Methods and compositions for increasing ror1 expression

Abstract

Disclosed are methods of increasing R0R1 in a cell comprising administering an EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof to the cell. Disclosed are methods of treating lymphoma in a subject comprising administering to the subject one or more EZH2 inhibitors, G9A inhibitors, DNMT1 inhibitors or combinations thereof, wherein the one or more EZH2 inhibitors, G9A inhibitors, DNMT1 inhibitors or combinations thereof increase expression of R0R1 in one or more B cells in the subject; and one or more R0R1 targeting therapeutics.

Description

METHODS AND COMPOSITIONS FOR INCREASING ROR1 EXPRESSIONBACKGROUND

[0001] ROR1 is an orphan cell-surface transmembrane receptor that plays an essential role in embry ogenesis but is also expressed in various types of malignant tumors. As such, ROR1 can be expressed in hematological malignancies such as chronic lymphocytic leukemia, mantle cell lymphoma and diffuse large B-cell lymphoma, with increased levels of ROR1 associated with worse patient outcomes. Clinical trials with anti-RORl antibody-drug conjugate are underway in lymphoid malignancies. In addition to hematologic malignancies, solid tumors such as breast and lung carcinomas often also express ROR1. ROR1 -targeted chimeric antigen receptor (CAR) T- cell therapy are currently being tested in patients with breast and lung cancer. Expression of the targeted ROR1, optimally high, uniform and sustained on tumor cells is believed to be crucial for therapeutic success of all targeted therapies.BRIEF SUMMARY

[0002] Disclosed are methods of increasing ROR1 in a cell comprising administering an EZH2 inhibitor, G9A inhibitor or a DNMT1 inhibitor to the cell.

[0003] Disclosed are methods of treating cancer in a subject comprising administering to the subject one or more EZH2 inhibitors, wherein the one or more EZH2 inhibitors increase expression of ROR1 in one or more cancer cells in the subject; and one or more ROR1 targeting therapeutics.

[0004] Disclosed are methods of treating lymphoid malignancies: lymphoma and leukemia in a subject comprising administering to the subject one or more EZH2 inhibitors, wherein the one or more EZH2 inhibitors increase expression of ROR1 in one or more B cells in the subject; and one or more ROR1 targeting therapeutics.

[0005] Disclosed are methods of treating cancer in a subject comprising administering to the subject one or more G9A inhibitors, wherein the one or more G9A inhibitors increase expression of ROR1 in one or more cancer cells in the subject; and one or more ROR1 targeting therapeutics.

[0006] Disclosed are methods of treating lymphoid malignancies (e.g., lymphoma and leukemia) in a subject comprising administering to the subject one or more DNMT1 inhibitors, wherein the one or more DNMT1 inhibitors increase expression of ROR1 in one or more cancer cells in the subject; and one or more ROR1 targeting therapeutics.

[0007] In some aspects, the methods of treating cancer can treat cancers that include, but are not limited to, lymphoid malignancies (e.g., lymphoma and leukemia), plasma cellmyeloma / neoplasm, ovarian cancer, bladder cancer, breast cancer, and colon cancer.

[0008] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary7and explanatory' only and are not restrictive of the invention as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, w'hich are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0010] FIGS. 1 A-1E show ROR1 expression in MCL patients-derived lymphoma cell lines detected by flow cytometry7analysis. FIG. IE shows ROR1 expression detected by Western blot. A) JeKo-1 cells, B) Maver cells, C) REC-1 cells, D (MCL)-RL cells. Right lower panels for A-D show ROR1 expression (red curves pointed by arrows). Left lower panels for A-B show expression of CD5 antigen serving as control. E: Western blot-detected ROR1 expression in the depicted MCL cell lines with GAPDH expression serving as a positive control.

[0011] FIGS. 2A-2D show ROR1 expression in primary MCL cells. A) ROR1 expression in the six patient-derived primary MCL cell population (the P series in the second and third row) with the depicted MCL cell lines serving as controls (first row). B-D) ROR1 expression in other 3 different index MCL patients. Right lower panels for B-D show ROR1 expression (red curves pointed by arrows). Left lower panels for B-B show7expression of CD5 antigen serving as control.

[0012] FIG. 3 shows Western blot results of both induced and markedly increased ROR1 expression in Granta MCL cells treated with an EZH2 inhibitor EPZ-6438 (tazemetostat), used at 2pM. Granta cells were cultured either unseparated (parental) or separated into ROR1 -negative and ROR1 -positive (+) subpopulation and cultured for 0, 6, or 9 days with EZH2 inhibitor.Expression of B actin (Bact) served as a control. Expression of H3K27me3. the target of EZH2 enzymatic activity, and of total H3 served as additional controls.

[0013] FIG. 4 shows ROR1 expression by MCL cell line (MCL)-RL treated with EZH2 inhibitor (EPZ-6438 (tazemetostat) at 2pM) and after subsequent withdrawal of EZH2 inhibitor. MCL-RL cells were cultured for 6 and 9 days (left and middle panel, respectively) in the presenceof an EZH2 inhibitor or medium alone and examined for R0R1 expression. In some experiments, MCL-RL cells were exposed to EZH2 inhibitor (EPZ-6438 (tazemetostat) at 2pM) for 6 days and then cultured for additional 3 days wi th medium alone after removal of the EZH2 inhibitor and examined for R0R1 expression.

[0014] FIG. 5 shows ROR1 expression by MCL cell line SP49 treated with EZH2 inhibitor and after subsequent withdrawal of EZH2 inhibitor. SP49 cells were cultured for 6 and 9 days (left and middle panel, respectively) in the presence of an EZH2 inhibitor or medium alone and examined for ROR1 expression. In some experiments, SP49 cells were exposed to EZH2 inhibitor for 6 days and then cultured for additional 3 days with medium alone after removal of the EZH2 inhibitor and examined for ROR1 expression.

[0015] FIG. 6 shows ROR1 expression by diffuse large B-cell lymphoma (DLBCL) LY8 cell line treated with EZH2 inhibitor (EPZ-6438 (tazemetostat) at 2pM) and after subsequent withdrawal of EZH2 inhibitor. DLBCL LY8 cells were cultured for 6 and 9 days (left and middle panel, respectively) in the presence of an EZH2 inhibitor or medium alone and examined for ROR1 expression. In some experiments, LY8 cells were exposed to EZH2 inhibitor for 6 days and then cultured for additional 3 days with medium alone after removal of the EZH2 inhibitor and examined for ROR1 expression.

[0016] FIG. 7 shows ROR1 expression by MCL cell line Granta treated with EZH2 inhibitor (EPZ-6438 (tazemetostat) at 2pM) and after subsequent withdrawal of EZH2 inhibitor. Granta cells were cultured either unseparated (parental) or separated into ROR1 -negative and ROR1- positive (+) subpopulation and cultured for 6, 9 or 13 days with EZH2 inhibitor or medium alone (left 3 panels) and examined for ROR1 expression. Right panel: after 13 days of culture with EZH2 inhibitor, the Granta cells were cultured with medium alone and tested for ROR1 expression. Lower right comer of the figure: Western blot and flow cytometry examination of the separated ROR1- and ROR1+ populations for ROR1 expression.

[0017] FIG. 8 shows the effect of cell treatment with EZH2 inhibitor on growth of index MCL cells: (MCL)-RL and SP49 cell lines. The RL and SP49 MCL cell lines were cultured in the presence or absence (CTRL) of EZH2 inhibitor (EPZ-6438 (tazemetostat) at 2pM) for 6 days and examined for their grow th capacity in the 48 hr WST assay.

[0018] FIG. 9 shows enhancement of ROR1 expression in SP-49 MCL cells by EZH2 and G9A inhibition on day 5.

[0019] FIG. 10 shows an induction / enhancement of ROR1 expression in MCL cells by DNMT1 inhibition and combined EZH2 / DNMT1 inhibition.

[0020] FIG. 11 shows EZH2 inhibition enhances ROR1 expression in plasma cellmyeloma / neoplasm. FIG. 11A) ROR1 expression in plasma cell myeloma / neoplasm (PCM) KMS11 cells. FIG. 1 IB) Enhancement of ROR1 expression in PCM KMS11 cells by EZH2 and G9A inhibition.

[0021] FIG. 12 shows expression of ROR1 mRNA in ovarian carcinoma cell lines (Cancer Atlas) and enhancement of ROR1 protein expression by EZH2 inhibition in the OVCAR3 cell line.

[0022] FIG. 13 Expression of ROR1 mRNA in bladder, breast, and colorectal carcinoma (Cancer Atlas).DETAILED DESCRIPTION

[0023] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0024] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0025] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids are discussed, each and every' combination and permutation of the peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary'. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E. and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosedfrom disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.A. Definitions

[0026] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0027] It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "an Enhancer of zeste homolog 2 (EZH2) inhibitor" includes a plurality of such inhibitors, reference to "the EZH2 inhibitor" is a reference to one or more peptide and equivalents thereof known to those skilled in the art, and so forth.

[0028] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0029] As used herein, the term "therapeutically effective amount" of a composition (e.g. EZH2 inhibitor) as provided herein is meant a sufficient amount of the composition to provide the desired therapeutic effect (e.g. induction of or an increase in ROR1 expression). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular composition used, its mode of administration, and the like. Thus, it is not possible to specify an exact “therapeutically effective amount.” However, an appropriate “therapeutically effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

[0030] The term “therapeutic” refers to a composition that treats a disease. For example, the therapeutics disclosed herein are compositions that treat hepatocellular carcinoma.

[0031] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, relieving, preventing or delaying onset of, inhibiting progression of, reducing severity- of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. For example, "treating" lymphoma may refer to inhibiting survival,growth, and / or spread of the cancer cells. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0032] "Peptide ’ as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A peptide is comprised of consecutive amino acids. The term “peptide” encompasses naturally occurring or synthetic molecules.

[0033] The terms “patient,” “subject.” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat. guinea pig, fruit fly, etc.). In one aspect, a subject is a mammal. In another aspect, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

[0034] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein andthe material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0036] Throughout the description and claims of this specification, the word “comprise’' and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.B. Methods

[0037] Disclosed are methods of using an EZH2 inhibitor to increase expression of ROR1 in cells. In some aspects, “increasing ROR1 expression” can mean increasing ROR1 expression in cells that already express ROR1. In some aspects, “increasing ROR1 expression” can mean increasing the amount of ROR1 expression in the cell that did express ROR1 prior to administration of the EZH2 inhibitor as well as inducing ROR1 expression in cells that did not express ROR1 prior to administration of the EZH2 inhibitor.

[0038] In some aspects, methods of increasing ROR1 expression can broaden the subject population that can be treated with a ROR1 targeting therapeutic. Thus, not only can subjects having cells with an increase in ROR1 expression be treated with ROR1 targeting therapeutic, but subjects with low levels of ROR1 expression be treated with ROR1 targeting therapeutic after increasing the ROR1 expression with EZH2 inhibitors.1. Methods of Increasing ROR1 Expression

[0039] Disclosed are methods of increasing ROR1 in a cell comprising administering an EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor, or a combination thereof to the cell.

[0040] Disclosed are methods of increasing ROR1 in a cell comprising administering an EZH2 inhibitor to the cell. EZH2 is a member of the family of poly comb group genes (PcGs). which is a group of important epigenetic regulators that repress transcription. Thus, in some aspects, if increased transcription is necessary, inhibition of EZH2 can be performed.

[0041] Disclosed are methods of increasing ROR1 in a cell comprising administering a G9Ainhibitor to the cell. G9A inhibitors, such as UNC0638 and BIX-01294, can inhibit cell proliferation by inducing cell cycle arrest, triggering apoptosis, or inducing autophagic cell death.

[0042] Disclosed are methods of increasing R0R1 in a cell comprising administering a DNA methyltransferase 1 (DNMT1) inhibitor to the cell. In some aspects, DNMT1 inhibitors can be used to lower DNA methylation levels.

[0043] In some aspects, the cell is in a subject. In some aspects, the subject has cancer. In some aspects, the subject has a lymphoid malignancy (e.g., lymphoma and leukemia), plasma cell myeloma / neoplasm, ovarian cancer, bladder cancer, breast cancer, lung cancer, colon cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or brain cancer.

[0044] Thus, in some aspects, the methods of increasing ROR1 are in vivo methods. In some aspects, the methods of increasing ROR1 can be in vitro methods.

[0045] In some aspects, the cell is a cancer cell. In some aspects, the cancer cell can be, but is not limited to, a lymphoma cell, plasma cell, breast cancer cell, lung cancer cell, bladder cancer cell, colon cancer cell, liver cancer cell, pancreatic cancer cell, brain cancer cell, or ovarian cancer cell. In some aspects, the cell is a lymphocyte. In some aspects, the lymphocyte is a B cell. In some aspects, the B cell is a B cell lymphoma cell. In some aspects, the B cell lymphoma cell is mantle cell lymphoma cell.

[0046] In some aspects, EZH2 inhibitor is a small molecule, protein, or nucleic acid. In some aspects, an EZH2 inhibitor can be any inhibitor that is EZH2 inhibition-dependent. For example, in some aspects, an EZH2 inhibitor is a direct inhibitor of EZH2 enzymatic activity. In some aspects, an EZH2 inhibitor is an inhibitor of expression of EZH2 at the protein or mRNA level. Thus, in some aspects, an EZH2 inhibitor can prevent or inhibit activity, gene expression, or protein expression of EZH2.

[0047] In some aspects, the EZH2 inhibitor can be, but is not limited to, tazemetostat (E7438 / EPZ6438), 3-deazaneplanocin A (DZNep), GSK126 (GSK2816126), EPZ005687, Ell, GSK343, GSK926. EPZ011989, CPI-1205, CPI-169, CPI-1205, ZLD1039, PF-06821497, UNC1999, (R)-OR-Sl. (R)-OR-S2, DS-3201b, stabilized alpha-helix of EZH2 (SAH-EZH2), Astemizole, Wedelolactone, apomorphine hydrochloride, oxyphenbutazone, nifedipine, ergonovine maleate, AZD9291 (Osimertinib, TAGRISSO), MAK683 / EED226, GNA022, long non-coding RNA (IncRNA) ANCR, F-box and WD repeat domain-containing 7 (FBW7), ZRANB1, KAN0439834, KAN0441571C. DB03208, strictinin, ARI-1, NVG-111, Cirtuzumab- based CAR-T, JCAR-024, or VLS-101. In some aspects, the EZH2 inhibitor can be, but is not limited to, a S-adenosyl-L-homocysteine (SAG) hydrolase inhibitor [3-deazaneplanocin A (DZNep)], or a S-adenosyl-methionine-competitive inhibitor [EIl], stabilized alpha-helix of EZH2(SAH-EZH2) [peptides], gambogenic acid (GNA) derivative [[GNA022]], long-coding RNA, small interfering RNA (siRNA) [ ZRANB1], In some aspects, the EZH2 inhibitor can be one or more of those described in / / jhoonline.biomedcentral.com / articles / 10.1186 / sl3045-020-00937-8, which is incorporated by reference in its entirety herein.

[0048] In some aspects, G9A inhibitors can be, but are not limited to, UNC0638, BIX-01294, Chaetocin, UNC0642, UNC0646, RK-701, A-366, or BRD4770

[0049] In some aspects, DNMT1 inhibitors can be, but are not limited to, GSK-3484862, Theaflavin, Glyburide and panobinostat, Aza-T-dCyd, SGI-1027, MC3343, Nanaomycin A, Hydralazine, and Procaine.

[0050] In some aspects, the cell does not express ROR1 prior to administering the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor. In some aspects, the cell is a ROR1 expressing cell prior to administering the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor. In some aspects, the cell expresses lower levels of ROR1 prior to administering the EZH2 inhibitor. G9A inhibitor, or DNMT1 inhibitor compared to levels after receiving EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor, respectively.

[0051] In some aspects, the disclosed methods further comprise ceasing the administration of the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor, wherein ROR1 expression decreases in the cell after administration of the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor is ceased. In some aspects, the disclosed methods further comprise ceasing the administration of the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor, wherein ROR1 expression is maintained in the cell after administration of the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor is ceased. In some aspects, in either scenario, the ROR1 no longer has an increase in expression once administration of the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor is ceased.

[0052] In some aspects, the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor is administered in a therapeutically effective amount. In some aspects, the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor can be administered as a single dose. In some aspects, the EZH2 inhibitor. G9A inhibitor, or DNMT 1 inhibitor can be administered in multiple doses. In some aspects, the stability of the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor determines the dosing regimen.

[0053] In some aspects, two or more of EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor can be administered together (in a single or separate compositions) in the disclosed methods. i. Combination Treatment

[0054] In some aspects, cells can be treated with an EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor, or a combination thereof and therapeutic. In some aspects, the therapeutic is a cancertherapeutic.

[0055] In some aspects, the an EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor, or a combination thereof can be administered in combination with a ROR1 targeting therapeutic. In some aspects, a ROR1 targeting therapeutic can be an immunotherapy. In some aspects, the immunotherapy targets the ROR1, which has increased expression due to the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor, or a combination thereof. In some aspects, the ROR1 targeting therapeutic targets ROR1 signaling, directly or indirectly. In some aspects, the immunotherapy can be, but is not limited to, a ROR1 -specific immunotoxin, a ROR1 antibody or a ROR1 chimeric antigen receptor T cell (CART cell). In some aspects, the ROR1 antibody can be, but is not limited to, Zilovertamab Vedotin (UC-961), 2A2, 2D11, 1A1, 1A7, R12, XBR1-402, clone F, 4A5, or Rl l.

[0056] In some aspects, an ROR1 antibody can be conjugated to a cytotoxic agent. In some aspects, a cytotoxic agent can be. but is not limited to, tubulin inhibitors (an antimicrotubule cytotoxic agent (e.g. monomethyl auristatin E (MMAE)), maytansinoids, tubulysins) or pseudomonas toxin. Saporin, DNA damaging agents (calicheamicins, duocarmycins, exatecans). In some aspects, the cytotoxic agent is a toxin.

[0057] In some aspects, the ROR1 CAR T cell can be one or more of those listed in / / www.frontiersin.org / articles / 10.3389 / fmed.2023.1121020 / full, which is hereby incorporated by reference in its entirety for its teaching of ROR1 CAR T cells. In some aspects, the ROR1 CAR T cell is LYL797.

[0058] In some aspects, the ROR1 -specific immunotoxin can be, but is not limited to, HuXBRl-402, VelosBiolOl. or LCB71 / ABL202. In some aspects, the ROR-1 specific immunotoxin can be one or more of those listed in Peng, Antibody Therapeutics, Volume 4, Issue 4, Oct 2021, pages 222-227, incorporated by reference in its entirety herein.2. Methods of Treating Cancer

[0059] Disclosed are methods of treating cancer in a subject comprising administering to the subject one or more EZH2 inhibitors. G9A inhibitors, DNMT1 inhibitors, or a combination thereof.

[0060] Disclosed are methods of treating cancer in a subject comprising administering to the subject one or more EZH2 inhibitors, wherein the one or more EZH2 inhibitors increase expression of ROR1 in a cell in the subject; and one or more ROR1 targeting therapeutics.

[0061] Disclosed are methods of treating cancer in a subject comprising administering to the subject one or more G9A inhibitors, wherein the one or more G9A inhibitors increase expression of ROR1 in a cell in the subject; and one or more ROR1 targeting therapeutics.

[0062] Disclosed are methods of treating cancer in a subject comprising administering to the subject one or more DNMT1 inhibitors, wherein the one or more DNMT1 inhibitors increase expression of ROR1 in a cell in the subject; and one or more ROR1 targeting therapeutics.

[0063] In some aspects, the cancer can be, but is not limited to, lymphoid malignancy (e g., lymphoma and leukemia), plasma cell myeloma / neoplasm, ovarian cancer, bladder cancer, breast cancer, lung cancer, colon cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or brain cancer.

[0064] In some aspects, the cell can be, but is not limited to, a lymphoma cell, plasma cell, breast cancer cell, lung cancer cell, bladder cancer cell, colon cancer cell, liver cancer cell, kidney cancer cell, cervical cancer cell, pancreatic cancer cell, brain cancer cell, or ovarian cancer cell. Disclosed are methods of treating lymphoma in a subject comprising administering to the subject one or more EZH2 inhibitors, G9A inhibitors, DNMT1 inhibitors or combinatiosn thereof, wherein the one or more EZH2 inhibitors, G9A inhibitors, or DNMT1 inhibitors or combination thereof increase expression of ROR1 in one or more B cells in the subject; and one or more ROR1 targeting therapeutics.

[0065] In some aspects, the lymphoma is a B cell lymphoma. In some aspects, the B cell lymphoma is mantle cell lymphoma.

[0066] In some aspects, EZH2 inhibitor is a small molecule, protein, or nucleic acid. In some aspects, an EZH2 inhibitor can be any inhibitor that is EZH2 inhibition-dependent. For example, in some aspects, an EZH2 inhibitor is a direct inhibitor of EZH2 enzymatic activity and not an inhibitor of expression of EZH2 at the protein or mRNA level. In some aspects, an EZH2 inhibitor can prevent or inhibit gene expression or protein expression of EZH2.

[0067] In some aspects, the EZH2 inhibitor can be, but is not limited to, tazemetostat (E7438 / EPZ6438), 3-deazaneplanocin A (DZNep), GSK126 (GSK2816126), EPZ005687, Ell, GSK343, GSK926, EPZ011989, CPI-1205, CPI-169, CPI-1205, ZLD1039, PF-06821497, UNC1999, (R)-OR-S1. (R)-OR-S2, DS-3201b, stabilized alpha-helix of EZH2 (SAH-EZH2), Astemizole, Wedelolactone, apomorphine hydrochloride, oxyphenbutazone, nifedipine, ergonovine maleate, AZD9291 (Osimertinib, TAGRISSO), MAK683 / EED226, GNA022, long non-coding RNA (IncRNA) ANCR, F-box and WD repeat domain-containing 7 (FBW7), ZRANB1, KAN0439834, KAN0441571C, DB03208, strictinin, ARI-1, NVG-111, Cirtuzumab- based CAR-T, JCAR-024, or VLS-101. In some aspects, the EZH2 inhibitor can be. but is not limited to, a S-adenosyl-L-homocysteine (SAH) hydrolase inhibitor [3-deazaneplanocin A (DZNep)], or a S-adenosyl-methionine-competitive inhibitor [Ell], stabilized alpha-helix of EZH2 (SAH-EZH2) [peptides], gambogenic acid (GNA) derivative [[GNA022]], long-coding RNA,small interfering RNA (siRNA) [ ZRANB1], In some aspects, the EZH2 inhibitor can be one or more of those described in / / jhoonline.biomedcentral.com / articles / 10.1186 / sl3045-020-00937-8, which is incorporated by reference in its entirety herein.

[0068] In some aspects, G9A inhibitors can be, but are not limited to, UNC0638, BIX-01294, Chaetocin, UNC0642, UNC0646, RK-701. A-366. or BRD4770

[0069] In some aspects, DNMT1 inhibitors can be, but are not limited to, GSK-3484862, Theaflavin, Glyburide and panobinostat, Aza-T-dCyd, SGI-1027, MC3343, Nanaomycin A, Hydralazine, and Procaine.

[0070] In some aspects, a ROR1 targeting therapeutic can be an immunotherapy. In some aspects, the immunotherapy targets the ROR1, which has increased expression due to the EZH2 inhibitor, G9A inhibitor, or DNMT1 inhibitor or combination thereof. In some aspects, the ROR1 targeting therapeutic targets ROR1 signaling, directly or indirectly. In some aspects, the immunotherapy can be, but is not limited to, a RORl-specific immunotoxin, a ROR1 antibody or a ROR1 chimeric antigen receptor T cell (CART cell). In some aspects, the ROR1 antibody can be, but is not limited to, Zilovertamab Vedotin (UC-961), 2A2, 2D11, 1A1, 1A7, R12, XBR1-402, clone F, 4A5, or R11.

[0071] In some aspects, an ROR1 antibody can be conjugated to a cytotoxic agent. In some aspects, a cytotoxic agent can be. but is not limited to, tubulin inhibitors (an antimicrotubule cytotoxic agent (e g. monomethyl auristatin E (MMAE)), maytansinoids, tubulysins) or pseudomonas toxin, Saporin, DNA damaging agents (calicheamicins, duocarmycins, exatecans). In some aspects, the cytotoxic agent is a toxin.

[0072] In some aspects, the ROR1 CAR T cell can be one or more of those listed in Osorio- Rodriguez et al. Front. Med., 17 February 2023 Sec. Gene and Cell Therapy, Volume 10 - 2023, which is hereby incorporated by reference in its entirety for its teaching og ROR1 CAR T cells. In some aspects, the ROR1 CAR T cell is LYL797.

[0073] In some aspects, the RORl-specific immunotoxin can be, but is not limited to, HuXBRl-402, VelosBiolOl. or LCB71 / ABL202. In some aspects, the ROR-1 specific immunotoxin can be one or more of those listed in Peng, Antibody Therapeutics, Volume 4, Issue 4, Oct 2021, pages 222-227, incorporated by reference in its entirety herein.

[0074] In some aspects, the disclosed methods further comprise ceasing the administration of the EZH2 inhibitor, G9A inhibitor. DNMT1 inhibitor or combination thereof, wherein ROR1 expression decreases in the cell after administration of the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof is ceased. In some aspects, the disclosed methods further comprise ceasing the administration of the EZH2 inhibitor, wherein ROR1 expression ismaintained in the cell after administration of the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof is ceased. In some aspects, in either scenario, the R0R1 no longer has an increase in expression once administration of the EZH2 inhibitor, G A inhibitor, DNMT1 inhibitor or combination thereof is ceased.

[0075] In some aspects, the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof is administered in a therapeutically effective amount. In some aspects, the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof can be administered as a single dose. In some aspects, the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof can be administered in multiple doses. In some aspects, the stability of the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof determines the dosing regimen. In some aspects the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof can be administered once daily for 1, 2, 3, 4, 5, 6, 7, 10, 14, 20, 30 or any number of days in between.

[0076] In some aspects, the B cell does not express ROR1 prior to administering the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof. In some aspects, the B cell is a ROR1 expressing cell prior to administering the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof. In some aspects, the B cell expresses lower levels of ROR1 prior to administering the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof compared to levels after receiving EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof, respectively.

[0077] In some aspects, two or more of EZH2 inhibitors, G9A inhibitors, or DNMT1 inhibitors can be administered together (in a single or separate compositions) in the disclosed methods of treating.C. Compositions

[0078] In some instances, the compositions can further comprise a pharmaceutically acceptable carrier. For example, disclosed are compositions comprising a EZH2 inhibitor, G9A inhibitor. DNMT 1 inhibitor or combination thereof and a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well know n to one of skill in the art. Examples of carriers include dimyristoylphosphatidyl (DMPC), phosphate buffered saline or a multi vesicular liposome. For example, PG:PC:Cholesterol:peptide or PCpeptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company,Easton, PA 1995. Typically, an appropriate amount of pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically- acceptable carrier include, but are not limited to, saline, Ringer’s solution and dextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0079] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, nucleic acid, vector of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0080] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0081] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0082] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions maypotentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and ary l amines and substituted ethanolamines.

[0083] The disclosed peptides can be formulated and / or administered in or with a pharmaceutically acceptable carrier. As used herein, the term '■pharmaceutically acceptable earner” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug (e.g. peptide) in biodegradable polymers such as polylactidepolyglycolide, poly (orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable mediajust prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0084] Thus, the compositions disclosed herein can comprise lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a peptide and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory tract to target cells of the respiratory tract. For example, a composition comprising a peptide or nucleic acid sequence described herein and a cationic liposome can be administered to a subject's lung cells. Regarding liposomes, see, e g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95 100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413 7417 (1987); U.S. Patent No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0085] In some instances, disclosed are pharmaceutical compositions comprising any of the disclosed peptides described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, buffer, or diluent. In various aspects, the peptide of the pharmaceutical composition is encapsulated in a delivery vehicle. In a further aspect, the delivery vehicle is a liposome, a microcapsule, or a nanoparticle. In a still further aspect, the delivery' vehicle is PEG-ylated.

[0086] In the methods described herein, delivery of the compositions to cells can be via a variety of mechanisms. As defined above, disclosed herein are compositions comprising any one or more of the peptides described herein and can also include a carrier such as a pharmaceutically acceptable carrier. For example, disclosed are pharmaceutical compositions, comprising the peptides disclosed herein, and a pharmaceutically acceptable carrier. In one aspect, disclosed are pharmaceutical compositions comprising the disclosed peptides. That is, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed peptide or at least one product of a disclosed method and a pharmaceutically acceptable carrier.

[0087] In certain aspects, the disclosed pharmaceutical compositions comprise the disclosed peptides (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for nasal, oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods wellknown in the art of pharmacy.

[0088] In practice, the peptides described herein, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and / or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and / or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0089] By ‘'pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. The peptides described herein, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.

[0090] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil. olive oil. and water. Examples of gaseous carriers include carbon dioxide and nitrogen. Other examples of carriers include dimyristoylphosphatidyl (DMPC), phosphate buffered saline or a multivesicular liposome. For example, PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro. Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically - acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically -acceptable carrier include, but are not limited to, saline, Ringer’s solution anddextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile w ater, saline, and buffered solutions at physiological pH.

[0091] In order to enhance the solubility and / or the stability of the disclosed peptides in pharmaceutical compositions, it can be advantageous to employ a-, |3- or y-cyclodextrins or their derivatives, in particular hydroxyalkyl substituted cyclodextrins, e.g. 2-hydroxypropyl-P- cyclodextrin or sulfobutyl-P-cyclodextrin. Also, co-solvents such as alcohols may improve the solubility and / or the stability of the compounds according to the invention in pharmaceutical compositions.

[0092] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended acti vity of the polypeptide, peptide, nucleic acid, vector of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical composition may be administered in a number of w ays depending on whether local or systemic treatment is desired, and on the area to be treated.

[0093] Because of the ease in administration, oral administration can be used, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystal line cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.

[0094] Compositions for oral administration include pow ders or granules, suspensions orsolutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0095] A tablet containing the compositions of the present invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0096] The pharmaceutical compositions of the present invention comprise a disclosed peptide(or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0097] Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as. for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental grow th of microorganisms.

[0098] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. Typically, the final injectable form should be sterile and should be effectively fluid for easy syringability . The pharmaceutical compositions should be stable under the conditions ofmanufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0099] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations.

[0100] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as. for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0101] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.

[0102] In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and / or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and / or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot on, as an ointment.

[0103] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dosesuppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0104] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be desirable.

[0105] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a disclosed peptide, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0106] The exact dosage and frequency of administration depends on the particular disclosed peptide, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be low ered or increased depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the compositions.

[0107] Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05 to 99 % by w eight, preferably from 0. 1 to 70 % by w eight, more preferably from 0.1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.D. Administration

[0108] The disclosed methods can include one or more of the types of administration disclosed herein.

[0109] In the methods described herein, administration or delivery of the therapeutics to a subject can be via a variety of mechanisms. For example, the EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof or the ROR1 targeting therapeutic can be formulated asa pharmaceutical composition.

[0110] Pharmaceutical compositions can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0111] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0112] Formulations for optical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0113] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions can be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyd and ary l amines and substituted ethanolamines.E. Kits

[0114] The compositions and materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits comprising an EZH2 inhibitor, G9A inhibitor, DNMT1 inhibitor or combination thereof and ROR1 targeting therapeutics.ExamplesA. Example 1

[0115] The current study shows a short-course treatment of lymphoma cell lines with the EZH2 inhibitor tazemetostat can either induce or further increase ROR1 expression. A combination of EZH2 inhibition and CAR T cell treatment may thus allow either improved efficacy or a broadening of the patient population that are eligible to receive ROR1 -targeted CAR T cells.

[0116] This study is focused on the impact of EZH2 inhibition on ROR1 expression in a wider set of heme and solid tumor cell lines in vitro, as well as healthy tissues in mice. Further, the experiments will study the mechanism of ROR1 induction by EZH2 inhibition. Finally, the study can measure the impact of EZH2 inhibition on CAR T cell function against ROR1+ tumor cell lines in vitro and in vivo.

[0117] One aspect of the study explores lymphoma-specificity of ROR1 expression including the examination of potential ROR1 induction by EZH2 inhibition in normal immune cells. Flow cytometry of lymphoma patients can be used for baseline ROR1 expression and ROR1 induction on tumor cells and subsets of healthy immune cells present in the sample after tazemetostat treatment using multi-marker flow cytometiy analysis. Flow cytometry of 10 RORl -f- (nonlymphoma) cases can be used to examine subsets of immune cells (B lymphocytes, T lymphocytes, and monocytes) for baseline ROR1 expression. This study can also evaluate the potential induction of ROR1 expression on reactive immune cells. Activation of PBMC from 5-7 healthy individuals with mitogenes (PHA, ConA, PKW, and LPS) can be performed in the presence or absence of tazemetostat, followed by multi-marker flow cytometry to detect ROR1 expression with ROR1 -expressing lymphoma cells serving as positive control.

[0118] Another aspect of the study can evaluate ROR1 expression induction by EZH2 inhibitors in selected solid tumors and healthy tissues: breast, ovarian, and lung carcinoma. A total of ~15 cell lines of breast, ovarian, and lung carcinoma can be treated with EZH2 inhibitors and analyzed for induction / enhancement of ROR1 expression by flow cytometry. Additionally, mice can be treated with tazemetostat and tissues can be analyzed for changes in ROR1 expression. Flow cytometi ' of cell lines can be used for baseline ROR1 and induced ROR1 expression by multi-marker flow cytometi ' analysis. An in vivo assessment of ROR1 induction in NSG and B6 mice can be performed.

[0119] Another aspect of the study focuses on the mechanisms of ROR1 expression induction by EZH2 inhibitors in lymphoma and solid tumor cells. Conformation of EZH2 activity can be determined by detecting H3K27me3 loss at ROR1 gene promoter in up to 3 lymphoma and 3 solid tumor cell lines. Single-cell RNA Seq of 2-4 lymphoma and solid tumor cell lines with EZH2inhibitor-inducible or “enhance-able” R0R1 expression and 2-4 EZH2 inhibitor-non-inducible lymphoma and solid tumor cell lines can be used to identify cell signaling pathway- and transcription factor candidates involved in R0R1 expression regulation. In silico R0R1 gene promoter and putative enhancer analysis can be used for transcription factor candidates potentially directly involved in R0R1 gene expression regulation. CRISPR / Cas9 or shRNA depletion of transcription factor candidates regulating R0R1 gene expression can be studied.

[0120] Another aspect of the study is directed to the CART-ROR1-EZH2 inhibitor combination in therapeutic models of lymphoma. In vitro repeated-stimulation cytotoxicity studies can be performed using 4-5 lymphoma cell lines (2-3 with inducible ROR1 and 2-3 with “enhance-able” ROR1, and 1 cell line without enhancement of ROR1 expression) and using predetermined EZH2 inhibitor concentration and two types of CART-ROR1 : high anti-RORl and low anti-RORl. Mock T cells, EZH2 inhibitor drug vehicle (medium) and single arms (EZH2i or CART-ROR1 alone serving as controls). In vitro repeated-stimulation cytotoxicity studies can be performed using 3-4 solid tumor cell lines (at least 1 without enhancement of ROR1 expression) and using pre-determined EZH2 inhibitor concentration and various E:T ratios. Mock T cells, EZH2i drug vehicle (medium) and single arms (EZH2i or CART-ROR1 alone serving as controls). In vivo xenotransplant lymphoma model with NSG MHCI / II KO mice and up to 3 lymphoma cell lines can be used with 2-3 dose levels of CAR T cells; untransduced T cells, CAR T and tazemetostat alone serve as control groups. In vivo xenotransplant solid tumor model with NSG MHCI / II KO mice and up to 3 solid tumor cell lines can be used with 2-3 dose levels of CAR T cells; untransduced T cells, CAR T and tazemetostat alone sen e as control groups. In vivo syngeneic tumor model in B6 mice implanted with s.c. B16-hRORl or KP-hRORl cells with murine CAR T cells expressing mouse cross-reactive R1 1 CAR T cells can be used to determine potential for toxicity of ROR1 induction in healthy tissues.B. Example 2

[0121] EZH2 inhibitor induces expression of cell-surface receptor ROR1 making malignant cells amenable to therapy targeting ROR1. This ROR1 expression induction is achieved by exposing cultured cells to the optimal dose of EZH2 inhibitor, e g. EPZ-6438 (tazemetostat) at 2 pM, followed by testing the cells for ROR1 expression using RT-qPCR (for mRNA detection), flow cytometry, Western blotting and / or immunohistochemistry (for protein detection) using ROR1 -specific antibodies. Prospectively, this approach can be applied to animal models such as cancer-transplanted mice or cancer patients.

[0122] While ROR1 is being targeted already clinically by RORl-specific immunotoxins or CAR T-cell therapies, the number of patients amenable to RORf targeting may be substantiallyincreased by applying EZH2 inhibitor. The increase in R0R1 caused by the EZH2 inhibitor would then allow ROR1 -specific therapies to be used in patients that did not originally have increased R0R1.

[0123] The combination therapy of EZH2 inhibitor and ROR1 immunotoxin or CAR-ROR1 T cells can be much more effective than either immunotherapy alone. The combination can prevent, or reverse, ROR1 loss by malignant cells and, hence prevent / reverse development of resistance to immunotherapy.

[0124] The combination therapy of EZH2 inhibitor and ROR1 immunotoxin or CAR-ROR1 T cells can also benefit from the anti-lymphoma activity of the EZH2 inhibitor, which is already FDA approved for therapy of advanced follicular lymphoma.

[0125] Expression of ROR1 can be examined on both rnRNA and protein levels by several complementary methods including, but not limited to, RT-(q)PCR, flow cy tometry, Western blotting, and immunohistochemistry.

[0126] A similar approach to increase expression of ROR1 as described above for lymphoma, can be done with malignant populations of plasma cells and breast colonic, and ovarian cancers to determine if EZH2 inhibition results in increase of ROR1 expression.C. Example 3

[0127] FIG. 9 shows a different epigenetic modifier (inhibitor of G9A: the H3K9mel / 2 methyltrasferase) induces ROR1 expression. FIG. 9 indicates that ROR1 induction is not confined to inhibition of EZH2 (H3K27me3 methyltransferase) but involved also at least another one epigenetic modification of histone 3 (loss of H3K9 mono- and demethylation, in this case).

[0128] To determine if the induction / enhancement of ROR1 expression is solely mediated by inhibition of EZH2, i.e. suppression of trimethylation of lysine 27 on histone 3 (H3K27me3) or can be also mediated by other epigenetic modifications, MCL SP-49 cells were treated with an inhibitor UNC0642 (2 pM) of G9A, i.e. suppression of mono- and dimethylation of lysine 9 on histone 3 (H3K9mel / 2). As show n in Fig. 9, inhibition of G9A also augmented ROR1 expression, indicating that the ROR1 expression can be upregulated by perturbation of epigenetic modification of H3K27 but also H3K9.

[0129] FIG. 10 shows that in addition to H3 methylation on selected lysines such as 27 and 9, as addressed in Fig. 9, DNA methylation, in particular of the promoter regions, also plays an important role in gene silencing. The DNA methylation is mediated by enzymes from the DNA methyltransferase (DNMT) family, including DNMT1.

[0130] To determine if the induction / enhancement of ROR1 expression can also by impacted by inhibition of the DNA methylation, MCL RL and Granta cells were treated with a directDNMT1 inhibitor GSK.36 (1 pM). As shown in Fig. 10, inhibition of DNMT1 induced / enhanced ROR1 expression. This induction / enhancement was the most pronounced when DNMT1 inhibitor was combined with EZH2 inhibitor, indicating that the joined inhibition of histone 3 and DNA methylation may prove more effective than either inhibition alone.

[0131] Although this induction / enhancement seems less pronounced than when inhibitors of H3K methyltransferases (EZH2 and G9A) are used, it is still significant. Furthermore, DNMT1 inhibition shows an at least additive effect with EZH2 inhibition with regard to ROR1 expression induction / enhancement, indicating that combination of inhibitors of histone 3 modifiers and DNA methylation can be clinically very effective.

[0132] EZH2 inhibition enhances ROR1 expression in plasma cell myeloma / neoplasm (FIG.11). This finding indicates that induction of ROR1 expression by EZH2 inhibition is not limited to lymphoma cells but also applies to plasma cell malignancy. To explore if ROR1 expression can be induced / enhanced in malignant non-lymphoma cells, 4 plasma cell myeloma / neoplasm cell lines (ANBL-6, RPMI-822, U266, and KMS11) have been evaluated for expression of ROR1. As shown in FIG. 11A, KMS11 cell line moderately expressed ROR1 protein, as detected by Western blotting and flow cytometry. This protein expression correlated with moderate expression of ROR1 mRNA (blue arrow) identified in the Human Cancer Atlas study (performed by others). Treatment of KMS11 (FIG. 1 IB) with EZH2 or G9A inhibitor but not DNMT1 inhibitor markedly augmented ROR1 expression, indicating that ROR1 expression could be enhanced not only in lymphoma but also plasma cell myeloma / neoplasm by inhibitors of the histone 3 modifiers.

[0133] EZH2 inhibition enhances ROR1 expression in plasma cell myeloma / neoplasm (FIG.12). This finding indicates that induction of ROR1 expression by EZH2 inhibition is not limited to lymphoma cells but also applies to plasma cell malignancy.

[0134] To explore if ROR1 expression can be induced / enhanced in also in non-hematopietic malignant cells, the Human Cancer Atlas study data has been evaluated for ROR1 mRNA expression in ovarian carcinoma. Western blotting was used to examine two selected ovarian carcinoma cell lines (marked by arrows) for expression of ROR1 protein in the absence or presence of EZH2 inhibitor. As shown in FIG. 12, both cell lines expressed to some degree ROR1 protein at the baseline. Noteworthy, there was a marked augmentation of ROR1 expression by EZH2 inhibition (EZH2i) in the OVCAR3 cell line. ROR1 expression can be enhanced by inhibition of the histone 3 modification not only in lymphoma and plasma cell myeloma / neoplasm, but also carcinoma represented here by ovarian carcinoma.

[0135] Diverse levels of expression of ROR1 RNA identified by Human Cancer Atlas study in several solid tumors such as carcinomas of bladder, breast, and colorectal (FIG. 13).

[0136] Given that mRNA expression profiles in cancers of bladder, breast, and colorectal closely resemble the profile of mRNA expression in ovarian cancer and that EZH2 inhibition resulted in marked augmentation of ROR1 expression in the ovarian carcinoma OVCAR3 cells (Fig. 12), inhibitor mediated epigenetic alteration can also induce / enhance ROR1 expression in other carcinoma types.

[0137] These results (generated by others) combined with our data showing ROR1 expression induction / enhancement in lymphoma, plasma cell myeloma / neoplasm, and, above all, ovarian carcinoma, suggest that ROR1 should by inducible by epigenetic modifiers such as EZH2 inhibitors in the whole spectrum of cancers.

[0138] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:

1. A method of increasing R0R1 in a lymphoma cell comprising administering an EZH2 inhibitor to the lymphoma cell.

2. The method of claim 1, wherein the lymphoma cell is a B cell.

3. The method of claim 2, wherein the B cell lymphoma cell is mantle cell lymphoma cell.

4. The method of any one of claims 1-3, wherein the lymphoma cell is in a subject.

5. The method of any one of claims 1-4, wherein the EZH2 inhibitor is a small molecule, protein, or nucleic acid.

6. The method of any one of claims 1-5, wherein the EZH2 inhibitor is a S-adenosyl-L- homocysteine (SAH) hydrolase inhibitor [3-deazaneplanocin A (DZNep)], or a S- adenosyl-methionine-competitive inhibitor [EIl], stabilized alpha-helix of EZH2 (SAH-EZH2) [peptides], gambogenic acid (GNA) derivative [[GNA022]], long- coding RNA, small interfering RNA (siRNA) [ ZRANB1]7. The method of any one of claims 1-6, wherein the EZH2 inhibitor is tazemetostat (E7438 / EPZ6438), 3-deazaneplanocin A (DZNep), GSK126 (GSK2816126), EPZ005687, Ell, GSK343, GSK926, EPZ011989, CPI-1205, CPI-169, CPI-1205, ZLD1039, PF-06821497, UNC1999, (R)-OR-S1, (R)-OR-S2, DS-3201b, stabilized alpha-helix of EZH2 (SAH-EZH2), Astemizole, Wedelolactone, apomorphine hydrochloride, oxyphenbutazone, nifedipine, ergonovine maleate, AZD9291 (Osimertinib, TAGRISSO), MAK683 / EED226, GNA022, long non-coding RNA (IncRNA) ANCR, F-box and WD repeat domain-containing 7 (FBW7), ZRANB1,KAN0439834, KAN0441571C, DB03208. strictinin, ARI-1, NVG-111. Cirtuzumab- based CAR-T, JCAR-024, or VLS-101.

8. The method of any one of claims 1-7, wherein the EZH2 inhibitor is administered in combination with an immune therapy.

9. The method of any one of claims 1 -8, wherein the lymphoma cell does not express R0R1 prior to administering the EZH2 inhibitor.

10. The method of any one of claims 1-9, further comprising ceasing the administration of the EZH2 inhibitor, wherein ROR1 expression decreases in the cell after administration of the EZH2 inhibitor is ceased.

11. The method of any one of claims 1-10, further comprising ceasing the administration of the EZH2 inhibitor, wherein ROR1 expression is maintained in the cell after administration of the EZH2 inhibitor is ceased.

12. The method of any one of claims 1-11. further comprising contacting the cell with a ROR1 targeting therapeutic.

13. The method of any one of claims 1 -12, wherein the cell is a ROR1 expressing cell.

14. A method of treating lymphoma in a subject comprising administering to the subject: a) one or more EZH2 inhibitors, wherein the one or more EZH2 inhibitors increase expression of ROR1 in one or more B cells in the subject; and b) one or more receptor tyrosine kinase-like orphan receptor 1 (ROR1) targeting therapeutics.

15. The method of claim 14, wherein the ROR1 targeting therapeutic is a RORl-specific immunotoxin, an ROR1 antibody or a ROR1 chimeric antigen receptor T cell (CART cell).

16. The method of claim 15, wherein the R0R1 targeting therapeutic is an R0R1 antibody.

17. The method of claim 16, wherein the R0R1 antibody is Zilovertamab Vedotin (UC- 961), 2A2, 2D1 1, 1A1, 1A7, R12, XBR1-402, clone F, 4A5, or Rl l.

18. The method of any of claims 14 -17, wherein the wherein the R0R1 antibody is conjugated to a toxin.

19. The method of claim 18, wherein the toxin is an antimicrotubule cytotoxic agent.

20. The method of claim 19, wherein the ROR1 targeting therapeutic is a ROR1 chimeric antigen receptor T cell (CART cell).

21. The method of claim 20, wherein the ROR1 targeting therapeutic is a ROR1 -specific immunotoxin.

22. The method of claim 21, wherein the RORl-specific immunotoxin is HuXBRl-402, VelosBiolOl, or LCB71 / ABL202.

23. The method of any one of claims 14-22, wherein the lymphoma is a B cell lymphoma.

24. The method of claim 23, wherein the B cell lymphoma is mantle cell lymphoma.

25. The method of any one of claims 14-24, wherein the EZH2 inhibitor is a small molecule, protein, or nucleic acid.

26. The method of any one of claims 14-25, wherein the EZH2 inhibitor is a S-adenosyl- L-homocysteine (SAH) hydrolase inhibitor [3-deazaneplanocin A (DZNep)], or a S- adenosyl-methionine-competitive inhibitor [Ell], stabilized alpha-helix of EZH2 (SAH-EZH2) [peptides], gambogenic acid (GNA) derivative [[GNA022]], long- coding RNA, small interfering RNA (siRNA) [ ZRANB1],27. The method of any one of claims 14-26, wherein the EZH2 inhibitor is tazemetostat (E7438 / EPZ6438), 3-deazaneplanocin A (DZNep), GSK126 (GSK2816126), EPZ005687, EIl. GSK343. GSK926, EPZ011989. CPI-1205, CPI-169, CPI-1205, ZLD1039, PF-06821497. UNC1999. (R)-OR-S1, (R)-OR-S2, DS-3201b, stabilized alpha-helix of EZH2 (SAH-EZH2), Astemizole, Wedelolactone. apomorphine hydrochloride, oxyphenbutazone, nifedipine, ergonovine maleate, AZD9291 (Osimertinib, TAGRISSO), MAK683 / EED226, GNA022, long non-coding RNA (IncRNA) ANCR, F-box and WD repeat domain-containing 7 (FBW7), or ZRANB1.

28. The method of any one of claims 14-27, wherein the EZH2 inhibitor is administered in combination with an immune therapy.

29. The method of any one of claims 14-28, further comprising ceasing the administration of the EZH2 inhibitor, wherein R0R1 expression decreases in the cell after administration of the EZH2 inhibitor is ceased.

30. The method of any one of claims 14-29, further comprising ceasing the administration of the EZH2 inhibitor, wherein ROR1 expression is maintained in the cell after administration of the EZH2 inhibitor is ceased.

31. The method of any one of claims 14-30, wherein the B cells of the subject do not express ROR1 prior to administering the EZH2 inhibitor.

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