assay. (n=2)

[0015] FIG. 4 shows the Western blot images of active / phosphorylated STAT3 (pSTAT3), inactive unphosphorylated STAT3, and Bcl-XL compared to the loading control GAPDH in the cell lines HH (JAK™-), H9 (JAKGOF) and H9-belino-R (belinosat- resistant H9) after 4-day exposure otYF2 (15 pM), ruxolitinib (10 pM) and combination.

[0016] FIG. 5 shows the expressions of selected markers of lymphomagenesis in SUCHL6 (9p+) cell line with JIB-04. Cells treated at concentration of [500k] per flask using increasing doses of JIB-04: 0, 100, 250, 500, 1000, 2000nM. Data shown represents treatment for 144 hours. Two series of cells were treated and compared.

[0017] FIG. 6 shows the expressions of selected markers of lymphomagenesis in Karpas1106p (9p+) cell line with JIB-04. Cells treated at concentration of [500k] perflask using increasing doses of JIB-04: 0, 100, 400, 1000, 2000, 4000nM. Data shown represents treatment for 72 hours. Two series of cells were treated and compared.

[0018] FIG. 7 shows the expressions of selected markers of lymphomagenesis in U2940 (9p-) cell line with JIB-04. Cells treated at concentration of [500k] per flask using increasing doses of JIB-04: 0, 100, 250, 500, 750, 1000, 2000nM. Data shown represents treatment for 144 hours. Two series of cells were treated and compared.

[0019] FIG. 8 shows the results of flow cytometry analysis for B cell line (SUDHL4) after four days treatment. Control: received 10 pg / mL PE M BRO; YF2: treated with 10 pM YF2 + 10 pg / mL PEMBRO; RUXO: treated with 2 pM RUXO + 10 pg / mL PEMBRO; COMBO: treated with 10 pM YF2 + 2 pM RUXO + 10 pg / mL PEMBRO.

[0020] FIG. 9A shows % dead, apoptotic and live per treatment group in SUDHL4 cell line. FIG. 9B shows the sum of apoptotic and dead cells (%) per treatment group in SUDHL4 cell line.

[0021] FIG. 10A shows % dead, apoptotic and live per treatment group in SUDHL6 cell line. FIG. 10B shows the sum of apoptotic and dead cells (%) per treatment group in SUDHL6 cell line.DETAILED DESCRIPTION OF THE DISCLOSURE

[0022] The present disclosure investigates the unique interplay of immune escape and epigenetic derangements that can be targeted by epigenetic modifiers, and which may restore MHC expression, thereby enhancing the effects of combination immunotherapies, such as pembrolizumab. This strategy could offer the opportunity to de-escalate intensive frontline chemotherapy, while maintaining a robust OS benefit, or to offer more treatment options in the R / R setting where outcomes are dismal. Ruxolitinib, a JAK1 / 2 inhibitor and histone 3 modifier6, has demonstrated efficacy in PMBCL lines11and xenograft mouse models but has failed to show single agent activity in clinical trials,12suggesting combination therapy may yield more promising results.

[0023] Therefore, if deranged histone modification and amplified expression of PD- 1 ligands cooperate to drive cell growth in 9p amplified lymphomas, then dual targeting with epigenetic modifiers and immune checkpoint blockade will lead to altered gene expression and enhanced immunogenicity inducing cell death.

[0024] The present disclosure also provides data in employing epigenetic primingto enhance effects of immunotherapy in immune-competent mouse models of lymphoma yielding synergistic responses. In one example disclosed herein, combining epigenetic modifier (YF2) with anti-PD-1 antibody in a syngeneic DLBCL mouse model significantly prolonged survival. In addition, investigation of the tumor microenvironment (TME) with scRNAseq using the Visium platform demonstrated distinct gene expression clustering and increased infiltration of activated T-cells. This preliminary data provides the proof of-concept that epigenetic priming of immune checkpoint blockade can be studied in a unique context in this proposal.

[0025] Accordingly, one aspect of the present disclosure relates to a method of treating or ameliorating the effect of a cancer in a subject. This method comprises administering to the subject an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor.

[0026] In some embodiments, the cancer is a solid tumor. As used herein, “a solid tumor” or “a solid cancer” refers to abnormal growths of cells that form a mass in a specific tissue or organ. They are distinct from liquid tumors, such as leukemias and lymphomas, which circulate in the bloodstream. In some embodiments, the solid tumor is selected from the group consisting of colorectal cancer, genitourinary tract cancer, sarcoma, melanoma, hepatocellular carcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, prostate cancer, pancreatic ductal adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC) , breast cancer, ovarian cancer, colon cancer, renal cancer, hepatic cancer, head-neck cancer, and gallbladder cancer. In certain embodiments, the solid tumor is breast cancer or lung cancer. In some embodiments, the solid tumor is advanced solid tumor. In some embodiments, the solid tumor is metastatic solid tumor. In some embodiments, the solid tumor is advanced and metastatic solid tumor. The term “advanced solid tumor” as used herein refers to a solid tumor that cannot be cured or grows beyond the initial site of origin, either locally advanced or metastatic. In some embodiments, the advanced solid tumor includes but is not limited to solid tumors in stage I II or stage IV. The term “metastatic” or “metastasis” as used herein refers to a tumor spread from an initial or primary site to a different or secondary site within the subject’s body. It is generally distinguished from cancer invasion, which is the direct extension and penetration by cancer cells into neighboring tissues.

[0027] In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is selected from the group consisting of Primary mediastinal B-celllymphoma (PMBCL), Cutaneous T-cell lymphoma (CTCL), Hodgkin lymphoma (HL), B-cell non-Hodgkin lymphoma, gray zone lymphoma (GZL), and diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is Primary mediastinal B-cell lymphoma (PMBCL).

[0028] As used herein, “an epigenetic agent” or “an epigenetic-targeted agent” refers to an agent that causes epigenetic modification that can alter the gene expression without altering the DNA sequence. Aberrant epigenetic regulations in cancer include DNA methylation, histone methylation, histone acetylation, non-coding RNA, and mRNA methylation. In some embodiments of the present disclosure, the epigenetic agent is selected from the group consisting of a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC), inhibitor, a bromodomain and extraterminal motif protein (BET) inhibitor, a methyltransferase like-3 (METTL3) inhibitor, a lysine-specific histone demethylase 1 (LSD1 ) inhibitor, an enhancer of zeste homolog 2 (EZH2) inhibitor, a DOT1-like histone lysine methyltransferase (DOT1 L) inhibitor, an isocitrate dehydrogenase (IDH) inhibitor, a Jumonji histone demethylase inhibitor, a histone acetyltransferase (HAT) activator, and combinations thereof.

[0029] In some embodiments, the epigenetic agent is an HAT activator. In some embodiments, the epigenetic agent is YF2.

[0030] In some embodiments, the epigenetic agent is a Jumonji histone demethylase inhibitor. For example, in one embodiment, the Jumonji histone demethylase inhibitor is JIB-04.

[0031] In some embodiments the epigenetic agent is an EZH2 inhibitor. For example, in one embodiment, the EZH2 inhibitor is tazemetostat.

[0032] As used herein, “an immunotherapeutic agent” is a compound that stimulate or enhance the body's immune system to fight diseases such as cancers. Non-limiting examples of immunotherapeutic agents include checkpoint inhibitors (e.g., pembrolizumab, nivolumab, ipilimumab, etc.); cytokines (e.g., interleukin-2 (IL-2), interferon-alpha (IFN-a), etc.); monoclonal antibodies (e.g., rituximab, cetuximab, etc.); cancer vaccines (e.g., sipuleucel-T, BCG vaccine, etc.); CAR-T cells; immune system modulators (e.g., lenalidomide, thalidomide, etc.) In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, and combinations thereof. For example, in some embodiments, the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab,nivolumab, atezolizumab, and combinations thereof. In one embodiment, the immunotherapeutic agent is pembrolizumab.

[0033] In some embodiments, the JAK1 / 2 inhibitor is selected from the group consisting of AG-490, abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclactinib, peficitinib, pacritinib, ruxolitinib, tofacifinib, upadacitinib, and combinations thereof. For example, in one embodiment, the JAK1 / 2 inhibitor is ruxolitinib.

[0034] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure include, for example, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.

[0035] In some aspects of this and other embodiments, the subject is a mammal. Preferably, the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals. More preferably, the mammal is a human. In some embodiments, the subject is a human.

[0036] In some embodiments, the subject has amplification of chromosome 9p24.1 (9p). As used herein, “amplification of chromosome 9p24.1 (9p)” refers to a genetic abnormality that results in an increase in the number of copies of this segment. This amplification frequently includes the JAK2 gene and the PD-L1 and PD-L2 genes, leading to the overexpression of these proteins. This overproduction can lead to immune evasion by tumors.

[0037] In some embodiments, the epigenetic agent, immunotherapeutic agent, and JAK1 / 2 inhibitor are administered to the subject serially or concurrently.

[0038] In some embodiments, the epigenetic agent is administered to the subject prior to the administration of the immunotherapeutic agent and JAK1 / 2 inhibitor.

[0039] In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of

[0040] In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is selected from the group consisting of Primary mediastinal B-cell lymphoma (PMBCL), Cutaneous T-cell lymphoma (CTCL), Hodgkin lymphoma (HL), B-cell non-Hodgkin lymphoma, gray zone lymphoma (GZL), and diffuse large B-cell lymphoma (DLBCL).

[0041] As used herein, the term “tumor microenvironment (TME)” refers to thecomplex and dynamic ecosystem surrounding a tumor. It consists of various cellular (tumor cells, immune cells (e.g., macrophages, lymphocytes), fibroblasts, endothelial cells, pericytes, etc.) and non-cellular (extracellular matrix (ECM), growth factors, cytokines, chemokines, metabolites, etc.) components that interact with tumor cells. TME promots tumor growth, facilitates invasion and metastasis, suppresses immune responses, and alters drug resistance.

[0042] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population may fail to respond or respond inadequately to treatment.

[0043] As used herein, the terms “ameliorate”, "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject, preferably a human.

[0044] As used herein, "administration," "administering" and variants thereof means introducing a composition, such as a synthetic membrane-receiver complex, or agent into a subject and includes concurrent and sequential introduction of a composition or agent. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. Administration includes self-administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject. Administration can be carried out by any suitable route.

[0045] Another aspect of the present disclosure relates to a composition for treating or ameliorating the effect of a cancer in a subject, comprising an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor.

[0046] In some embodiments, the epigenetic agent is YF2, the immunotherapeuticagent is pembrolizumab, and the JAK1 / 2 inhibitor is ruxolitinib.

[0047] Another aspect of the present disclosure relates to a kit for treating or ameliorating the effect of a cancer in a subject, comprising an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor, packaged with instructions of use.

[0048] A further aspect of the present disclosure relates to a method for treating or ameliorating the effects of hypoxia in the tumor microenvironment (TME) in a subject having a cancer. This method comprises administering to the subject an effective amount of a Jumonji histone demethylase inhibitor.

[0049] As used herein, the term “hypoxia” or “tumor hypoxia” is a condition where cancer cells experience a lack of oxygen (O2) supply. It can arise due to rapid tumor growth, abnormal blood vessels (disorganized and / or leaky), increased oxygen consumption, or compression of blood vessels. Tumor hypoxia can lead to increased cell survival and resistance to therapy, enhanced tumor angiogenesis, promotion of tumor metastasis, altered metabolism, immune suppression, etc.

[0050] In some embodiments, the Jumonji histone demethylase inhibitor downregulates the activity of histone demethylase JMJD2D and / or JMJD2C. For example, in one embodiment, the Jumonji histone demethylase inhibitor is JIB-04.

[0051] In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of colorectal cancer, genitourinary tract cancer, sarcoma, melanoma, hepatocellular carcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, prostate cancer, pancreatic ductal adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC) , breast cancer, ovarian cancer, colon cancer, renal cancer, hepatic cancer, head-neck cancer, and gallbladder cancer. In certain embodiments, the solid tumor is breast cancer or lung cancer.

[0052] In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is selected from the group consisting of Primary mediastinal B-cell lymphoma (PMBCL), Cutaneous T-cell lymphoma (CTCL), Hodgkin lymphoma (HL), B-cell non-Hodgkin lymphoma, gray zone lymphoma (GZL), and diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is Primary mediastinal B-cell lymphoma (PMBCL).Additional Definitions

[0053] As used herein, the term "increase," "enhance," "stimulate," and / or "induce"(and like terms) generally refers to the act of improving or increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

[0054] As used herein, the term "inhibit," "suppress," "decrease," "interfere," and / or "reduce" (and like terms) generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

[0055] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0056] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0057] The following examples are provided to further illustrate certain aspects of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.EXAMPLESExample 1JAK1 / 2 Inhibition Modulates Key Markers of Lymphomagenesis in 9p24.1 Amplified Lymphoma, Priming for Enhanced Antigenicity

[0058] Primary Mediastinal B-cell Lymphoma (PMBCL) and Hodgkin Lymphoma (HL) are frequently dened by chromosome 9p24.1 amplification, leading to upregulation of PDL-1 / -2 and JAK2. JAK2, in addition to its known role in the JAK / STAT pathway, has also been shown to act as an epigenetic modifier by phosphorylation of H3Y41 , enabling an euchromatin state and expression of MYC. When these epigenetic modifications are coupled with upregulation of PDL-1 / -2, the 9p amplicon leads to immune escape, unchecked cell proliferation, and tumorigenesis. We hypothesize that if deranged histone modification and amplified expression of PD- 1 ligands cooperate to drive cell growth in 9p amplified PMBCL and HL, then dualtargeting with JAK inhibitor and immune checkpoint blockade would lead to altered gene expression and enhanced immunogenicity inducing synergistic cell death. The following abstract demonstrates preliminary data using ruxolintib monotherapy in cell lines, with goal of combining immunotherapy in mouse models.

[0059] In order to understand how ruxolitinib modulates key drivers of lymphomagenesis in 9p24.1 amplified lymphoma, human lymphoma cell lines with 9p amplification, (n=2): Karpas1106p (PMBCL) and L540 (HL) were compared to cell lines lacking 9p24.1 amplification, (n=2): Farage (PMBCL) and U2940 (PMBCL). All samples were treated for 3- and 6-day intervals. The ICso concentrations were established for ruxolintib using Cell TiterGlo assay: Karpasl 106p (5.72 pM), L540 (22 pM), Farage (29.46 pM), and U2940 (32.99 pM). All cells were treated for 48 hours with control or ruxolitnib and probed by Western blot, flow cytometry, and RNA- sequencing. The expression of key determinants of survival including JAK / STAT, pro- and anti-apoptotic proteins (SOCS1 and MYC, respectively) and MHCII were measured by Western blot. PD-1 / -2 ligands were measured by flow cytometry. Transcriptom ic changes were evaluated by RNA-seq and data was presented as Log2 fold change compared to vehicle controls.

[0060] Pharmacodynamic effects were most prominent in the 9p amplified Karpasl 106p cell line, demonstrating increased markers of immunogenicity by protein quantification: increased CIITA by 3.5-fold (SEM ± 1.15) and increased MHCII by 2.67- fold (SEM ±0.33). Expression of tumor suppressor SOCS1 was increased by 2.67-fold (SEM ± 0.68) and expression of oncogene MYC was decreased by 6.06-fold (SEM ± 4.66). The 9p amplified L540 cell line similarly demonstrated increased CIITA by 3.22- fold (SEM ± 1.59) and decreased MYC by 2.69-fold (SEM ± 0.65), however, no signicant change in MHCII or SOCS1 was observed. Signicant reduction of STAT3p and / or STAT6p was demonstrated across all cell lines (p < 0.01 ). Cell lines without 9p amplification, Farage and U2940, did not demonstrate consistent increase nor decrease in markers of immunogenicity or oncogenes, suggesting ruxolitinib preferentially modulates downstream markers of survival and immune recognition driven by the 9p amplicon.

[0061] Flow cytometry demonstrated significantly reduced expression of PDL-1 (p = 0.01 ) and PDL-2 (p = 0.001 ) in 9p amplified cell lines (Karpasl 106p and L540), and non-amplified cell lines (Farage and U2940), as shown in FIG. 1. Down-regulation of PDL-1 / -2 was more prominent in the 9p amplified cell lines compared to those without9p amplification (p = 0.055 and p = 0.18, respectively), suggesting that ruxolitinib may prime for increased immunogenicity by reducing PDL-1 / -2, especially in 9p amplified cell lines.

[0062] RNA-seq of the 9p amplied Karpasl 106p cell line demonstrated increased MHCII: HLA-DQ (1.12, p < 0.001 ), HLA-DO (0.70, p <0.001 ), HLA DM (0.14, p = 0.001 ), and decreased PDL-1 (-1.04, p < 0.009), PDL-2 (-1.70, p < 0.001 ), C-MYC (- 0.95, p < 0.001 ), and STAT1 / 3 / 4 (all p < 0.001 ). Gene ontology (GO) analysis demonstrated increased lymphocyte differentiation (p < 0.001) and immune response activating pathways (p = 0.003) (FIG. 2).

[0063] Altogether, data suggests that ruxolitinib enhances immunogenicity primarily in 9p amplified cell lines and supports the hypothesis that ruxolitinib may prime for lymphoma cell death when combined with immunotherapy. These findings provide a foundation for in vivo assessment in humanized mouse models treated with combination anti-PDL-1 and JAK1 / 2 inhibitor, targeting tumorigenesis driven by 9p amplification.

[0064] Lymphoma organoids were also generated with SUDHL6 (DLBCL) and Karpasl 106p (PMBCL) cell lines as previously described,12-14to understand the effects of combination treatment on T-cell infiltration and function within the TME (see Examples 2 and 3).

[0065] This data provides the pre-clinical groundwork for translating epigenetic priming of checkpoint blockade for treatment of 9p24.1 amplified lymphomas. The potential impact of this combination strategy in PMBCL could be to reduce the number of cycles of frontline chemotherapy, or to improve outcomes in the relapsed / refractory setting. Furthermore, this data may also be broadly applied to other 9p24.1 amplified lymphomas including gray zone lymphoma (GZL) and Hodgkin lymphoma (HL).Example 2Combination JAK inhibition and Immune Checkpoint Blockade for the Treatment of 9p Amplified Lymphomas

[0066] Amplification of 9p24.1 (9p) upregulates PDL-1 / 2 and JAK2, leading to immune-privileged tumor microenvironment (TME) and lymphomagenesis. The 9p amplicon is a key driver of disease in lymphomas that exist on a similar clinicopathologic spectrum found in 100% of Hodgkin Lymphoma (HL), and 75% of Primary Mediastinal B-cell lymphoma (PMBCL). We hypothesize that ifoverexpression of PDL-1 / 2 and JAK / STAT cooperate to lead to immune escape and unchecked proliferation, then dual targeting with ruxolitinib (RUX, JAK2 inhibitor) and pembrolizumab (PEM, PD-1 inhibitor) would target complementary pathways and lead to altered gene expression and enhanced immunogenicity inducing cell death.

[0067] The IC50 values were established for RUX in four 9p positive (9p+) and negative (9p-) human lymphoma cell lines: Karpasl 106p (PMBCL, 9p+) 5.72 pM, L540 (HL, 9p+) 22 pM, Farage (PMBCL, 9p-) 29.46 pM, and U2940 (PMBCL, 9p-) 32.99 pM (Cell TiterGlo). To understand how RUX modulates markers of lymphomagenesis, Western blot and flow cytometry was performed in all cell lines. Transcriptom ic changes were evaluated by RNA-seq, presented as Log2 fold change compared to vehicle control.

[0068] To understand the effects of combination treatment on T-cell infiltration and function within the TME, PMBCL, DLBCL and HL organoids were developed and treated with RUX (0.5 pM, 2.5 pM, 5 pM) and PEM (10 pg / mL) and compared to monotherapy and vehicle controls. Following co-culture with donor CD8+ T-cells, organoids were stained with fluorescent calcein (live), ethidium (apoptotic), and CD3+ dyes and imaged on Leica SP8-DLC. Mean fluorescent values were calculated by Fiji. Reserved organoids were analyzed for T-cell function / activation and apoptosis (flow cytometry). Preliminary in vivo experiments were completed with partially H LA- matched humanized mice (huMice) xenografted with Karpasl 106p.

[0069] The effects of RUX are most prominent in 9p+ cell lines. The 9p+ Karpasl 106p (n=5) demonstrated increased markers of immunogenicity: CIITA 4.83- fold (SEM ± 1.15) and MHCII 2.67-fold (SEM ± 0.33). Tumor suppressor SOCS1 increased 3.33-fold (SEM ± 0.68), and MYC decreased 8.58-fold (SEM ± 4.66). The 9p+ L540 (n=5) paralleled these findings. In all four cell lines, with and without 9p, RUX reduced expression of PDL-1 / 2 (p < 0.01 ) and MYC (p<0.05), (n=4). In Karpasl 106p treated with RUX, RNA-seq showed increased lymphocyte differentiation (p < 0.001 ), immune response activating pathways (p = 0.003), increased MHCII (p=0.001 ), decreased PDL-1 / 2 (-1.04, p < 0.009), and decreased MYC (-0.95, p < 0.001 ).

[0070] The 9p+ organoids (n=80) had the most significant decrease in live mean fluorescence with RUX+PEM compared to RUX alone (p=0.01 ) and to vehicle control (p=0.02). Intra-organoid T-cells (n=16) demonstrated increased CD3+ infiltration with RUX+PEM (p=0.01 ) compared to controls. There was significant decrease in intra-organoid PDL-1 / 2 ligands (p < 0.0001 ) with RUX treatment, suggesting a mechanism for synergy with RUX+PEM. Combination RUX+PEM demonstrated 43% reduction in total B-lymphocytes (p <0.001 ) with decreased live cells (p < 0.001 ) and increased dead cells (p=0.01 ) compared to controls (n=3). To confirm that RUX did not have a negative effect on T-cell viability and function, RUX treated T-cells demonstrated no change in live (p=0.71 ), dead (p=0.91 ), or apoptotic (p=0.6) T-cells, and no change T- cell Ki-67 (p=0.78), granzyme-A (p=0.96), or granzyme-B (p=0.59), (n=3). In Karpas1106p huMice, there was significant difference in tumor volume between control, and both treatment groups: RUX, and RUX+PEM (p=0.007, n=10), with stable weight (p=0.32) over 25 days of treatment. All drugs were safe and well tolerated. Doses have been optimized and there were no signs of graft vs. host disease in control or treated groups.

[0071] Altogether, data suggests that RUX enhances immunogenicity and modulates lymphomagenesis primarily in 9p+ cell lines with increased CIITA, MHCII, and decreased MYC. In 9p+ organoids, combination treatment with RUX+PEM enhanced cell death via reduction in PDL-1 / 2 and increased T-cell infiltration. Deleterious effects of RUX on T-cell function were not observed.Example 3Ruxolitinib increases immunogenicity and primes the tumor microenvironment for immune checkpoint blockade in 9p24.1 amplified Lymphomas

[0072] Amplification of 9p24.1 (9p+) upregulates PDL-1 / 2 and JAK2, leading to an immune-privileged tumor microenvironment (TME) and lymphomagenesis. This biology is relevant for lymphomas that harbor 9p+ or rely on JAK / STAT and immune escape pathways for growth, including Hodgkin Lymphoma (HL) and primary B-cell mediastinal lymphoma (PMBCL). As noted in Examples 1 and 2 above, ruxolitinib (RUXO), a JAK1 / 2 inhibitor, enhances immunogenicity by increasing CIITA, MHCII, and decreasing c-MYC in cell lines, while also increasing T-cell infiltration in lymphoma organoids, priming the TME for PD-1 inhibition with pembrolizumab (PEMBRO). We hypothesize that RUXO disrupts the immune-privileged TME by increased T-cell activation and immune surveillance, thereby increasing the efficacy of PEMBRO in 9p+ and JAK / STAT mutated lymphomas to enhance tumor death when combined with PEMBRO.

[0073] To discern the effects of RUXO+PEMBRO on the peripheral immune milieuin patients, isolated peripheral blood mononuclear cells (PBMCs) were treated with vehicle, RUXO (1 pM), PEMBRO (1 mg / ml), or combination (COMBO); samples were analyzed by flow cytometry (HL = 7, PMBCL = 3). Compared to control, RUXO and COMBO increased peripheral immune surveillance via increased NK cells, decreased Tregs, and decreased immune suppressive myeloid derived suppressor cells (MDSCs) with all p<0.05; altogether suggesting an activated peripheral immune compartment.

[0074] To investigate effects of RUXO+PEMBRO on tumor growth and survival, immunized BALB / c mice were engrafted with the syngeneic A20 cell line (JAK3, STAT 2 / 4 mutated). Mice (n=72) were treated with vehicle control, RUXO (90 mg / kg), PEMBRO (5 mg / kg every 4 days), or COMBO until tumor volumes>2000 mm3. A significant decrease in tumor volume was observed with COMBO compared to vehicle and monotherapy controls. With median follow up of 30 days, Kaplan-Meier analysis showed significant differences in median survival for COMBO (not reached) compared to vehicle (17 days), RUXO (23 days), and PEMBRO (25 days) with p<0.0001. Treatment was well tolerated and there was no significant decrease in weight. To understand the impact of RUXO+PEMBRO on the peripheral immune milieu, n=6 mice were sacrificed from each cohort on Day 7 for PBMC analysis by flow cytometry. Immune activating effects via decreased MDSCs and increased NK cells were observed with COMBO compared to control. RUXO and COMBO increased markers of activation (CD25+) while decreasing markers of exhaustion (LAG3 and PD-1 ) in both CD4+ and CD8+ T-cells (all p <0.05).

[0075] To understand the impact of RUXO+PEMBRO on the TME, humanized mice (huMice) partially HLA-matched to the human Karpasl 106p cell line (9p+) were treated with vehicle control, RUXO (30 mg / kg daily), PEMBRO (10 mg / kg Day 1 , then 5 mg / kg every 5 days), and COMBO; n=6 mice from each cohort were sacrificed at Day 7 and analyzed by flow cytometry. PBMCs demonstrated increased markers of CD4+ and CD8+ T-cell activation (CD40L) with RUXO and COMBO. In tumor samples, RUXO and COMBO decreased PDL-1 expression. Tumor-infiltrating CD3+ T-cells were increased with RUXO and COMBO with increased markers of activation in CD4+ (CD40L) and CD8+ (CD40L, CD69) T-cells. A decreased CD4:CD8 ratio was observed in both PBMCs and tumor samples with RUXO and COMBO, shifting toward higher levels of activated CD8+ T cells.

[0076] huMice tumor samples were further analyzed by spatial Multiomics Single-Cell Imaging (CosMx™) for protein tissue microarray. After QC, normalization, and scaling, five batch-correction methods (CLR, SCTransform, Harmony, BANKSY, InsituType) were compared. Cohorts demonstrated distinct treatment-specific remodeling of the TME with increased clusters of activated CD3+T-cells, dendritic cells, plasmablasts, macrophages, and antigen-presenting B-cells. Specifically, COMBO (n=9) increased tumor-infiltrating activated macrophages, NK cells, and activated CD3+ T-cells compared to control (n=7).

[0077] RUXO induces a shift toward an activated immune milieu, priming the 9+ TME for PEMBRO. RUXO+PEMBRO leads to synergistic anti-tumor activity, modulates the TME via increased infiltration of cytotoxic immune cells, and promotes a shift toward an activated CD8+T cell-dominant TME. These data underscore the potential of JAK / PD-1 axis co-targeting to overcome immune evasion and enhance immunotherapeutic efficacy, leading to the development of an investigator-initiated trial in 9p+ and JAK / STAT mutated lymphomas.Example 4First-in-Class HAT Activator (YF2) Combined with JAK / STAT Inhibitor (ruxolitinib) Unveils Potential Novel Treatment Approach for HDAC Inhibitor- Resistant CTCL

[0078] T-cell lymphomas are both aggressive and challenging to treat. This is in part due to multiple epigenetic derangements found in T-cell lymphomas. One treatment strategy includes HDAC inhibitors; however, the duration of response is short for most patients which is consistent with many treatments for T-cell lymphoma. As a result, alternative treatments are required. YF2, a first-in-class histone acetyltransferase (HAT) activator, was developed. YF2 increases acetylation of histones by augmenting the activity of HAT enzymes.

[0079] The JAK / STAT pathway contributes to CTCL lymphomagenesis through a number of cellular processes including cell cycle, proliferation, apoptosis, etc. JAKs activate STAT3, which activates the transcription of the anti-apoptotic Bcl-2-like protein, Bcl-XL. Additionally, HATs acetylate the pro-apoptotic p53 protein, increasing its stability and activity. Given the dependence on both the JAK / STAT pathway and epigenetic derangements in CTCL, we hypothesize that if the JAK inhibitor, ruxolitinib, induces apoptosis via modulation of Bcl-XL, and YF2 induces apoptosis via acetylationand activation of p53 , then dual targeting of these complementary pathways could lead to synergistic apoptosis in CTCL.

[0080] To study this hypothesis, we used two CTCL cell lines: HH [JAK wildtype (JAKm)], and H9 [JAK1 / 3 gain-of-function mutated (JAK1 / 3GOF)]. In addition, we generated a belinostat-resistant H9 cell line (H9-belino-R) by incrementally exposing H9 to increasing concentrations of belinostat. H9-belino-R resistance was observed at a 20-fold (IC50 = 896.7nM, SEM ± 185.3) increase over that of the parental H9 (IC50 = 43.8nM, SEM ± 4.61 ) (FIG. 3). H9-belino-R retained significant resistance to other HDAC inhibitors such as romidepsin [(H9: IC50 = 0.97nM (SEM ± 0.030), H9-belino-R: IC50 = 1 ,38nM (SEM ± 0.028)] and panobinostat [H9: IC50 = 3.11 nM (SEM ± 0.19), H9- belino-R: IC50 = 9.00nM (SEM ± 1 .55)] as measured by the CellTiter-Glo Viability Assay. Interestingly, the IC50 of the HAT activator, YF2, was retained between the resistant (14.84pM, SEM ± 0.46) and parental (12.45pM, SEM ± 0.27) cell lines (FIG. 3).

[0081] HH (JAK™-) showed significant cytotoxicity (63.5% live, 30.7% apoptotic, and 5.4% dead) after 4-day exposure to 10pM YF2 compared to limited cytotoxicity (93.3% live, 4.6% apoptotic, and 2.0% dead) in H9 (JAKGOF) as measured by flow cytometry with Annexin V. At baseline, active STAT3 (pSTAT3) was not detected in HH possibly contributing to its susceptibility to YF2 as the absence of pSTAT3 reduces the transcription Bcl-XL. As illustrated by FIG. 4, Western blot analysis confirmed ruxolitinib-induced inhibition of active STAT3 (pSTAT3) with associated reduction in Bcl-XL expression in H9 and H9-belino-R. This may explain the observed synergistic cytotoxicity with YF2 and ruxolitinib in HH, H9 and H9-belino-R (Excess over Bliss scores: 4.85, 14.23, and 5.38 respectively) as determined by flow cytometry.

[0082] In conclusion, the novel first-in-class HAT activator, YF2, retains activity in HDAC inhibitor-resistant CTCL. YF2’s activity may be blunted by JAK1 / 3GOFmutation; however, when combined with the JAK-inhibitor ruxolitinib, the response mirrored that of the HH (JAKwr) cell line. The combination of HAT activator and JAK inhibitor led to enhanced reduction of the anti-apoptotic protein Bcl-XL, potentially explaining synergistic apoptosis in CTCL. These findings provide us with evidence that suggests that the combination of YF2 and ruxolitinib can serve as a novel treatment combination for CTCL.Example 5Targetting Hypoxia with JIB-04

[0083] Due to the rapid growth of tumors, most of the TME is characterized and affected by hypoxia (low oxygen levels). Hypoxia could induce TAM polarization, promote the accumulation of MDSCs and Tregs and downregulate the activity of NK cells. Hypoxia-inducible factor 1 (HIF-1 ) is a transcription factor that responds to decreases in available oxygen in the cellular environment. HIF-1 is a heterodimer, composed of two subunits: the stable, constitutively expressed HIF-1 beta (HIF-1 p) and the oxygen-regulated HIF-1 alpha (HIF-1 a). Under hypoxic conditions, HIF-1 activates genes involved in energy metabolism, angiogenesis (new blood vessel growth), and cell survival, which is crucial for adapting to low oxygen and plays a key role in diseases like cancer. HIF-1a synthesis is regulated by PI3K or mitogen- activated protein kinase (MAPK) signal pathways. High expression of HIF-1a induces tumor cells to adapt to hypoxia and grow rapidly.

[0084] JMJD2D (or KDM4D) increased the promoter activities of genes regulated by p-catenin (MYC, CCND1 , MMP2, and MMP9). It was found that JMJD2D and p- catenin interacted physically and that JMJD2D demethylated H3K9me3 at promoters of p-catenin target genes. JMJD2D-knockout mice developed fewer colitis-associated colorectal tumors than control mice, and their tumor tissues had lower levels of p- catenin, MYC, cyclin D1 , and proliferating cell nuclear antigen than tumors from control mice.

[0085] JMJD2C (or KDM4C) selectively stimulates HIF-1-mediated transactivation, which promotes metabolic reprogramming (in which cells switch from oxidative to glycolytic metabolism) in breast cancer cells through the activity of GLUT 1 , LDHA, and PDK1 , and increases breast tumor growth and spontaneous metastasis through the activity of LOXL2 and L1 CAM. The current Example explored potential therapeutics targeting hypoxia in cancers.

[0086] The ICso values were established for JIB-04 (CAS No. : 199596-05-9) (a pan-selective Jumonji histone demethylase inhibitor, which increases methylation and gene suppression) in six 9p positive (9p+) and negative (9p-) human lymphoma cell lines: Karpasl 106p (PMBCL, 9p+) 1 15 ± 80 nM, L540 (HL, 9p+) 1743.33 ± 262.05 nM, Farage (PMBCL, 9p-) 1017.53 ± 650 nM, U2940 (PMBCL, 9p-) 625.33 nM, SUDHL6 (DLBCL, 9p+) 911 ± 214 nM, and HDLM2 (HL, 9p+) 4957.66 ± 1317.95 nM (Cell TiterGlo). ICso values were also established for tazemetostat (an EZH2 inhibitor) and ruxolitinib (RUXO). The results were summarized and compared in the table below.

[0087] To understand how JIB-04 modulates markers of lymphomagenesis, Western blot and flow cytometry were performed in selected cell lines (SLIDHL6, Karpas1106p, and LI2940). Flow cytometry results of the expression fold change analysis on selected markers (HLADM, HLA DR, DP, DQ, MHC I, PDL1 , PDL2) were shown in FIGS. 5-7.Example 6 Combination of YF2, Pembrolizumab and Ruxolitinib

[0088] Given the promising results of the combination of pembrolizumab + ruxolitinib and the combination of YF2 + ruxolitinib, as described in the previous Examples, we hypothesize that if the JAK inhibitor, ruxolitinib, induces apoptosis via modulation of Bcl-XL, the immune checkpoint inhibitor, pembrolizumab, suppresses the amplified expression of PD-1 ligands, and YF2 induces apoptosis via acetylation and activation of p53, then triple targeting of these complementary pathways could lead to more synergistic apoptosis in BCL. Thus, a three-component combination of YF2 + pembrolizumab (PEMBRO) + ruxolitinib (RUXO) was also tested.

[0089] To study this hypothesis, two B cell lines SLIDHL4 (DLBCL, 9p-) and SLIDHL6 (DLBCL, 9p+) were used. Each cell line was treated in four groups with (1) control (no YF2 or RUXO treatment); (2) 10 pM YF2 only, (3) 2 pM RUXO only, and (4) 10 pM YF2 + 2 pM RUXO, respectively, for three days. All four groups were then each combined with healthy T cells, and further treated with 10 pg / mL PEMBRO, and co-cultured for 24 hours before assessed for B cell specific apoptosis via flow cytometry with Annexin V (FIG. 8).

[0090] As illustrated in FIG. 9B, SUDHL4 cell line treated with the three-componentcombination of YF2 + PEMBRO + RUXO showed significant cytotoxicity (26% apoptotic or dead) after 4-day exposure to the combination compared to the control (12.5%), and also more cytotoxicity than YF2-only group (20%) and RUXO-only group (17.5%). Similar results were observed in SUDHL6 cell line (FIG. 10B). The three- component combination caused 23.5% apoptosis or death, compared to 12% (control), 16.5% (YF2-only), and 13.5% (RUXO-only).

[0091] In conclusion, the combination of HAT activator, immune checkpoint inhibitor and JAK inhibitor led to enhanced apoptosis in DLBCL, suggesting a novel therapy for lymphoma.CITED DOCUEMNTS1. Dunleavy K, Pittaluga S, Maeda LS, et al. Dose-Adjusted EPOCH-Rituximab Therapy in Primary Mediastinal B-Cell Lymphoma. New England Journal of Medicine 2013;368(15):1408-16.2. Hodgson DC. Long-term toxicity of chemotherapy and radiotherapy in lymphoma survivors: optimizing treatment for individual patients. Clin Adv Hematol Oncol 2015; 13(2): 103-12.3. Savage KJ. Primary mediastinal large B-cell lymphoma. Blood 2022;140(9):955-70.4. Dunleavy K, Wilson WH. Primary mediastinal B-cell lymphoma and mediastinal gray zone lymphoma: do they require a unique therapeutic approach? Blood 2015;125(1 ):33-39.5. Green MR, Monti S, Rodig SJ, et al. Integrative analysis reveals selective 9p24.1 amplification, increased PD-1 ligand expression, and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma. Blood 2010;116(17):3268-77.6. Rui L, Emre NT, Kruhlak MJ, et al. Cooperative epigenetic modulation by cancer amplicon genes. Cancer ce / / 2010;18(6):590-605.7. Kuruvilla J, Armand P, Hamadani M, et al. Pembrolizumab for patients with nonHodgkin lymphoma: phase 1 b KEYNOTE-013 study. Leukemia & lymphoma 2022:1-10.8. Chen R, Zinzani PL, Lee HJ, et al. Pembrolizumab in relapsed or refractory Hodgkin lymphoma: 2-year follow-up of KEYNOTE-087. Blood2019;134(14):1144-53.9. Zinzani PLL, Thieblemont C, Melnichenko V, et al. Final Analysis of Keynote- 170: Pembrolizumab in Relapsed or Refractory Primary Mediastinal Large 13- Cell Lymphoma (PMBCL). Blood 2021 ; 138(Supplement 1):306-06.10. Herrera AF, LeBlanc M, Castellino SM, et al. NIVOLUMAB(N)-AVD IMPROVES PROGRESSION-FREE SURVIVAL COMPARED TO BRENTUXIMAB VEDOTIN(BV)-AVD IN ADVANCED STAGE (AS) CLASSIC HODGKIN LYMPHOMA (HL): RESULTS OF SWOG S1826. Hematological Oncology 2023;41 (S2):33-35.11. Steidl C, Gascoyne RD. The molecular pathogenesis of primary mediastinal large B-cell lymphoma. Blood 2011 ;118(10):2659-69.12. Matsuzawa-lshimoto Y, Hine A, Shono Y, et al. An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury. Blood 2020;135(26):2388-401.13. Teijeira A, Migueliz I, Garasa S, et al. Three-dimensional colon cancer organoids model the response to CEA-CD3 T-cell engagers. Theranostics 2022; 12(3): 1373-87.14. Shah SB, Carlson CR, Lai K, et al. Combinatorial treatment rescues tumourmicroenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas. Nature / Wate / 7a / s 2023;22(4):511-23.

[0092] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety as if recited in full herein.

[0093] The disclosure being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the disclosure and all such modifications are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or ameliorating the effect of a cancer in a subject, comprising administering to the subject an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor.

2. The method of claim 1 , wherein the cancer is a solid tumor.

3. The method of claim 1 , wherein the cancer is a lymphoma.

4. The method of claim 3, wherein the lymphoma is selected from the group consisting of Primary mediastinal B-cell lymphoma (PMBCL), Cutaneous T-cell lymphoma (CTCL), Hodgkin lymphoma (HL), B-cell non-Hodgkin lymphoma, gray zone lymphoma (GZL), and diffuse large B-cell lymphoma (DLBCL).

5. The method of claim 1 , wherein the cancer is Primary mediastinal B-cell lymphoma (PMBCL).

6. The method of claim 1 , wherein the epigenetic agent is selected from the group consisting of a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC), inhibitor, a bromodomain and extra-terminal motif protein (BET) inhibitor, a methyltransferase like-3 (METTL3) inhibitor, a lysine-specific histone demethylase 1 (LSD1 ) inhibitor, an enhancer of zeste homolog 2 (EZH2) inhibitor, a DOT1-like histone lysine methyltransferase (DOT1 L) inhibitor, an isocitrate dehydrogenase (IDH) inhibitor, a Jumonji histone demethylase inhibitor, a histone acetyltransferase (HAT) activator, and combinations thereof.

7. The method of claim 1 , wherein the epigenetic agent is an HAT activator.

8. The method of claim 1 , wherein the epigenetic agent is YF2.

9. The method of claim 1 , wherein the epigenetic agent is a Jumonji histone demethylase inhibitor.

10. The method of claim 9, wherein the Jumonji histone demethylase inhibitor is JIB-04.11 . The method of claim 1 , wherein the epigenetic agent is an EZH2 inhibitor.

12. The method of claim 11 , wherein the EZH2 inhibitor is tazemetostat.

13. The method of claim 1 , wherein the immunotherapeutic agent is an immune checkpoint inhibitor selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, and combinations thereof.

14. The method of claim 13, wherein the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, atezolizumab, and combinations thereof.

15. The method of claim 1 , wherein the immunotherapeutic agent is pembrolizumab.

16. The method of claim 1 , wherein the JAK1 / 2 inhibitor is selected from the group consisting of AG-490, abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclactinib, peficitinib, pacritinib, ruxolitinib, tofacifinib, upadacitinib, and combinations thereof.

17. The method of claim 1 , wherein the JAK1 / 2 inhibitor is ruxolitinib.

18. The method of claim 1 , wherein the subject is a human.

19. The method of claim 1 , wherein the subject has amplification of chromosome 9p24.1 (9p).

20. The method of claim 1 , wherein the epigenetic agent, immunotherapeutic agent, and JAK1 / 2 inhibitor are administered to the subject serially or concurrently.

21. The method of claim 1 , wherein the epigenetic agent is administered to the subject prior to the administration of the immunotherapeutic agent and JAK1 / 2 inhibitor.

22. A composition for treating or ameliorating the effect of a cancer in a subject, comprising an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor.

23. The composition of claim 22, wherein the epigenetic agent is YF2, the immunotherapeutic agent is pembrolizumab, and the JAK1 / 2 inhibitor is ruxolitinib.

24. A kit for treating or ameliorating the effect of a cancer in a subject, comprising an effective amount of (i) an epigenetic agent, (ii) an immunotherapeutic agent, and (iii) a Janus kinase 1 / 2 (JAK1 / 2) inhibitor, packaged with instructions of use.

25. A method for treating or ameliorating the effects of hypoxia in the tumor microenvironment (TME) in a subject having a cancer, comprising administering to the subject an effective amount of a Jumonji histone demethylase inhibitor.

26. The method of claim 25, wherein the Jumonji histone demethylase inhibitor downregulates the activity of histone demethylase JMJD2D and / or JMJD2C.

27. The method of claim 25, wherein the Jumonji histone demethylase inhibitor is JIB-04.

28. The method of claim 25, wherein the cancer is a solid tumor.

29. The method of claim 28, wherein the solid tumor is breast cancer or lung cancer.

30. The method of claim 25, wherein the cancer is a lymphoma.

31. The method of claim 30, wherein the lymphoma is selected from the group consisting of Primary mediastinal B-cell lymphoma (PMBCL), Cutaneous T-cell lymphoma (CTCL), Hodgkin lymphoma (HL), B-cell non-Hodgkin lymphoma, gray zone lymphoma (GZL), and diffuse large B-cell lymphoma (DLBCL).

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