MTA-cooperative PRMT5 inhibitors for use in the treatment of cancer - Patent Application 20070122999

Abstract

The present disclosure relates to a method for treating wild-type MTAP gene cancers, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof.

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 397,996, filed August 15, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] This document relates to a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has a tumor with silenced wild-type MTAP gene, i.e., a tumor that has wild-type MTAP gene but still accumulates methylthioadenosine (MTA).One of the cancer types that has been found to frequently exhibit this profile is Hodgkin's lymphoma (HL).This document also relates to a method for identifying cancer patients who will benefit from treatment with an MTA synergistic PRMT5 inhibitor. [Background technology]

[0003] Protein arginine methyltransferase 5 (PRMT5) is a member of the PRMT family of arginine methyltransferase enzymes that catalyzes the addition of methyl groups to guanidine motifs of arginine residues using S-adenosyl-L-methionine (SAM) as the methyl donor. PRMT5 is a type II arginine methyltransferase that symmetrically dimethylates the guanidine group of arginine residues, thus converting the guanidine NH2 group of arginine to an NMe2 group. PRMT5 methylates a large number of diverse substrates, including histone and non-histone proteins, and in so doing regulates processes such as RNA splicing, cell proliferation, and DNA repair. Importantly, PRMT5 is overexpressed in various cancer types and has been identified as a candidate for therapeutic intervention by the expression of small molecules that inhibit PRMT5 methyltransferase activity (see, for example, Non-Patent Document 1).

[0004] Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a tumor suppressor that is homozygously deleted in approximately 15% of cancers. Loss of the 9p21 chromosomal locus (where CDKN2A resides) results in the concomitant deletion of additional genes, including MTAP, which encodes methylthioadenosine phosphorylase (MTAP). MTAP is a metabolic enzyme involved in methionine salvage. Loss of MTAP results in increased concentrations of the MTAP substrate methylthioadenosine (MTA) in CDKN2A / MTAP-deficient cancer cells. MTA itself functions as a weak PRMT5 inhibitor, and therefore, MTA accumulation in CDKN2A / MTAP-deficient cancer cell lines leads to partial suppression of PRMT5 activity. Defective PRMT5 activity renders CDKN2A / MTAP-deficient cancer cells susceptible to further targeting of PRMT5, for example, using short hairpin RNA (shRNA). "Collateral vulnerabilities" in cancer have been identified, where CDKN2A / MTAP-deficient tumors can be selectively targeted by PRMT5 inhibition (see Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4).

[0005] Recently, reports have emerged of MTA-synergistic PRMT5 inhibitors, i.e., PRMT5 inhibitors that preferentially bind to PRMT5 in the presence of MTA (see, e.g., Patent Documents 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15). These MTA-synergistic PRMT5 inhibitors are designed to exploit the "collateral vulnerability" resulting from the CDKN2A / MTAP gene deletion described in the literature. Importantly, MTA-synergistic PRMT5 inhibitors exert greater inhibitory effects on PRMT5 in the presence of relatively high concentrations of MTA, such as those found in tumor cells lacking CDKN2A / MTAP, but do not exert their effects in healthy tissues where PRMT5 inhibition causes toxic side effects. Consequently, MTA-synergistic PRMT5 inhibitors should have a high therapeutic index (and low off-target toxicity) because their antiproliferative activity is selectively expressed in the MTA-rich environment targeted in CDKN2A / MTAP-deficient tumor cells.

[0006] Hodgkin's lymphoma (HL) is a type of B-cell lymphoma that accounts for approximately 15% of all lymphomas. The incidence of HL is low in the general population, at approximately 2-3 cases per 100,000 people of European descent (see, for example, Non-Patent Document 5), and it is one of the most common types of cancer in young adults. HL can also be seen in older adults, but less frequently.

[0007] Histopathologically, 90-95% of HL cases are classified as classical HL (cHL), and the remaining 5-10% are classified as nodular lymphocyte-predominant HL (NLPHL). There are four subtypes of cHL: nodular sclerosing (NSHL), mixed cell type (MCHL), lymphocyte-rich (LRHL), and lymphocyte-depleted (LDHL). LDHL is the most common subtype observed in all age groups.

[0008] HL is characterized by the presence of a small number of malignant cells surrounded by numerous immune effector cells in the tumor microenvironment. HL malignant cells are large mononuclear or multinuclear cells with a characteristic morphology and are derived from B cells. The malignant cells in cHL are called Hodgkin and Reed-Sternberg (HRS) cells, while those in NLPHL are called lymphocyte-predominant (LP) cells. HRS cells are characterized by CD30 expression. On the other hand, LP cells are CD30-negative but CD20-positive.

[0009] Malignant cells, either HRS or LP, account for only approximately 1% of the HL tumor cell composition. The remainder of the HL tumor is primarily composed of noncancerous immune and stromal cells. The low frequency of malignant cells in HL tumors makes analyzing genomic alterations in HRS and LP cells extremely challenging. Recently, methods such as laser capture microdissection and fluorescence-associated cell sorting have enabled efficient enrichment and genomic analysis of HRS and LP cells by several research groups. Additionally, methods based on ctDNA capture have also been successfully used to analyze the HL genomic landscape.

[0010] First-line treatment for HL involves radiation therapy and multiagent chemotherapy, which has a high probability of cure. For relapsed or refractory (R / R) disease, high-dose chemotherapy and autologous hematopoietic stem cell transplantation are commonly used. Immunotherapeutic approaches using immune checkpoint inhibitors and antibody-drug conjugates have shown promising results in the treatment of R / R HL patients. Nevertheless, alternative and improved approaches for the treatment of HL are needed. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2022 / 026892A1 Brochure [Patent Document 2] International Publication No. 2022 / 115377 Brochure [Patent Document 3] International Publication No. 2021 / 163344 Brochure [Patent Document 4] International Publication No. 2021 / 050915 Brochure [Patent Document 5] International Publication No. 2022 / 192745 Brochure [Patent Document 6] International Publication No. 2023 / 278564 Brochure [Patent Document 7] International Publication No. 2022 / 132914 Brochure [Patent Document 8] International Publication No. 2022 / 14619948 Brochure [Patent Document 9] International Publication No. 2023 / 036974 Brochure [Patent Document 10] International Publication No. 2023 / 081367 Brochure [Patent Document 11] CN202310191381 specification [Patent Document 12] CN116462676 specification [Patent Document 13] CN116462677 specification [Patent Document 14] International Publication No. 2023 / 098439 Brochure [Patent Document 15] International Publication No. 2021 / 086879 Brochure [Non-patent literature]

[0012] [Non-Patent Document 1] Kim et al.,(2020)Cell Stress 4(8)199-2151 [Non-patent document 2] Marjon et al.,(2016)Cell Reports 15, 574-587 [Non-patent document 3] Mavrakis et al.,(2016)Science 11;351(6278):1208-13 [Non-patent document 4] Kryukov et al.,(2016)Science 11;351(6278):1214-8 [Non-Patent Document 5] JMConnors et al,Nature Rev Disease Primers,6,Art.:61(2020) Summary of the Invention [Means for solving the problem]

[0013] As described herein, it has been discovered that in certain tumor types that express wild-type MTAP gene and have not lost the MTAP gene, hypermethylation at or around the MTAP gene can reduce or eliminate MTAP mRNA levels, resulting in the accumulation of MTA in the tumor due to the absence of MTAP protein. This has revealed a previously unrealized opportunity to treat patients with certain MTAP wild-type tumors. Therefore, it is an object of the present specification to provide a new approach to treating tumors that have a wild-type MTAP gene but nonetheless accumulate MTA. Furthermore, the present specification also provides a method for identifying a patient eligible for treatment with an MTA synergistic PRMT5 inhibitor, the method comprising the step of identifying, from a sample obtained from the patient, that the patient has a tumor that has a wild-type MTAP gene but nonetheless accumulates MTA in tumor cells.

[0014] According to a first aspect of the present specification, a treatment method is provided, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor in which the wild-type MTAP gene is silenced. A tumor in which the wild-type MTAP gene is silenced is a tumor that has an intact wild-type MTAP gene but nevertheless has reduced or no expression of MTAP mRNA or MTAP protein. As a result of the silencing of the wild-type MTAP gene, such tumor cells have reduced or no ability to phosphorylate and accumulate MTA. The MTA synergistic PRMT5 inhibitor binds to and inhibits PRMT5 in cooperation with MTA, which is enhanced in an MTA-rich environment, presenting a new opportunity for "collateral vulnerability."

[0015] In a further aspect herein, there is provided a method of treatment comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor that has a wild-type MTAP gene and characteristically accumulates MTA.

[0016] In a further aspect herein, there is provided a method of treatment comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor that has a wild-type MTAP gene and characteristically accumulates MTA due to silencing of the MTAP gene.

[0017] In a further aspect herein, a method of treatment is provided comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor that has a wild-type MTAP gene and characteristically accumulates due to MTAP gene silencing mediated by MTAP hypermethylation.

[0018] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has a tumor that has a wild-type MTAP gene but accumulates MTA due to downregulation of MTAP at the protein level.

[0019] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has a tumor that has a wild-type MTAP gene but accumulates MTA due to down-regulation of MTAP protein expression.

[0020] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor retains a wild-type MTAP gene but characteristically accumulates MTA due to epigenetic downregulation of MTAP mRNA.

[0021] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor retains a wild-type MTAP gene but accumulates MTA due to partial or complete silencing of MTAP protein expression.

[0022] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor retains a wild-type MTAP gene but accumulates MTA due to partial or complete silencing of MTAP protein expression by epigenetic modification of the MTAP gene.

[0023] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor retains a wild-type MTAP gene but accumulates MTA due to partial or complete silencing of MTAP protein expression by hypermethylation of the MTAP gene.

[0024] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor retains a wild-type MTAP gene but accumulates MTA due to downregulation of MTAP mRNA.

[0025] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor retains a wild-type MTAP gene but accumulates MTA due to downregulation of MTAP mRNA caused by hypermethylation at or around the MTAP gene.

[0026] In a further aspect, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor retains a wild-type MTAP gene but accumulates MTA due to downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or nearby genes such as CDKN2A, or other genomic locations.

[0027] In a further aspect, the present specification provides a method for identifying a patient who will benefit from treatment with an MTA synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient to confirm that the tumor has a wild-type MTAP gene but is nevertheless prone to accumulating MTA, optionally by performing an immunohistochemistry assay that shows that the relevant cell population is deficient in MTAP protein.

[0028] In the above embodiments, accumulation of MTA may be present in both the nucleus and cytoplasm of the relevant cells, or may be localized to the nucleus of the relevant cells.

[0029] In the above embodiment, the accumulation of MTA can be determined by performing immunochemical staining of MTAP in a sample obtained from the patient.

[0030] In a further aspect, the present specification provides a method for identifying a patient who will benefit from treatment with an MTA synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient and identifying relevant tumor cells as having wild-type MTAP silenced.

[0031] In a further aspect, the specification provides a method for identifying a patient who will benefit from treatment with an MTA synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient and identifying relevant tumor cells as having reduced levels of MTAP protein or mRNA expression, optionally as determined by an immunohistochemistry assay.

[0032] In a further aspect, the present specification provides a method for identifying a patient who will benefit from treatment with an MTA synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient and confirming that the tumor i) has a wild-type MTAP gene and ii) is MTAP-deficient or MTAP-null.

[0033] In a further aspect, the present specification provides a method for identifying a patient who will benefit from treatment with an MTA synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient and confirming that the tumor i) has a wild-type MTAP gene and ii) is MTAP mRNA null or defective.

[0034] In a further aspect, the present specification provides a method for identifying a patient who will benefit from treatment with an MTA synergistic PRMT5 inhibitor, the method comprising performing immunohistochemistry for MTAP on a tumor sample obtained from the patient and identifying relevant tumor cells as MTAP null or deficient.

[0035] In a further aspect, the present specification provides a method for treating cancer, comprising the steps of: i) identifying a patient as having a tumor that accumulates MTA, as determined by performing an immunohistochemical assay for MTAP; and ii) administering to the patient an MTA synergistic PRMT5 inhibitor.

[0036] In a further aspect, the present specification provides an MTA synergistic PRMT5 inhibitor for use in treating cancer, wherein the cancer has a wild-type MTAP gene and accumulates MTA.

[0037] In a further aspect, the present disclosure provides an MTA synergistic PRMT5 inhibitor for use in the treatment of cancer, wherein the tumor has a wild-type MTAP gene, yet is MTAP null or defective at the protein level.

[0038] In a further aspect, the present specification provides a kit comprising an MTA synergistic PRMT5 inhibitor and instructions for its use in treating cancers that have a wild-type MTAP gene but are nevertheless MTAP null or defective at the protein level.

[0039] In order that this specification may be more readily understood, reference is made to the following figures: [Brief explanation of the drawings]

[0040] [Figure 1]Graph of MTAP mRNA expression and MTAP copy number in tumor cells in the Cancer Cell Line Encyclopedia (CCLE, https: / / sites.broadinstitute.org / ccle / ). Figure 1A shows an overall plot of tumor cells in CCLE. Samples within the boxed area have at least one copy of the wild-type MTAP gene. Figure 1B shows the region of the plot in Figure 1A that includes cell lines that have the wild-type MTAP gene but have reduced MTAP gene expression, as reflected by low MTAP mRNA expression. Figure 1C shows a plot of the seven Hodgkin's lymphoma cell lines shown in Figure 1A. [Figure 2] Figure 2A shows a plot of MTAP DNA methylation (y-axis) versus MTAP mRNA expression in seven HL cell lines during CCLE. Figure 2B shows methylation across the transcriptional site of the MTAP gene. The x-axis shows the chromosomal location of the reduced-representation bisulfite sequencing (RRBS) promoter methylation CpG clusters in the MTAP promoter region (data obtained from the Broad Institute at https: / / data.broadinstitute.org / ccle / ). Figure 2C shows Western blots of MTAP and GAPDH in four HL cell lines (L540, L1236, KMH2, and HDLM2) that have wild-type MTAP genes but are gene-silenced, as well as the HL cell line L428 in which MTAP is not silenced. The HCT116 colorectal cell line is included as a positive (MTAP wild-type) and negative (MTAP KO) control. [Figure 3] Plot showing 84% of HL histopathology tumor samples stained for nuclear and cytoplasmic MTAP protein. Of the 46 HL samples lacking nuclear MTAP, 14 also lacked MTAP in the cytoplasm, 27 had faint MTAP staining in the cytoplasm, and 5 had MTAP staining above +1 in the cytoplasm. [Figure 4]Histopathology slides of normal tonsil tissue (Figure 4A) and NSCLC (Figure 4B). In Figure 4A, MTAP staining is seen throughout the cells. In contrast, in Figure 4B, MTAP staining is restricted to tumor-infiltrating lymphocytes (right, dark staining area), while NSCLC cells extending from the upper left corner to the lower right corner of the image lack MTAP staining. [Figure 5] Histopathology image taken from Kueppers, R. and Hansmann, M.-L., Int J Biochem & Cell Biol., 37(3), 2005 p 511-17, showing CD30-stained Hodgkin-Reed / Sternberg (HRS) cells, a tumor clone characteristic of Hodgkin lymphoma, among a large population of lymphoma cells. [Figure 6] Histopathological images of sample number 243969-LN-1 (HL subtype: MC interfollicular) obtained with MTAP antibody and casein-containing diluent (Figure 6A, 2 μg / mL mAb) or standard diluent without casein (Figure 6B, 0.5 μg / mL). HRS cells lack MTAP staining in both the nuclear and cytoplasmic compartments. [Figure 7] Histopathological images of sample number 243957-LN-1 (HL subtype: NS) obtained with MTAP antibody and casein-containing diluent (Figure 7A, 2 μg / mL mAb) or casein-free standard diluent (Figure 7B, 0.5 μg / mL). Nuclear staining is completely absent in HRS cells, with slight cytoplasmic staining of MTAP observed. [Figure 8] Histopathological images of sample number 243958-LN-1 (HL subtype: NS syncytial) obtained with MTAP antibody and casein-containing diluent (Figure 8A, 2 μg / mL mAb) or casein-free standard diluent (Figure 8B, 0.5 μg / mL). Nuclear staining is completely absent in HRS cells, with slight cytoplasmic staining of MTAP observed. [Figure 9]Histopathological images of sample number 243965-LN-1 (HL subtype: MC) obtained with MTAP antibody and casein-containing diluent (Figure 9A, 2 μg / mL mAb) or casein-free standard diluent (Figure 9B, 0.5 μg / mL). Nuclear staining is completely absent in HRS cells, with only slight cytoplasmic staining of MTAP observed. [Figure 10] Histopathology images obtained for sample number 243970-LN-1 (HL subtype: NS syncytial). Here, HRS cells are easily identified as "bright" areas, designated as having 1+ cytoplasmic staining with no nuclear staining present in HRS cells. Staining was performed with the MTAP antibody and casein-containing diluent (Figure 10A, 2 μg / mL mAb) or the standard diluent without casein (Figure 10B, 0.5 μg / mL). [Figure 11] Histopathology images obtained for sample number 243963-LN-1 (HL subtype: LRHL). Here, HRS cells are easily identified as "bright" areas, designated as having 1+ cytoplasmic staining with no nuclear staining present in HRS cells. Staining was performed with the MTAP antibody and a casein-containing diluent (Figure 11A, 2 μg / mL mAb) or a standard diluent without casein (Figure 11B, 0.5 μg / mL). [Figure 12] Histopathology images of sample number 243959-LN-1 (HL subtype: NS) show that HRS cells are stained for MTAP in the nucleus and cytoplasm of HRS cells. Staining was performed with MTAP antibody and casein-containing diluent (Figure 12A, 2 μg / mL mAb) or casein-free standard diluent (Figure 12B, 0.5 μg / mL). [Figure 13] Histopathology images of sample number 243961-LN-1 (HL subtype: NLPHL) show MTAP staining in the nuclei and cytoplasm of lymphocyte-predominant (LP) cells. Staining was performed with MTAP antibody and casein-containing diluent (Figure 13A, 2 μg / mL mAb) or casein-free standard diluent (Figure 13B, 0.5 μg / mL). [Figure 14]Plot showing the effect of treatment with Compound C on relative tumor volume in the L540HL xenograft model. [Figure 15] Plot showing the effect of treatment with Compound C on mouse body weight in the L540HL xenograft model. [Figure 16] Plot showing the effect of treatment with Compound C on SDMA protein levels in the L540HL xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0041] The opportunity to apply MTA-synergic PRMT5 inhibitors to treat tumors without CDKN2A / MTAP gene deletion arose from the novel observation that certain tumors possess wild-type MTAP genes but nonetheless accumulate MTA due to complete or partial silencing of the MTAP gene. Based on the studies described herein, this particular phenotype appears to be due to hypermethylation of or around the MTAP gene in certain tumors, resulting in significant or complete silencing of the MTAP gene or downregulation of MTAP gene expression, leading to reduced intratumoral concentrations of MTAP protein. Functionally, hypermethylation of or around the MTAP gene reduces MTAP gene expression, resulting in low or eliminated MTAP mRNA levels, i.e., silencing the wild-type MTAP gene. This results in reduced or absent MTAP protein in tumor cells. MTAP is an enzyme that plays a key role in polyamine metabolism and is important for the salvage of both adenine and methionine. In the context of cancer treatment strategies, the absence of the MTAP protein eliminates the clearance mechanism for methylthioadenosine (MTA), leading to the accumulation of MTA. Tumor cells or tumors containing relevant cell populations that have wild-type MTAP genes but accumulate MTA are identified herein as tractable targets for treatment with MTA-synergistic PRMT5 inhibitors (PRMT5 inhibitors that bind to PRMT5 in combination with MTA). More specifically, MTA-synergistic PRMT5 inhibitors exhibit optimal activity only in cells with high MTA concentrations, thereby utilizing a selective cytotoxic effect that avoids or significantly reduces the off-target toxicity associated with non-MTA-selective PRMT5 inhibitors observed in clinical settings.

[0042] The realization of the opportunity to selectively target specific tumors with wild-type MTAP genes with MTA-synergic PRMT5 inhibitors arose from an analysis of the Cancer Cell Line Encyclopaedia (CCLE, https: / / sites.broadinstitute.org / ccle / ). More specifically, a search was conducted in the CCLE for tumor cell lines with low MTAP mRNA levels. This characteristic, prior to the studies described herein, had been associated with cells with homozygous deletion of CDKN2A / MTAP. Therefore, MTAP mRNA levels (confirmed by RNA sequencing) were plotted against MTAP (gene) copy number (see Figure 1A). As expected, the search revealed a cluster of MTAP-deficient cell lines in the lower left corner of Figure 1A, which showed significantly reduced MTAP mRNA compared with cells with wild-type MTAP (cells with a log2-1 MTAP copy number of -1), clustered in the upper right corner of Figure 1A. (Note: The copy number scale on the x-axis in Figures 1A, 1B, and 1C is the log2-1 of copy number; therefore, cells with a log2-1 copy number of -1 or greater express at least one copy of wild-type MTAP, while cells with a log2-1 copy number of -2 or less are MTAP-null, i.e., do not express MTAP.) Unexpectedly, a group of cells within CCLE was found to have wild-type MTAP and, therefore, appears on the right side of the plot, yet nevertheless exhibits MTAP mRNA levels comparable to tumor cells with CDKN2A / MTAP deletion (see the cluster of cells in the lower right corner within the boxed region in Figure 1A (MTAP copy number ≥ -1, MTAP mRNA ≤ 0)). Cells that have a wild-type MTAP gene but nevertheless have reduced expression of MTAP mRNA are referred to as cells in which the wild-type MTAP gene has been silenced, and tumors containing clonal tumor cells of this phenotype are referred to herein as tumors in which the wild-type MTAP gene has been silenced.

[0043] Figure 1B shows a magnified view of the population of MTAP-silenced tumor cells, i.e., tumor cells that retain the wild-type MTAP gene and have low or no MTAP mRNA levels. Table 1 provides a table correlating the types of MTAP-silenced tumor cells with the total number of models classified by tissue of origin or tumor type, and the prevalence of the MTAP-silenced phenotype per tissue of origin. As can be seen from inspection of Figure 1B and Table 1, 23 tumor cell lines from CCLE were identified as possessing the wild-type MTAP gene and MTAP-silenced. While many of the tumor cells in this panel were outliers in terms of prevalence for their tissue of origin or tumor type, the majority of Hodgkin lymphoma (4 / 7) and non-Hodgkin lymphoma (5 / 27) tumors exhibited this MTAP-silenced profile. Data for all seven CCLE Hodgkin lymphoma cell lines are shown in Figure 1C.

[0044] [Table 1]

[0045] This observation prompted further investigation into the origins of MTAP gene silencing, with the aim of determining whether the opportunity for “collateral vulnerability” presented by the availability of MTA-synergistic PRMT5 inhibitors could be exploited beyond tumors lacking CDKN2a / MTAP.

[0046] To understand the origin of MTAP gene silencing in tumor cell lines with wild-type MTAP, we looked for unifying features present across 4 / 7 HL cell lines in which the MTAP gene was silenced, compared with Hodgkin lymphoma cell lines with wild-type MTAP but without the remaining three MTAP genes silenced. Four of the seven gene-silenced HL cell lines, namely HDLM2, L540, KMH2, and L1236 (see Figure 2A), were also methylated throughout the MTAP gene transcription site (Figure 2B), confirming that the MTAP gene was hypermethylated. In contrast, the three HL cell lines with unmethylated MTAP expressed normal levels of MTAP mRNA and therefore should express MTAP at the protein level. Western blot analysis of MTAP protein is shown in Figure 2C, which confirms that MTAP protein is present only in the L428 cell line, which expresses MTAP mRNA, while the HDLM2, L540, KMH2, and L1236 cell lines, in which the MTAP gene is hypermethylated, are MTAP protein null. Thus, the data suggest that MTAP gene hypermethylation causes MTAP gene silencing in many tumors.

[0047] After confirming the likely epigenetic origin of MTAP gene silencing in HL cell lines, experiments were performed to confirm that an MTA-synergistic PRMT5 inhibitor could inhibit the proliferation of MTAP gene-silenced HL cell lines and accumulate MTA accordingly. Therefore, the activity of the MTA synergistic PRMT5 inhibitors Compound A, (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione, and Compound C, (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1'-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3'-pyrrolidine]-2',3-dione, was evaluated for their ability to inhibit the growth of HDLM2, L540, and L1236 (all MTAP genes silenced), and L428 (intact MTAP expression), as well as wild-type and MTAP knockout HCT116 cells. The activity of Compound B (GSK3326595, described in WO 2015 / 198229 A1, available commercially from medchemexpress.com under catalog number HY-101563), a non-selective PRMT5 inhibitor, was evaluated in parallel against the same cells. The results from these experiments are shown in Table 2.

[0048] [Table 2]

[0049] As can be seen in Table 2, all cell lines are sensitive to treatment with the nonselective PRMT5 inhibitor Compound B. In contrast, the MTA synergistic PRMT5 inhibitor Compound A primarily exhibits activity in cell lines with silenced MTAP genes (HDLM2, L540, and L1236) and the MTAP knockout HCT116 cell line, which has low or absent MTAP mRNA levels and accumulates MTA due to the absence or reduced levels of MTAP protein. Similarly, Compound C exhibits preferential activity in the same cell lines as Compound A, i.e., cell lines that accumulate MTA due to the absence or reduced levels of MTAP protein. Thus, the potential for targeting tumors that have wild-type MTAP genes but nonetheless accumulate MTA with MTA synergistic PRMT5 inhibitors has been demonstrated in vitro.

[0050] To investigate whether the prevalence of the MTAP wild-type / MTAP-silenced phenotype in CCLE is representative of clinical HL, we obtained a sample set of Hodgkin lymphoma and attempted to assess whether the majority of clinical Hodgkin lymphoma samples lack MTAP protein expression, resulting in MTAP accumulation. To perform this analysis, an immunohistochemistry (IHC) approach to detect MTAP at the protein level was developed and applied to the analysis of clinical samples. The results from these experiments are summarized in Figure 3 and Tables 3a and 3b.

[0051] [Table 3]

[0052] [Table 4]

[0053] [Table 5]

[0054] As seen in Tables 3a and 3b, IHC analysis revealed loss of nuclear MTAP in 46 of 55 primary HL samples (84%). In other words, as judged by expert histopathologists, MTAP protein was completely absent from the nuclei of Hodgkin and Reed / Sternberg cells in these 46 Hodgkin lymphoma samples. Interestingly, of the 46 samples in which MTAP was lost, 32 expressed some MTAP in the cytoplasm and were assigned a score of +1 or slight cytoplasmic MTAP. Based on this sample set, it is clear that MTAP expression is reduced in the majority of Hodgkin lymphomas, which correlates well with the data from the CCLE described above and shown in Figure 1.

[0055] To illustrate the IHC studies performed to obtain the above data, Figure 4 shows staining of a normal tonsil tissue sample with MTAP antibody (Figure 4A), which shows staining throughout the plate, while staining of an NSCLC sample (Figure 4B) shows darkly stained areas (due to tumor-infiltrating lymphocytes) and tumor cells that are virtually devoid of MTAP staining and show slightly brighter blue areas revealing the nuclei and cytoplasm in the color image.

[0056] To understand the significance of the histopathological images provided in Figure 5 and subsequent figures, it is important to first recall that Hodgkin lymphoma tissue consists of a relatively small number of clonal tumor cells within a broader population of normal cells (see above). More specifically, Hodgkin and Reed / Sternberg cells (hereafter referred to as HRS cells) are characteristic cells of Hodgkin lymphoma. They are often large, multinucleated cells with distinctive morphology and an abnormal immunophenotype, dissimilar to any normal cells in the body (see, e.g., Kueppers, R. and Hansmann, M.-L., Int J Biochem & Cell Biol., 37(3), 2005, pp. 511-17). Hodgkin cells are characteristically mononucleated, whereas Reed / Sternberg cells are multinucleated. While rare in HL tissue, HRS cells are the clonal tumor cells of HL. In almost all cases of HL, HRS cells are derived from B cells and, rarely, from T cells. In particular, the pattern of somatic mutations in rearranged immunoglobulin V genes suggests that they originate from preapoptotic germinal center B cells. Although the pathogenesis of HL remains largely unknown, it is now clear that the aberrant activation of several signaling pathways (e.g., the NFκB pathway) is crucial for the survival of HRS cells. HRS or HRS-like cells are also found in several other diseases, e.g., in some non-Hodgkin's lymphomas and infectious mononucleosis, where they are rarely found as intermixed cells. To demonstrate the prevalence of HRS cells in HL, a literature image of an HL sample stained for CD30 is shown in Figure 5A, and a magnified image is shown in Figure 5B.

[0057] For individual IHC analysis data, Figure 6 shows histopathological images obtained for sample number 243969-LN-1 (HL subtype: MC interfollicular) and MTAP antibody and casein-containing diluent (Figure 6A, 2 μg / mL mAb) or casein-free standard diluent (Figure 6B, 0.5 μg / mL). All IHC images referenced in Figure 6 and subsequent sections were acquired under the same conditions, i.e., using the MTAP antibody and casein-containing diluent (Figure Xa, X = 7-13) or casein-free standard diluent (Figure Xb) at the same concentrations. In this image, HRS cells are the bright areas of the stained histopathological slide, and IHC analysis reveals the complete absence of MTAP in the nuclear and cytoplasmic compartments of HRS cells.

[0058] Figure 7 shows data from sample number 243957-LN-1 (HL subtype: NS). Again, no nuclear staining of MTAP is observed in HRS nuclei, but slight MTAP staining is observed in the cytoplasmic compartment of HRS cells. Figure 8 (sample number 243958-LN-1, HL subtype: NS syncytial) and Figure 9 (sample number 243965-LN-1, HL subtype: MC) similarly show the complete absence of nuclear staining and slight cytoplasmic staining in HRS cells.

[0059] Figure 10 shows data for sample number 243970-LN-1 (HL subtype: NS syncytial). Here, HRS cells are easily identified as "bright" areas, designated as 1+ cytoplasmic staining with no nuclear staining present in HRS cells. Results for LRHL subtype sample number 243963-LN-1 (see Figure 11) similarly show 1+ cytoplasmic staining with no nuclear staining present in HRS cells.

[0060] In contrast, IHC slides of sample number 243959-LN-1, HL subtype: NS and sample number 243961-LN-1, HL subtype: NLPHL are shown in Figures 12 and 13, respectively, and in both cases MTAP staining was observed in HRS cells.

[0061] Thus, based on the 55 clinical samples analyzed, and consistent with observations from CCLE, we identified that MTA accumulates in the majority of Hodgkin lymphomas and that this MTA accumulation is due to MTAP gene silencing. Although loss of nuclear MTAP protein expression is highly common (seen in 46 of 55 HL samples), the observation that residual levels of MTAP protein expression are present in 32 of 46 samples that do not express MTAP in the nucleus clearly indicates MTAP gene silencing, rather than MTAP gene deletion. Data from the Human Protein Atlas confirm that MTAP protein expression is observed in both the cytoplasmic and nuclear compartments of most tissues (see https: / / www.proteinatlas.org / ENSG00000099810-MTAP / tissue). Nevertheless, Protein Subcellular Localization Prediction Tool (PSORTII) analysis did not reveal any nuclear localization signal for MTAP protein, predicting that MTAP protein is distributed proportionally in the cytoplasm (78.3%), nucleus (17.4%), and endoplasmic reticulum (4.3%) (see https: / / psort.hgc.jp / form2.html). Interpolation from this prediction suggests that when MTAP protein levels are low, as indicated by partial MTAP gene silencing, detection of MTAP protein in the cytoplasm may still be possible even if nuclear MTAP protein levels are reduced below the detection limit. In other words, if the % reduction of MTAP protein is uniform throughout the cell, it is more likely that nuclear MTAP protein levels will be below the detection limit compared to the cytoplasm.

[0062] The broader scientific literature reports that when MTAP protein is expressed in cells, depending on the expression level, MTAP protein is expected to be observed in both the cytoplasm and nucleus (if expression levels are high) or only in the cytoplasm (if expression levels are low), but not only in the nucleus.

[0063] In the context of the data reported herein, we conclude that nuclear loss of MTAP protein in HL samples detected by IHC is a strong indicator of global MTAP protein deficiency, leading to intracellular MTA accumulation, and can be used as a surrogate marker of sensitivity to MTA-synergistic PRMT5 inhibitors.

[0064] Thus, the IHC experiments reported herein confirmed that a significant proportion of HL clinical tumor samples are deficient in MTAP. Because these tumors inevitably accumulate high concentrations of MTA, and because MTA-synergic PRMT5 inhibitors are available, this new approach to treating HL, which characteristically accumulates MTA, is uncovered for the first time. This new "collateral vulnerability" opportunity may provide an effective option for treating HL with a characteristically favorable side effect profile, as significant targeting of PRMT5 in healthy MTAP protein-expressing cells is likely to be minimized. Furthermore, this opportunity may be equally applicable to other tumor types, selected from those tumor types in which MTAP gene silencing is present, e.g., those disclosed in Table 1, that characteristically retain the wild-type MTAP gene but nonetheless exhibit the MTAP gene silencing phenotype described here for the first time.

[0065] In vivo experiments were performed to evaluate tumor growth inhibition and pharmacodynamic changes following treatment with the MTA synergistic PRMT5 inhibitor Compound C in the MTAP-silenced L540HL xenograft model (see Biological Example 1).

[0066] As described above, in a first embodiment, the present specification provides a method of treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a cancer in which the wild-type MTAP gene is silenced.

[0067] In embodiments, MTAP gene silencing results in partial or complete loss of MTAP gene expression protein in relevant tumor cells, which can be established by immunohistochemical analysis or other suitable techniques, such as RT-qPCR, that allow for quantification of MTAP protein or mRNA in relevant cells or cell compartments.

[0068] In embodiments, MTAP gene silencing results in partial or complete loss of MTAP protein in the nucleus of the relevant tumor cells.

[0069] In embodiments, MTAP gene silencing results in partial or complete loss of MTAP mRNA in relevant tumor cells, which can be confirmed by RNA-Seq, in situ hybridization, or other suitable techniques.

[0070] In embodiments, MTAP gene silencing reduces MTAP protein expression in the nuclei of tumor cells. In embodiments, MTAP gene silencing leads to reduced MTAP protein expression in the nuclei of clonal tumor cells. In embodiments, the tumor cells are Hodgkin-Reed / Sternberg cells and the cancer is Hodgkin lymphoma.

[0071] In embodiments, a method of treating cancer is provided, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor that has a wild-type MTAP gene and characteristically accumulates MTA.

[0072] In embodiments, a method of treating cancer is provided, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor that has a wild-type MTAP gene and characteristically accumulates MTA due to silencing of the MTAP gene.

[0073] In embodiments, a method of treating cancer is provided, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor that has a wild-type MTAP gene and characteristically accumulates MTA in associated tumor cells due to MTAP gene silencing mediated by MTAP hypermethylation.

[0074] In embodiments, a method of treating cancer is provided, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor that has a wild-type MTAP gene and that characteristically accumulates MTA in associated tumor cells due to epigenetic modifications of the MTAP gene.

[0075] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has a tumor that has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to downregulation of MTAP at the protein level.

[0076] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has a tumor that has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to downregulation of MTAP protein expression.

[0077] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor has a wild-type MTAP gene but characteristically accumulates MTA in associated tumor cells due to epigenetic downregulation of MTAP mRNA.

[0078] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to partial or complete silencing of MTAP protein expression.

[0079] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to partial or complete silencing of MTAP protein expression by epigenetic modification of the MTAP gene.

[0080] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to partial or complete silencing of MTAP protein expression due to hypermethylation of the MTAP gene.

[0081] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to downregulation of MTAP mRNA.

[0082] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to downregulation of MTAP mRNA caused by hypermethylation of or around the MTAP gene.

[0083] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor has a wild-type MTAP gene but accumulates MTA in associated tumor cells due to downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or adjacent genes such as CDKN2A.

[0084] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, the method comprising analyzing a sample obtained from the patient to identify that the tumor has a wild-type MTAP gene but is nevertheless prone to accumulating MTA based on an immunohistochemistry assay showing that an associated tumor cell population is deficient in MTAP protein.

[0085] In embodiments, the present specification provides a method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein MTA accumulation may be present in both the nucleus and cytoplasm of associated tumor cells, or may be localized in the nucleus of associated cells.

[0086] In embodiments, accumulation of MTA may be present in both the nucleus and cytoplasm of the relevant cells, or may be localized or substantially localized in the nucleus of the relevant cells.

[0087] In embodiments, the identification of a tumor's propensity to accumulate MTA may be determined by performing immunochemical staining of MTAP in a sample obtained from the patient.

[0088] In embodiments, there is provided the use of an MTA synergistic PRMT5 inhibitor for the treatment of cancer in which the wild-type MTAP gene is silenced.

[0089] In embodiments, there is provided a use of an MTA synergistic PRMT5 inhibitor for the treatment of cancer in which the wild-type MTAP gene is silenced, wherein the MTAP gene silencing is (a) results in partial or complete loss of MTAP protein in the relevant tumor cells; or (b) results in partial or complete loss of MTAP protein in the nuclei of the involved tumor cells; or (c) leading to a decrease in MTAP protein expression in the nuclei of tumor cells; or (d) leading to a decrease in MTAP protein expression in the nuclei of clonal tumor cells; or (e) characteristically causes accumulation of MTA in associated tumor cells; or (f) MTAP gene silencing mediated by MTAP hypermethylation characteristically leads to the accumulation of MTA in the associated tumor cells; or (g) epigenetic modification of the MTAP gene characteristically leads to the accumulation of MTA in the associated tumor cells; or (h) downregulation of MTAP at the protein level, characteristically leading to accumulation of MTA in the associated tumor cells; or (i) downregulation of MTAP protein expression, characteristically leading to accumulation of MTA in the associated tumor cells; or (j) causes epigenetically driven downregulation of MTAP mRNA; or (k) partial or complete silencing of MTAP protein expression, leading to the accumulation of MTA in the associated tumor cells; or (l) epigenetic modification of the MTAP gene resulting in partial or complete silencing of MTAP protein expression, leading to the accumulation of MTA in the associated tumor cells; or (m) partial or complete silencing of MTAP protein expression due to hypermethylation of the MTAP gene, resulting in tumors that characteristically accumulate MTA; or (n) Downregulation of MTAP mRNA caused by hypermethylation of or around the MTAP gene, resulting in tumors that characteristically accumulate MTA; or (o) Downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or neighboring genes such as CDKN2A leads to tumors that characteristically accumulate MTA.

[0090] In embodiments, there is provided the use of an MTA synergistic PRMT5 inhibitor for the manufacture of a medicament for the treatment of a cancer characterized by silencing of the wild-type MTAP gene.

[0091] In embodiments, there is provided a use of an MTA synergistic PRMT5 inhibitor for the manufacture of a cancer in which the wild-type MTAP gene is silenced, wherein the MTAP gene silencing is (a) results in partial or complete loss of MTAP protein in the relevant tumor cells; or (b) results in partial or complete loss of MTAP protein in the nuclei of the involved tumor cells; or (c) leading to a decrease in MTAP protein expression in the nuclei of tumor cells; or (d) leading to a decrease in MTAP protein expression in the nuclei of clonal tumor cells; or (e) characteristically causes accumulation of MTA in associated tumor cells; or (f) MTAP gene silencing mediated by MTAP hypermethylation characteristically leads to the accumulation of MTA in the associated tumor cells; or (g) epigenetic modification of the MTAP gene characteristically leads to the accumulation of MTA in the associated tumor cells; or (h) downregulation of MTAP at the protein level, characteristically leading to accumulation of MTA in the associated tumor cells; or (i) downregulation of MTAP protein expression, characteristically leading to accumulation of MTA in the associated tumor cells; or (j) causes epigenetically driven downregulation of MTAP mRNA; or (k) partial or complete silencing of MTAP protein expression, leading to the accumulation of MTA in the associated tumor cells; or (l) epigenetic modification of the MTAP gene resulting in partial or complete silencing of MTAP protein expression, leading to the accumulation of MTA in the associated tumor cells; or (m) partial or complete silencing of MTAP protein expression due to hypermethylation of the MTAP gene, resulting in tumors that characteristically accumulate MTA; or (n) Downregulation of MTAP mRNA caused by hypermethylation of or around the MTAP gene, resulting in tumors that characteristically accumulate MTA; or (o) Downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or neighboring genes such as CDKN2A leads to tumors that characteristically accumulate MTA.

[0092] As used herein and above, the term "associated tumor cells" refers to cells that drive tumor growth and maintain tumor survival. In embodiments referring to "associated tumor cells," examples of associated tumor cells include Hodgkin-Reed / Sternberg cells found in Hodgkin lymphoma.

[0093] In embodiments, there is provided a method for identifying a patient who would benefit from treatment with an MTA synergistic PRMT5 inhibitor, comprising identifying relevant tumor cells in a sample obtained from the patient that exhibit reduced MTAP protein in their nuclei and optionally cytoplasm by performing an immunohistochemical assay of MTAP protein on the sample.

[0094] In embodiments, methods are provided for identifying patients with Hodgkin's lymphoma who would benefit from treatment with an MTA synergistic PRMT5 inhibitor, comprising analyzing MTAP expression in tumor samples obtained from the patient and identifying relevant tumor cells as having wild-type MTAP gene silencing. In such embodiments, identification of wild-type MTAP gene silencing status is achieved by immunohistochemistry, which demonstrates reduced or absent MTAP protein expression in the nuclei and / or cytoplasm of Hodgkin-Reed / Sternberg cells.

[0095] In embodiments of the above-described methods of treatment, the methods can also include analyzing a sample obtained from a patient with a cancer in which the wild-type MTAP gene is silenced. In such embodiments, determining whether wild-type MTAP gene silencing is present can be based on an immunochemical assay of MTAP protein, revealing reduced or absent expression in the nuclei and / or cytoplasm of relevant tumor cells, e.g., in the case of Hodgkin's lymphoma in Hodgkin-Reed / Sternberg cells.

[0096] In embodiments relating to the use of an MTA synergistic PRMT5 inhibitor for the treatment of cancer, use may be indicated based on results obtained from an analysis of a sample obtained from a patient in need of treatment indicating that the patient has a cancer in which the wild-type MTAP gene is silenced. In such embodiments, determination of wild-type MTAP gene silencing may be based on an immunochemical assay of MTAP protein, revealing reduced or absent expression of MTAP protein in the nuclei and / or cytoplasm of relevant tumor cells, e.g., in the case of Hodgkin lymphoma in Hodgkin-Reed / Sternberg cells.

[0097] In embodiments relating to the use of an MTA synergistic PRMT5 inhibitor for the manufacture of a medicament for the treatment of cancer, use of the resulting medicament may be prescribed based on the patient being identified as having a tumor in which the wild-type MTAP gene is silenced after analysis of a sample taken from the patient. In such embodiments, the determination of wild-type MTAP gene silencing may be based on an immunochemical assay of MTAP protein, revealing reduced or absent expression of MTAP protein in the nuclei and / or cytoplasm of relevant tumor cells, e.g., in the case of Hodgkin's lymphoma in Hodgkin-Reed / Sternberg cells.

[0098] In embodiments, a kit is provided that includes an MTA synergistic PRMT5 inhibitor and instructions for use in treating cancer in which the wild-type MTAP gene is silenced. In such embodiments, the instructions include instructions for treating cancer in which the MTAP gene silencing is (a) results in partial or complete loss of MTAP protein in the relevant tumor cells; or (b) results in partial or complete loss of MTAP protein in the nuclei of the involved tumor cells; or (c) leading to a decrease in MTAP protein expression in the nuclei of tumor cells; or (d) leading to a decrease in MTAP protein expression in the nuclei of clonal tumor cells; or (e) characteristically causes accumulation of MTA in associated tumor cells; or (f) MTAP gene silencing mediated by MTAP hypermethylation characteristically leads to the accumulation of MTA in the associated tumor cells; or (g) epigenetic modification of the MTAP gene characteristically leads to the accumulation of MTA in the associated tumor cells; or (h) downregulation of MTAP at the protein level, characteristically leading to accumulation of MTA in the associated tumor cells; or (i) downregulation of MTAP protein expression, characteristically leading to accumulation of MTA in the associated tumor cells; or (j) causes epigenetically driven downregulation of MTAP mRNA; or (k) partial or complete silencing of MTAP protein expression, leading to the accumulation of MTA in the associated tumor cells; or (l) epigenetic modification of the MTAP gene resulting in partial or complete silencing of MTAP protein expression, leading to the accumulation of MTA in the associated tumor cells; or (m) partial or complete silencing of MTAP protein expression due to hypermethylation of the MTAP gene, resulting in tumors that characteristically accumulate MTA; or (n) Downregulation of MTAP mRNA caused by hypermethylation of or around the MTAP gene, resulting in tumors that characteristically accumulate MTA; or (o) Cancers in which the wild-type MTAP gene is silenced can be characterized based on the fact that downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or neighboring genes such as CDKN2A leads to tumors that characteristically accumulate MTA.

[0099] Thus, by way of example, the kit may provide instructions for the use of an MTA-synergistic PRMT5 inhibitor in the treatment of cancers in which the wild-type MTAP gene is silenced, which characteristically accumulate MTA due to MTAP gene silencing mediated by hypermethylation of the MTAP gene, as specified in use (f).

[0100] In an embodiment, the cancer in which the wild-type MTAP gene is silenced is a lymphatic cancer, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma. In an embodiment, the cancer in which the wild-type MTAP gene is silenced is Hodgkin's lymphoma (HL), which may be classical HL (cHL) classified into nodular sclerosing lymphoma (NSHL), mixed cellularity lymphoma (MCHL), lymphocyte-rich lymphoma (LRHL), or lymphocyte-depleted lymphoma (LDHL), or may be nodular lymphocyte-predominant HL. In an embodiment, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0101] In embodiments, the cancer in which the wild-type MTAP gene is silenced is a cancer selected from bladder cancer, breast cancer, kidney cancer, leukemia, lung cancer, ovarian cancer, pancreatic cancer, sarcoma, or skin cancer.

[0102] In embodiments, the cancer in which the wild-type MTAP gene is silenced is Hodgkin's lymphoma.

[0103] In embodiments, the cancer in which the wild-type MTAP gene is silenced is Hodgkin's lymphoma, and the determination of gene silencing status is based on an immunohistochemical assay of MTAP protein showing reduced or null levels of MTAP protein in the nuclei of Hodgkin-Reed / Sternberg (HRS) cells compared to normal cells, such as non-HRS cells, in the sample.

[0104] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor described in WO 2021 / 163344. In such embodiments, the inhibitor is represented by the general formula I [ka] a tautomer thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt of any of the above (In the formula, [ka] represents a single or double bond; X 1 and X 2 is independently in each instance N or C; X 1 If C, then X 1 teeth, Optionally, halo or C 1~6 optionally substituted with alkyl; Ar is a 6-membered aromatic ring having 0 to 2 N atoms, and each Ar independently contains 0 to 2 R a optionally substituted with a group; R ais independently, at each instance, cyano, halo, optionally substituted C 1~6 Alkyl, C 1~6 Haloalkyl, OR\NR c R d , -C(O)NR c R d , =S, -SO2, -SO2C 1~6 Alkyl, -C(O)H, -C(O)C 1~6 Alkyl, C(O)OC 1~6 alkyl, difluoropyrrolidinyl, and 4-6 membered heterocycles (0-2 heteroatoms independently selected from O and N, and the heterocycles further independently selected from 0-2 halogen, C 1~6 Alkyl, -C(O)H, -C(O)C 1~6 optionally substituted with alkyl or optionally substituted cycloalkoxyl; Each R b are each independently H, optionally substituted C 1~6 alkyl (substituents selected from halo) or oxetanyl; Here, each R e and R d is H, C 1~3 Alkyl, C 1~3 independently selected from haloalkyl or -CO; R e In each case, H or C 1~6 alkyl; R f and R g is, independently in each instance, H and C 1~6 independently selected from alkyl; R is H or halo; R 1 and R 2 is independently in each instance H, optionally substituted C 1~6 Alkyl, optionally substituted C 1~6 Alkynyl, -C(OR e ), optionally substituted monocyclic and bicyclic rings having 0-3 N, S or O atoms; the substituents are selected from halo, optionally substituted C 1~6 Alkyl, -C(O)NRf R g , OH, and an optionally substituted 5-membered ring having 0-3 N atoms; or R 1 and R 2 and the carbon atoms to which they are attached are optionally substituted monocyclic or bicyclic carbocyclic or heterocyclic rings (which may be saturated, partially saturated, or aromatic), wherein the heterocyclic rings contain 1, 2, or 3 heteroatoms independently selected from N, 0, and S; The substituents are optionally substituted C 1~6 Alkyl, Halo, CN, OR e and C(OR e ) and However, R 1 and R 2 and H cannot both be H at the same time; R 3 and R 4 is independently in each instance H, halogen, alkynyl, cyano, and C 1~6 alkyl (optionally substituted with halo or deuterium).

[0105] In such embodiments, the compound may be selected from the list of compounds described in claim 19 of WO 2021 / 163344, shown on pages 267 to 305 of the same publication.

[0106] In embodiments, the compound may be a compound of Formula II below, as set forth in Claim 1 of WO 2022 / 026892 A1 and shown on pages 2309-2311 of WO 2022 / 026892 A1. In such embodiments, the compound may be selected from the compounds set forth in Table 1 of WO 2022 / 026892 A1, as set forth on pages 122-470 of WO 2022 / 026892 A1. [ka]

[0107] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is N-(6-amino-5-methylpyridin-3-yl)-2-((2R,5S)-2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxoacetamide: [ka] or a pharmaceutically acceptable salt thereof.

[0108] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is N-(6-amino-5-methylpyridin-3-yl)-2-((2R,5S)-2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxoacetamide: [ka] is.

[0109] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is N-(6-amino-5-methylpyridin-3-yl)-2-((2R,5S)-2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxoacetamide: [ka] is a pharmaceutically acceptable salt of

[0110] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor described in WO 2022 / 115377 A1.

[0111] In such embodiments, the MTA synergistic PRMT5 inhibitor is a compound of Formula III: [ka] a tautomer thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt of any of the above, wherein R is a group represented by formulas IA and IB: [ka] is a tricyclic ring independently selected from where: [ka] is a single or double bond, X 1 , X 2 , X 6 , and X 7 is N or C in each instance, and X 1 and X 2 cannot be N at the same time, and X 1 If C, then X 1 is optionally substituted with halo; X 3 , X 4 , and X 5 is independently selected at each instance from optionally substituted C, O, N, and S; and the substituents are C 1~3 Alkyl, C 1~3 independently selected from alkyl(OH), wherein alkyl is optionally substituted with halo; R 3 is, independently in each instance, H or C 1~3 independently selected from alkyl; Ar 1 teeth, [ka] a 6-membered optionally substituted aryl or heteroaryl independently selected from 1~3 Alkyl, -OC 1~3 alkyl or halo); R in each example 1 are independently selected from H, halo, optionally substituted C 1~3 alkyl (substituents selected from halo)-CN, optionally substituted-OC 1~3 alkyl (substituents selected from halo), —C(O)OC 1~3 Alkyl (C 1~3alkyl is selected from (optionally substituted with halo) and morpholinyl; R in each example 2 are independently optionally substituted C 1~8 Alkyl (substituents include halo, hydroxy, amino, -OC 1~3 alkyl or -CN; optionally substituted with hydroxy, amino, C 1~6 5- or 6-membered ring or heterocycle substituted with alkyl (substituents selected from halo); optionally substituted C 1~6 Alkyl-OC 1~3 Alkyl (substituents selected from halo); 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridinyl; C 1~3 alkyl-heterocyclyl (heterocyclyl is selected from optionally substituted 3,4-dihydro-2H-pyrano[2,3-c]pyridinyl); pyradazinyl, triazolyl, pyrimidinyl, tetrahydrofuranyl, 1H-pyrrolo[2,3-b]pyridinyl, cyclohexyl (substituents are C 1~3 Alkyl, -CN, and halo, or optionally substituted C 1~6 Alkyl-OC 1~3 alkyl (substituents selected from halo); optionally substituted phenyl (substituents selected from halo or C 1~3 alkyl).

[0112] In such embodiments, the compound may be selected from the compounds set forth in claim 20 of WO 2022 / 115377 A1, set forth on pages 331-378 thereof. In such embodiments, in one embodiment, the MTA synergistic PRMT5 inhibitor is selected from those set forth in claim 21 of WO 2022 / 115377, set forth on pages 377 and 378 thereof.

[0113] In embodiments, the MTA synergistic PRMT5 inhibitor is (P)-2-[4-[4-(aminomethyl)-1-oxo-2H-phthalazin-6-yl]-2-methyl-pyrazol-3-yl]-4-chloro-6-(cyclopropoxy)-3-fluoro-benzonitrile, as described in Smith et al, https: / / doi.org / 10.1016 / j.bmc.2022.116947): [ka] is.

[0114] In embodiments, the MTA synergistic PRMT5 inhibitor is (P)-2-[4-[4-(aminomethyl)-1-oxo-2H-phthalazin-6-yl]-2-methyl-pyrazol-3-yl]-4-chloro-6-(cyclopropoxy)-3-fluoro-benzonitrile: [ka] or a pharmaceutically acceptable salt thereof.

[0115] In embodiments, the MTA synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt of (P)-2-[4-[4-(aminomethyl)-1-oxo-2H-phthalazin-6-yl]-2-methyl-pyrazol-3-yl]-4-chloro-6-(cyclopropoxy)-3-fluoro-benzonitrile: [ka] is.

[0116] In embodiments, the MTA synergistic PRMT5 inhibitor is (P)-2-[4-[4-(aminomethyl)-1-oxo-2H-phthalazin-6-yl]-2-methyl-pyrazol-3-yl]-4-chloro-6-(cyclopropoxy)-3-fluoro-benzonitrile hydrochloride: [ka] is.

[0117] In embodiments, the MTA synergistic PRMT5 inhibitor is a compound of formula (IV) as described in WO 2023 / 036974, or a pharmaceutically acceptable salt thereof: [ka] (In the formula, the ring containing X and Y is pyrrole, where X is NH and Y is CH, or X is CH and Y is NH; Z is selected from CH, CF, CCl or, if Q is not N, N; Q is selected from CH, CF, CCl or, if Z is not N, N; m is 0, 1 or 2; n is 0, 1, or 2; p is 1 or 2; R 1 is, at each occurrence, independently selected from F, Cl, CN, Me, CF3, C1-C3 alkyl, cyclopropyl, C1-C3 fluoroalkyl, OMe, or C1-C3 alkoxy; R 2 is, at each occurrence, independently selected from F, Cl, Me, MeO, and CF3; R 3 is H, Me, C1-C3 alkyl or C1-C3 fluoroalkyl; R 4 is H, M or C1-C3 alkyl; R 5 is H, Me, C1-C3 alkyl, C1-C3 fluoroalkyl, CHOMe, CHOCHF, CHOCF, CHO(C1-C3 alkyl), CHO(C1-C3 fluoroalkyl), C(CHCH)R 6 , CCR 7 , CH2R 8 , R 9 or CH2R 10 and; R 6 is H, Me, CHF, CHF, CF, CHOH or CHOMe; R 7is H, Me, cyclopropyl, C1-C3 alkyl, C1-C3 fluoroalkyl, C3-C6 cycloalkyl, or a 5-membered heteroaryl group optionally substituted with Me, C1-C3 alkyl, F, or Cl; R 8 is a 5-membered heteroaryl optionally substituted with Me, C1-C3 alkyl, F or Cl; R 9 is an optionally substituted phenyl, 5- or 6-membered heteroaryl, or bicyclic heteroaryl group; R 10 is an optionally substituted phenyl, 5- or 6-membered heteroaryl, or bicyclic heteroaryl group).

[0118] In embodiments, the MTA synergistic PRMT5 inhibitor is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1′-(4-fluorobenzyl)spiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: [ka] or a pharmaceutically acceptable salt thereof.

[0119] In embodiments, the MTA synergistic PRMT5 inhibitor is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1′-(4-fluorobenzyl)spiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: [ka] is.

[0120] In embodiments, the MTA synergistic PRMT5 inhibitor is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1′-(4-fluorobenzyl)spiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: [ka] is a pharmaceutically acceptable salt of

[0121] In embodiments, the MTA synergistic PRMT5 inhibitor is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1′-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: [ka] or a pharmaceutically acceptable salt thereof.

[0122] In embodiments, the MTA synergistic PRMT5 inhibitor is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1′-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: [ka] is.

[0123] In embodiments, the MTA synergistic PRMT5 inhibitor is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1′-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: [ka] is a pharmaceutically acceptable salt of

[0124] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula V as described in claim 1 of WO 2021 / 050915, as shown on pages 321 and 322 thereof: [ka] is.

[0125] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 88 of WO 2021 / 050915, as shown on pages 331-349 of the same.

[0126] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 89 of WO 2021 / 050915, as shown on pages 349 and 350 of WO 2021 / 050915.

[0127] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula VI-a, VI-b, VI-c, VI-d, VI-e or VI-f as described in claim 1 of WO 2022 / 192745, as shown on pages 512 and 513 thereof: [ka] is.

[0128] In such an embodiment, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 25 of WO 2022 / 192745, as shown on pages 523-536 of the same.

[0129] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula VI-g, VI-h, VI-i, VI-j, VI-k, or VI-l as described in claim 26 of WO 2022 / 192745 and shown on pages 536 and 537 of the same: [ka] is.

[0130] In such an embodiment, in an embodiment, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 27 of WO 2022 / 192745, as shown on page 538 of WO 2022 / 192745.

[0131] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula VII as set forth in claim 1 of WO 2023 / 278564, as shown on pages 145-147 thereof: [ka] is.

[0132] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those set forth in claim 19 of WO 2023 / 278564, as shown on pages 149-154 of WO 2023 / 278564.

[0133] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula VIII as described in claim 1 of WO 2022 / 132914, as shown on pages 188 and 189 thereof: [ka] is.

[0134] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 19 of WO 2022 / 132914, as shown on pages 192 and 193 of WO 2022 / 132914.

[0135] In such an embodiment, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 20 of WO 2022 / 132914, as shown on pages 194 and 195 thereof.

[0136] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl]methanone: [ka] or a pharmaceutically acceptable salt thereof.

[0137] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl]methanone: [ka] is.

[0138] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl]methanone: [ka] is a pharmaceutically acceptable salt of

[0139] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is (R)-(4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)(2-(4-(trifluoromethyl)phenyl)piperidin-1-yl)methanone: [ka] or a pharmaceutically acceptable salt thereof.

[0140] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is (R)-(4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)(2-(4-(trifluoromethyl)phenyl)piperidin-1-yl)methanone: [ka] is.

[0141] In such embodiments, the MTA synergistic PRMT5 inhibitor is (R)-(4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)(2-(4-(trifluoromethyl)phenyl)piperidin-1-yl)methanone [ka] is a pharmaceutically acceptable salt of

[0142] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula IX as described in claim 1 of WO 2022 / 169948, as shown on pages 240 and 241 thereof: [ka] is.

[0143] In such an embodiment, the MTA synergistic PRMT5 inhibitor is selected from those set forth in claim 23 of WO 2022 / 169948, as shown on pages 243 and 244 thereof.

[0144] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula X as described in claim 1 of WO 2023 / 081367, as shown on pages 161 and 162 thereof: [ka] is.

[0145] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XA as set forth in claim 6 of WO 2023 / 081367, as shown on pages 164 and 165 thereof: [ka] is.

[0146] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 17 of WO 2023 / 081367, as shown on pages 168-181 of WO 2023 / 081367.

[0147] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 18 of WO 2023 / 081367, as shown on pages 181-185 of WO 2023 / 081367.

[0148] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 19 of WO 2023 / 081367, as shown on pages 185-188 of WO 2023 / 081367.

[0149] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 20 of WO 2023 / 081367, as shown on pages 188-189 of WO 2023 / 081367.

[0150] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 21 of WO 2023 / 081367, as shown on pages 189-190 of WO 2023 / 081367.

[0151] In an embodiment, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XI as set forth in claim 1 of CN116178347, as shown on page 2 of the specification: [ka] is.

[0152] In such an embodiment, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 10 of CN116178347, as shown on pages 6 and 7 of the specification.

[0153] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XII as described in claim 1 of WO 2023 / 098439, as shown on pages 55-59 thereof: [ka] is.

[0154] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those set forth in claim 11 of WO 2023 / 098439, as shown on pages 68 and 69 thereof.

[0155] In embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XIII as described in claim 1 of WO 2021 / 086879, as shown on pages 497 and 498 thereof: [ka] is.

[0156] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XIII-a as described in claim 5 of WO 2021 / 086879, as shown on pages 499 and 500 thereof: [ka] is.

[0157] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XIII-b as set forth in claim 63 of WO 2021 / 086879, as shown on pages 507-509 thereof: [ka] is.

[0158] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XIII-c as set forth in claim 65 of WO 2021 / 086879, as shown on pages 509 and 510 thereof: [ka] is.

[0159] In such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is selected from those described in Table 1 of WO 2021 / 086879, shown on pages 103-114 of the same pamphlet.

[0160] In an embodiment, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XIV as described in claim 1 of CN116462676, as shown on pages 2-4 of the specification: [ka] is.

[0161] In such an embodiment, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 14 of CN116462676, as shown on pages 12 to 17 of the specification.

[0162] In an embodiment, the MTA synergistic PRMT5 inhibitor is an inhibitor of formula XV as described in claim 1 of CN116462677, as shown on pages 2 to 5 of the specification: [ka]

[0163] In such an embodiment, the MTA synergistic PRMT5 inhibitor is selected from those described in claim 18 of CN116462677, as shown on pages 17 to 22 of the specification.

[0164] In embodiments, the present disclosure provides a pharmaceutical composition comprising an MTA synergistic PRMT5 inhibitor for use in treating cancer characterized by wild-type MTAP gene silenced.In such embodiments, the MTA synergistic PRMT5 inhibitor can be selected from the list of inhibitors disclosed above.

[0165] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0166] Units, prefixes, and symbols are expressed in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers delimiting the range.

[0167] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient and does not contain any additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile. The pharmaceutical composition according to the present specification comprises an MTA synergistic PRMT5 inhibitor and at least one pharmaceutically acceptable excipient. The one or more pharmaceutically acceptable excipients may be selected from the group including fillers, binders, diluents, etc.

[0168] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow, alleviate the symptoms, and / or halt the progression of a diagnosed condition or disorder, and (2) prophylactic or preventative measures that prevent and / or slow the onset of the targeted condition or disorder. Thus, those in need of treatment include those already with the disorder, those prone to having the disorder, and those in whom the disorder is to be prevented.

[0169] The term "subject" refers to a human being who receives a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to human subjects.

[0170] The MTA synergistic PRMT5 compound or its pharmaceutically acceptable salt will usually be administered orally in the form of a pharmaceutical preparation containing the active ingredient or its pharmaceutically acceptable salt or solvate, or a solvate of such a salt, in a pharmaceutically acceptable dosage form. Depending on the cancer and patient to be treated and the route of administration, the composition can be administered in various dosages.

[0171] Pharmaceutical formulations of the MTA synergistic PRMT5 inhibitors can be conveniently administered in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical arts, for example, as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA. (1985).

[0172] Pharmaceutical formulations suitable for oral administration may contain one or more physiologically compatible carriers and / or excipients and may be in solid or liquid form. Tablets and capsules can be prepared using binders, fillers, lubricants, and surfactants. Liquid compositions may contain conventional additives such as suspending agents, emulsifying agents, and preservatives. Liquid compositions may be encapsulated, for example, in gelatin to provide a unit dosage form. Solid oral dosage forms include tablets, two-piece hard-shell capsules, and soft elastic gelatin (SEG) capsules.

[0173] To facilitate understanding of the therapeutic applicability of MTA synergistic inhibitors for use in treating cancers in which the wild-type MTAP gene has been silenced, an example is provided below. [Example]

[0174] Proliferation assay For Hodgkin's lymphoma cell lines L428, L540, L1236, HDLM2, and KM-H2 (purchased from DSMZ, http: / / www.dsmz.de): Assay plates were prepared by adding compounds to 384-well plates (Corning #3712) using an Echo Liquid Handler. On day 0, 1200 L428, L540, or KMH2 cells in 60 μl of growth medium (RPMI 1640 + 10% FBS + 1% L-Glu + 1% P / S) were dispensed into the assay plates at 1:3 dilution at a final top concentration of 10 μM, for a total of 10 replicates. The same volume was also dispensed into one blank plate for the day 0 control plate. 30 μl / well of CellTiter Glo reagent (Promega #7573) was added to the day 0 plate, which was then incubated in the dark for 20 minutes at room temperature. Luminescence was measured using a Tecan M200 plate reader with an integration time of 100 ms, decay and stabilization times of 0 ms. Assay plates were incubated in a 37°C, 90% humidity, 5% CO2 incubator for 7 days. Luminescence was measured as a day 0 control plate. IC 50 was calculated using nonlinear regression (curve fitting) analysis in GraphPad Prism 8.

[0175] Alternatively, for HDLM2 (purchased from DSMZ): cells were grown as adherent cells in growth medium (phenol red-free RPMI + 10% FCS + 2 mM glutamine). Cells were seeded at 1000 cells / well in 90 μl of growth medium into 96-well clear-bottom black tissue culture-treated plates and placed in a 37°C, 5% CO2 incubator. Compounds were obtained from liquid banks as solutions at a concentration of 10 mM in DMSO, and then half-log serial dilutions were performed in DMSO to make 1000x stock solutions.

[0176] Concentrations were then diluted 10x in DMSO, followed by 10x dilutions in growth medium to obtain an additional plate of 10x final concentrations in 10% DMSO. Then, 10 μl of compound was added to 90 μl of cells (10x dilutions) to obtain a concentration range of 0.1 nM to 10 μM in 1% DMSO. CellTiter Glo readings were taken at the time of dosing and on day 6. CellTiter Glo reagent was added to the volume of medium currently on the cells (100 μl). Mix on a plate shaker for 2 minutes at room temperature to induce cell lysis. Transfer 150 μl of lysate to a white 96-well plate. Incubate at room temperature for 10 minutes to stabilize the luminescence signal (cover with a silver plate seal). Read luminescence on an Envision F (ultrasensitive luminescence 96-well protocol with a 384-well opening). IC 50 was calculated using nonlinear regression (curve fitting) analysis in GraphPad Prism 8.

[0177] For the HCT116 isogenic cell line (parental model purchased from ATCC, MTAP KO clone generated in-house using CRISPR technology): Cells were harvested at a density of 400 cells per well (cCoys 5A + 10% FCS + 1% Glutamax) and seeded into 384-well plates (Greiner, Kremsmunster, Austria; 781090) at 40 μl / well using a Multidrop Combi. To the day 0 plate, immediately add 4 μl of Alamar Blue reagent (Thermo; DAL1100) using a Multidrop Combi and incubate for 3 hours at 37°C, 5% CO2. Day 0 cell plates were measured using an Envision plate reader with a fluorescence excitation wavelength of 540-570 nm (peak excitation at 570 nm) and a fluorescence emission wavelength of 580-610 nm (peak emission at 585 nm). Test compounds were added using an Echo 555 and placed in an incubator at 37°C with 5% CO2 for an additional 4 days. On day 5, 4 μl of Alamar Blue reagent was added using a Multidrop Combi and incubated at 37°C with 5% CO2 for 3 hours. Cell plates on day 4 were measured using an EnVision plate reader with a fluorescence excitation wavelength of 540-570 nm and a fluorescence emission wavelength of 580-610 nm. Growth rates (IC50 values) were determined by assessing the total cell counts of plates on day 0 and day 4 from the EnVision plate reader using Genedata screener software.

[0178] Western blotting experiments Cell pellets were washed twice with ice-cold PBS, lysed in 1x SDS lysis buffer (100 mM Tris-HCl buffer, pH 7.4, 10% glycerol, 1% SDS), and frozen at -80°C. Samples were thawed and heated at 95°C for 5 minutes. After spinning at 14,000 rpm for 10 minutes, the supernatant was transferred to a new tube. Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Pierce catalog number 23225). 25 μg of protein from each cell line was loaded onto a NuPAGE™ 4-12% Bis-Tris gel (Invitrogen catalog number WG1403BOX10) and run at 120 V for 1.5 hours. The gel was then transferred to a nitrocellulose membrane using a BioRad Semi-Dry Transfer System (BioRad, model number Trans-Blot SD Cell). The following primary and secondary antibodies were used to blot the membranes: MTAP (Cell Signaling, 4158), GAPDH (Cell Signaling, 2118), and HRP-conjugated anti-rabbit IgG (Cell Signaling, 7074).

[0179] Evaluation of MTAP protein expression in tumor samples of Hodgkin's lymphoma by immunohistochemistry IHC analysis was performed on the Ventana Benchmark platform (Roche Diagnostics) using the Ventana human immunohistochemistry staining protocol provided by the instrument supplier.

[0180] Formalin-fixed, paraffin-embedded (FFPE) Hodgkin's lymphoma samples were obtained from Tristar Technology Group LLC, Washington DC USA.

[0181] Antigen retrieval was performed at pH 8.55 and 100°C for 24 min.

[0182] Positive controls: HCT116 cells (human MTAP wild-type colon cancer cell line), human tonsil cells (tonsil FFPE block (ID68282B2(4)-4) commercially obtained from ProteoGenex, Inglewood, CA90301, USA).

[0183] Negative controls: MCF7 cells (human MTAP-deficient metastatic breast adenocarcinoma cell line) and xenograft tumors based on MCF7 MTAP-null human breast cancer cells (FFPE blocks from the AZ Archives Test Bank).

[0184] The following equipment was used for IHC analysis: Ventana Benchmark Ultra and Prep Kit dispenser (Roche Diagnostics); Leica XL Autostainer (ST5010), and Leica CV5030 Coverslipper (leicabiosystems.com).

[0185] The following reagents were used: Ventana bulk reagents: Benchmark Ultra LCS [Roche: 05424534001 (650-210)]; 10x EZ Prep Solution [Roche: 05279771001 (950-102)]; 10x Reaction Buffer Concentrate [Roche: 05353955001 (950-300)]; 10x SSC [Roche: 05353947001 (950-110)]; ULTRA Cell Conditioning (ULTRA CC1) [Roche: 05424569001 (950-224)] Ventana dispenser reagents: Optiview DAB IHC detection kit [Roche: 06396500001 (760-700)]; hematoxylin [Roche: 05266726001 (760-2021)]; cyanide reagent [Roche: 05266769001 (760-2037)]; primary antibody: MTAP (clone A8N9F) rabbit IgG monoclonal antibody, CST #62765S (www.cellsignal.com) Staining kit: OptiView DAB IHC Detection Kit #760-700 (Roche) Additional reagents: deionized water, Fairy's solution (detergent, Proctor & Gamble).

[0186] procedure: 1. Refer to the BenchMark ULTRA IHC / ISH System (Roche) user manual for instructions on how to operate the Ventana, fill bulk reagents, empty the waste container, and print slide labels. Note: EZ preparation solutions and reaction buffers are diluted to 1X with deionized water before use. 2. In a Ventana Prep Kit dispenser, MTAP(A8N9F) rabbit mAb [CST#62765S] was prepared to a working concentration of 0.5 μg / ml in Ventana Diluent [Roche: 05261899001 (251-018)] or 2 μg / ml in Ventana Diluent with Casein [Roche: 06440002001 (760-219)]. 3. Register and fill the Ventana Option dispenser with Dako Serum Free Protein Block [Agilent: X090930-2]. 4. Print labels for the slides (see the Bench Mark ULTRA IHC / ISH System User Manual). 5. Label the slides, place the slides and reagent dispenser into the Ventana, and run the machine. 6. After the Ventana run is complete, remove the slides from the slide tray and place them in the coverslipper rack. 7. Wash the slides with soapy water to remove the LCS oil and rinse under running tap water. 8. Repeat the wash and load the rack into the Leica XL autostainer and select program 3 "Ventana Clearing" to run the slides through running water, graded ethanol, and xylene. 9. Once finished, coverslip the slides with a CV5030 coverslipper using a 24 x 50 mm coverslip. 10. Prior to manual pathology scoring, samples were scanned at 40X magnification using a Leica Aperio AT2 scanner (https: / / www.leicabiosystems.com / ). Manual scoring was performed by an experienced pathologist. H-scoring was adapted and used for nuclear MTAP staining of tumor cells. The presence and intensity of cytoplasmic MTAP staining of tumor cells was also determined and recorded for each sample. For a description of H-scoring and its application, see D.A. Budwit-Novotny et al. Cancer Res. 1986;46:5419-5425; F. Aeffner et al. Archives of Pathology & Laboratory Medicine 141(9), 1267-1275; Meyerholz et al., Laboratory Investigation 98(7):844-855.

[0187] Preparation of (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione (Compound A) Compound A may be prepared according to the methods disclosed in WO 2023 / 036974, such as those disclosed herein.

[0188] 2-(2-Bromo-4-fluorophenyl)acetic acid methyl ester [ka] Thionyl chloride (31.3 mL, 429.1 mmol) was carefully added dropwise to (2-(2-bromo-4-fluorophenyl)acetic acid (CAS No. 61150-59-2) (100 g, 429.1 mmol) in MeOH (400 mL) at rt. The reaction mixture was stirred at 60 °C for 4 h, cooled, and the solvent removed in vacuo. The residue was partitioned between EtOAc (250 mL) and saturated NaHCO (200 mL). The organic phase was washed with water (100 mL), brine (100 mL), passed through a phase separation filter paper, and the solvent removed in vacuo to give the title compound (105 g, 99%) as a colorless oil. 1 H NMR(400MHz,DMSO-d6,30℃)3.64(3H,s),3.83(2H,s),7.25(1H,td),7.48(1H,dd),7.58(1H,dd));m / z MH + was not observed.

[0189] 5-Fluoro-2-(2-methoxy-2-oxoethyl)benzoic acid methyl ester [ka] Methyl 2-(2-bromo-4-fluorophenyl)acetate (45.0 g, 182.14 mmol) and triethylamine (27.90 mL, 200.35 mmol) were placed in a steel pressure vessel along with MeOH (300 mL). [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (complex with dichloromethane) (4.46 g, 5.46 mmol) was added and the vessel was sealed. The vessel was purged with carbon monoxide and then charged to 7 bar with carbon monoxide. The pressure vessel was heated to 100 °C and stirred for 2 h. The reaction mixture was allowed to cool, vented, and filtered to remove the catalyst. The solvent was removed in vacuo, and the residue was dissolved in EtOAc (250 mL) and washed with water (2 × 200 mL) and brine (100 mL). The organic phase was passed through a phase separation filter paper, and the solvent was removed in vacuo. The crude product was purified by flash silica chromatography (elution gradient 0 to 50% EtOAc in heptane). Pure fractions were evaporated to dryness to afford the title compound (38.40 g, 93%) as a pale yellow oil. 1H NMR(400MHz,DMSO-d6,30℃)3.60(3H,s),3.80(3H,s),3.99(2H,s),7.42-7.49(2H,m),7.66(1H,ddd);m / z MH + 227.

[0190] Methyl rac-2-(1-bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoate [ka] Methyl 5-fluoro-2-(2-methoxy-2-oxoethyl)benzoate (47.0 g, 207.8 mmol) was dissolved in chloroform (450 mL). 1-Bromopyrrolidine-2,5-dione (55.5 g, 311 mmol) was added, followed by 2,2'-azabis(2-methylpropionitrile) (3.41 g, 20.8 mmol), and the reaction mixture was stirred at reflux for 72 hours. The reaction mixture was cooled, washed with water (2 x 250 mL), brine (100 mL), passed through a phase separation filter, and the solvent was removed in vacuo. The crude product was purified by flash silica chromatography, eluting with a gradient of 0 to 40% EtOAc in heptane. Pure fractions were evaporated to dryness to give the title compound (50.50 g, 80%) as a colorless oil. 1 m / z MH + was not observed.

[0191] rac-5-Fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate methyl ester [ka] 4-Methoxybenzylamine (23.5 g, 171 mmol) was placed in a flask with MeCN (300 mL) and sodium bicarbonate (23.9 g, 285 mmol) was added. Methyl rac2-(1-bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoate (43.5 g, 142 mmol) dissolved in MeCN (100 mL) was slowly added via a dropping funnel while the reaction mixture was heated to 80 °C. The reaction mixture was stirred at 80 °C for 3 h. The reaction mixture was allowed to cool, most of the MeCN was removed in vacuo, and the residue was partitioned between EtOAc (400 mL) and water (400 mL). The aqueous phase was re-extracted with EtOAc (100 mL), and the organic phases were combined and washed with brine (50 mL). The organic phase was passed through a phase separation filter paper, and the solvent was removed in vacuo. The crude product was purified by flash silica chromatography, eluent gradient 0 to 50% EtOAc in heptane. Pure fractions were evaporated to dryness to afford the title compound (45.3 g, 96%) as a pale yellow oil. 1 H NMR(400MHz,DMSO-d6,30℃)3.69(3H,s),3.73(3H,s),4.31(1H,d),5.04(1H,d),5.18(1H, m / z M.H. + 330.

[0192] rac-1-Allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate methyl ester [ka] Methyl rac-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate (24.0 g, 72.9 mmol), allyl acetate (11.8 mL, 109 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.67 g, 1.82 mmol), and N,N'-((1R,2R)-cyclohexane-1,2-diyl)bis(2-(diphenylphosphanyl)benzamide) (2.52 g, 3.64 mmol) were stirred in THF (400 mL) under nitrogen at 5°C. 1,1,3,3-tetramethylguanidine (13.7 mL, 109 mmol) was then added dropwise. The reaction mixture was stirred at 5°C for 5 minutes. The THF was removed in vacuo. The reaction mixture was partitioned between EtOAc (400 mL) and water (400 mL) and the organic phase was passed through a phase separator filter. The solvent was removed in vacuo to give an orange oil. The crude product was purified by flash silica chromatography, eluent gradient 0 to 50% EtOAc in heptane. Pure fractions were evaporated to dryness to give the title compound (25.8 g, 96%) as a cream solid. 1 H NMR(400MHz,DMSO-d6,30℃)3.04-3.20(2H,m),3.26(3H,s),3.73(3H,s),4.52(1H,d),4.71(1H,d ),4.74-4.94(3H,m),6.82-6.96(2H,m),7.28-7.39(2H,m),7.45-7.58(2H,m),7.63(1H,dd);m / z M.H. + 370.

[0193] (S)-1-Allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate methyl ester [ka] Methyl rac-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate (approximately 70:30 in favor of the desired (S) enantiomer) (25.8 g, 69.7 mmol) was purified by SFC chromatography (column: Phenomenex C1, 30 × 250 mm, 5 micron, mobile phase: 10% IPA + 0.1% DEA / 90% scCO2; flow rate: 90 ml / min, BPR: 120 bar; column temperature: 40 °C; UVmax 210 nm). Pure fractions were evaporated to dryness to give the title compound (15.1 g, 56%) as a white solid. 1 H NMR(400MHz,DMSO-d6,30℃)3.04-3.20(2H,m),3.26(3H,s),3.73(3H,s),4.52(1H,d),4.71(1H,d ),4.74-4.94(3H,m),6.82-6.96(2H,m),7.28-7.39(2H,m),7.45-7.58(2H,m),7.63(1H,dd);m / z M.H. + 370.

[0194] (S)-5-Fluoro-2-(4-methoxybenzyl)-3-oxo-1-(2-oxoethyl)isoindoline-1-carboxylate methyl ester [ka] To a solution of (S)-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-methyl carboxylate (60.0 g, 162 mmol) in 1,4-dioxane (800 mL) and water (200 mL) was added osmium(VIII) oxide (4% in water) (5.16 mL, 0.81 mmol), sodium periodate (87.0 g, 406 mmol), and 2,6-dimethylpyridine (37.8 mL, 324 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered to remove salts and washed with dichloromethane (DCM, 500 mL). The filtrate was placed in a separatory funnel with water (500 mL) and partitioned. The aqueous phase was extracted with DCM (300 mL), and the organic phases were combined, passed through a phase-separating filter paper, and the solvent was removed in vacuo. The crude product was purified by flash silica chromatography, eluent gradient 0 to 50% EtOAc in heptane. Pure fractions were evaporated to dryness to afford the title compound (50.1 g, 83%) as a white crystalline solid. 1 H NMR(400MHz,DMSO-d6,30℃)3.40(3H,s),3.42-3.56(2H,m),3.72(3H,s),4.58(1H,d),4.74(1H,d), 6.80-6.92(2H,m),7.19-7.27(2H,m),7.52(1H,ddd),7.60(1H,dd),7.69(1H,dd),9.07(1H,t);m / z M.H. + 372.

[0195] (S)-5-Fluoro-1'-(4-fluorobenzyl)-2-(4-methoxybenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione [ka] (S)-5-Fluoro-2-(4-methoxybenzyl)-3-oxo-1-(2-oxoethyl)isoindoline-1-methyl carboxylate (45 g, 121.2 mmol) and 4-fluorobenzylamine (22.75 g, 181.8 mmol) were placed in a flask with 1,2-dichloroethane (600 mL) and stirred for 1 hour. The reaction mixture was placed in an ice bath, and acetic acid (13.87 mL, 242.4 mmol) was added, followed by sodium triacetoxyborohydride (51.4 g, 242.4 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was neutralized with 2 M NaOH, diluted with water (200 mL), and extracted with DCM (2 x 200 mL). The combined organic phases were passed through a phase separator filter, and the solvent was removed in vacuo to give the title compound as a pale yellow oil. The crude product was used in the next reaction and was considered to be in 100% yield. m / z MH + 449.

[0196] Step 2: (S)-5-Fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione [ka] (S)-5-Fluoro-1'-(4-fluorobenzyl)-2-(4-methoxybenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione (54.30 g, 121.08 mmol) was placed in a flask with MeCN (500 mL) and water (250 mL). Ammonium cerium(IV) nitrate (199.0 g, 363.2 mmol) was added, and the reaction mixture was stirred at rt for 1 h. The reaction mixture was partitioned between DCM (500 mL) and water (500 mL). The organic phase was washed with water (200 mL) and brine (200 mL), passed through a phase separation filter, and the solvent was removed in vacuo. The crude product was purified by flash silica chromatography (elution gradient 0 to 100% (10% MeOH in EtOAc) in heptane. Pure fractions were evaporated to dryness to afford the title compound (30.50 g, 77%) as a cream solid. 1H NMR(400MHz,DMSO-d6,30℃)2.37-2.44(1H,m),2.45-2.49(1H,m),3.48(1H,ddd),3.60(1 m / z M.H. + 329.

[0197] Step 3: (S)-2-((5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione [ka] (S)-5-Fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione (27 g, 82.24 mmol) and 5-chloro-2-(chloromethyl)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine (30.20 g, 86.35 mmol) were placed in a flask along with dry dimethylformamide (DMF, 120 mL). Cesium carbonate (67.00 g, 205.6 mmol) was added, and the reaction mixture was stirred at 50 °C for 1 hour. The reaction mixture was partitioned between water (500 mL) and EtOAc (500 mL), and the aqueous phase was re-extracted with EtOAc (250 mL). The organic phases were combined, washed with water (3 x 250 mL), brine (200 mL), passed through a phase separation filter, and the solvent removed in vacuo. The residue was triturated with diethyl ether (200 mL), and the resulting solid was filtered, washed with ether, and dried to give the title compound (42.20 g, 80%) as a cream-colored solid. 1H NMR(400MHz,DMSO-d6,30℃)-0.13(9H,s),0.55-0.80(2H,m),2.32-2.41(1H,m ),2.52(1H,d),3.30-3.38(1H,m),3.41-3.51(2H,m),3.57-3.69(1H,m),4.22 -4.36(2H,m),4.75(1H,d),5.11(1H,d),5.53(1H,d),5.61(1H,d),6.53(1H,s ),7.13-7.23(4H,m),7.45-7.54(2H,m),7.57-7.64(1H,m),8.28(1H,dd);m / z M.H. + 641.

[0198] (S)-2-((5-((diphenylmethylene)amino)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione [ka] (S)-2-((5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1′-(4-fluorobenzyl)spiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione (41.80 g, 65.19 mmol), diphenylmethanimine (14.18 g, 78.23 mmol), and sodium 2-methylpropan-2-olate (12.53 g, 130.4 mmol) were placed in a flask along with toluene (300 mL), and the reaction mixture was degassed by bubbling nitrogen through the mixture for 10 minutes. tBuXPhos (2.77 g, 6.52 mmol) and tris(dibenzylideneacetone)dipalladium(0) (2.99 g, 3.26 mmol) were added, and the reaction mixture was then stirred at 65° C. for 30 min. The reaction mixture was allowed to cool and partitioned between EtOAc (600 mL) and water (600 mL). The organic phase was separated, washed with brine (200 mL), passed through a phase separation filter, and the solvent was removed in vacuo. The crude product was purified by flash silica chromatography, eluting with a gradient of 0 to 100% EtOAc in heptane. Pure fractions were evaporated to dryness to afford the title compound (49.50 g, 97%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6,30℃)-0.15(9H,s),0.57-0.76(2H,m),2.29-2.39(1H,m),2. 39-2.48(1H,m),3.26-3.29(1H,m),3.34-3.45(2H,m),3.53-3.66(1H,m),4.24(2H ,s),4.67(1H,d),5.04(1H,d),5.44(2H,q),6.32(1H,s),7.11(2H,dd),7.17-7.26 (7H,m),7.43-7.54(4H,m),7.55-7.61(2H,m),7.68-7.76(2H,m),7.81(1H,d);m / z M.H. + 786.

[0199] (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione [ka] (S)-2-((5-((diphenylmethylene)amino)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione (49.50 g, 62.98 mmol) was placed in a flask along with 2,2,2-trifluoroacetic acid (96 mL, 1259.63 mmol). 0.50 mL of water was added, and the reaction mixture was stirred at 40 °C for 4 h. The 2,2,2-trifluoroacetic acid was removed in vacuo, and the residue was dissolved in MeCN (75 mL). Ammonium hydroxide (28-30% in water) (73.60 mL, 1889.45 mmol) was added, and the reaction mixture was stirred at 40 °C for 4 h, then at room temperature overnight. The resulting solid was filtered and washed with MeCN (100 mL) to yield approximately 20 g of the desired compound. The filtrate was reduced to approximately 200 mL and purified by reverse-phase chromatography (Interchim C18-HP Flash column, 2 x 415 g, loading 100 mL of solution per run) using a gradually decreasing polarity mixture (30-60% gradient) of water (containing 1% NH4OH (28-30% by volume) in HO) and MeCN as the eluent. Fractions containing the desired compound were combined and added to the previously obtained solid (approximately 20 g). The slurry was stirred for 1 h, and then the MeCN was removed in vacuo, resulting in a pale yellow precipitate. The solid was filtered off and dried under vacuum for 2 h. The solid was then suspended in MeCN (150 mL), and the slurry was gently refluxed for 2 h, after which it was allowed to cool overnight. The solid was filtered off and dried under vacuum to give the title compound (19.54 g, 63%) as a cream-coloured crystalline solid. 1H NMR(400MHz,DMSO-d6,30℃)2.34-2.40(2H,m),3.36(1H,ddd),3.60(1H,dt),4.29(1H,d),4.39-4.52(2H,m),5.03(1H,d ),5.48(2H,s),6.02(1H,d),7.18-7.27(2H,m),7.27-7.39(3H,m),7.46-7.55(2H,m),7.59(1H,ddd),10.69(1H,d);m / z M.H. + 492.

[0200] Preparation of (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1'-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3'-pyrrolidine]-2',3-dione (Compound C) [ka] Compound C was prepared according to the method disclosed in WO 2023 / 036974.

[0201] Biological Example 1: Evaluation of the effects of Compound C treatment on in vivo tumor growth inhibition and target binding in the L540 HL xenograft model This study was conducted to evaluate tumor growth inhibition and pharmacodynamic changes after treatment with the MTA synergistic PRMT5 inhibitor Compound C in an MTAP-silenced L540HL xenograft model. Three doses of Compound C (dose level 1, dose level 2, and dose level 3) were tested. Pharmacodynamic changes were assessed by Western blot for the reduction of SDMA (a target binding marker) after treatment with Compound C.

[0202] [Table 6]

[0203] [Table 7]

[0204] Xenograft transplantation Xenografts were established by subcutaneous (SC) injection of 5 x 10e6 cells suspended in 0.1 mL of PBS into the right front flank of 6- to 8-week-old animals. Tumors were allowed to grow to 100-200 mm before randomization. 3 The tumors were measured with a caliper, and the tumor volume was calculated using the following formula: Volume (mm3) = (tumor length) × (tumor width) × (tumor width) / 2. Tumor length (the longest dimension of the tumor); tumor width (the longest dimension of the tumor perpendicular to the length).

[0205] Randomization Animals were randomized into groups based on tumor size. Randomization was performed based on the "matched distribution" method (StudyDirector™ software, version 3.1.399.19). The date of randomization is designated as day 0. There was no substitution of animals.

[0206] Group assignment and dose level Control animals were orally administered vehicle (5% v / v DMSO / 20% v / v Kolliphor HS15 / 75% v / v purified water (pH 3.0-3.2)), while vehicle- and Compound C-treated animals were dosed according to Table 6. Dosing began one day after selection and randomization. On day 21 of dosing, all animals in each group received their final dose in the morning. Mice were sacrificed 6 hours later, and flash-frozen tumors were collected for PD analysis.

[0207] [Table 8]

[0208] Tumor measurements and weight Tumors were measured twice weekly with a vernier caliper and calculated as follows: volume (mm 3 ) = (tumor length) × (tumor width) × (tumor width) / 2

[0209] Tumor volume was calculated using the formula: RTV on day X = (tumor volume on day X) / (tumor volume on day 0). was calculated using

[0210] The anticancer effect of Compound C was expressed as the percentage of tumor growth inhibition (TGI) calculated on the last day of the study using the following formula: Percentage of TGI on day X for treatment group = (((vehicle geometric mean of RTV on day X) - (treatment group geometric mean of RTV on day X)) / ((vehicle geometric mean of RTV on day X) - 1) x 100

[0211] Animals were weighed daily during the dosing period and twice weekly during the other periods. The percentage of weight change was calculated using the formula: Percentage weight change on day X = (((weight on day X) - (weight on selected day) x 100)). was calculated using

[0212] Assay Vehicle Formulation Kolliphor HS15 was dissolved in hot water (approximately 40°C) and vortexed to ensure a homogeneous solution. DMSO (5% of the final solvent volume) was then added to the glass vial. Kolliphor HS15 (20% of the final vehicle volume) was then added to the glass vial and mixed thoroughly by vortexing. Up to 80% of the final solvent volume was made up with purified water and mixed thoroughly by vortexing. The pH was adjusted to 3.0-3.2 using hydrochloric acid (1 M) and mixed thoroughly by vortexing. Up to 100% of the final vehicle volume was made up with purified water and mixed thoroughly by vortexing.

[0213] Compound C formulation Kolliphor HS15 was dissolved in hot water (approximately 40°C) and vortexed to ensure a homogeneous solution. The appropriate amount of compound was weighed into a glass vial. DMSO (5% of the final solvent volume) was then added to the glass vial and thoroughly vortexed to completely dissolve the compound. Next, Kolliphor HS15 (20% of the final vehicle volume) was added to the glass vial and thoroughly vortexed. Up to 80% of the final solvent volume was made up with purified water and thoroughly vortexed. The pH was adjusted to 3.0-3.2 using 1M hydrochloric acid and thoroughly vortexed. Up to 100% of the final vehicle volume was made up with purified water and thoroughly vortexed.

[0214] Pharmacodynamic analysis Western blot To determine the levels of proteins of interest in tumor samples, tumor fragments snap-frozen at the end of the PD study were used to extract proteins. 600–1000 μl of lysis buffer was added to small and large tumors, respectively. The lysis buffer contained RIPA buffer (Thermo, #89901), Complete Protease Inhibitor Tablets (Roche, #58880600, 2 tablets / 50 ml), Phosphatase Inhibitor Cocktails 2 and 3 (Sigma, #P5726, #P0044), and Benzonase Nuclease (Sigma, #E1014). Samples were homogenized in a high-speed homogenizer at 6.5 m / s for three 30-second cycles. The lysates were then sonicated for one 30-second cycle in a chilled Diagenode Bioruptor and then chilled on ice for 30 minutes. The lysates were centrifuged twice, 13,000 rpm for 10 minutes, with tube changes between runs to remove debris. Lysates were transferred to deep-well plates, and protein in the supernatants was measured using the BCA normalization method (Thermofisher, #23225). Protein concentrations were normalized to 45µg by diluting with 4X sample buffer (Invitrogen, #NP007), 10X reducing agent (Invitrogen, #NP0009), and HO. Samples were then boiled at 95°C for 5 minutes. Proteins were separated on a 4-12% Bis-Tris gel, and then transferred to a nitrocellulose membrane (Thermofisher, #IB21001) using Iblot2. Primary antibodies recognizing SDMA or vinculin were diluted in 0.05% Tween (TBST) + 5% Marvel and incubated overnight at 4°C. The membrane was washed three times with 20mL of TBST for 15 minutes each. Secondary rabbit (CST #7074) or mouse (CST #7076) horseradish peroxidase (HRP)-conjugated antibodies were diluted 1:2000 in TBST + 5% Marvel and incubated for 1 hour at room temperature. Membranes were washed three times for 15 minutes with 20 mL of TBST, and signals were detected using the chemiluminescent SuperSignal West Dura long-lasting substrate (Thermofisher, #34075) and quantified using Syngene software.The 30 kDa molecular weight band of SDMA, a PRMT5 substrate, was quantified using Syngene software. The 110 kDa molecular weight band of vinculin was also quantified. Statistical analysis was performed using a one-way analysis of variance on values ​​normalized to vinculin compared to vehicle control. SDMA (SDMA #13222, 1:1000 dilution, obtained from CST) and Vinculin (#V9131, 1:10,000 dilution) were obtained from Sigma.

[0215] statistical methods In vivo Tumor volumes were plotted as geometric means with SEM. Percentage body weight changes were plotted as means with SEM. Significant p-values ​​for TGI vs. vehicle-treated controls (relative tumor volume) on the last day of treatment were obtained from a one-tailed Mann-Whitney test and calculated in GraphPadPrism 8.4.3.

[0216] Pharmacodynamic analysis (PD) Primary analysis was performed using Excel, with raw data first normalized to vinculin, then normalized to the geometric mean of the vehicle control, and then multiplied by 100. Statistical analysis was performed using GraphPad Prism 8.4.3, where data were log-transformed (Y = Log(Y)) and a conventional one-way ANOVA test adjusted for multiple comparisons (Dunnett's) was performed. The mean difference was obtained from Prism and used to calculate the percent inhibition according to the following formula: Mean difference values ​​were converted to percentages = 1-((-1 x mean difference) x 10) x 100. Significant p-values ​​(if present) obtained from ANOVA tests were quantified using GraphPad Prism 8.4.3.

[0217] SDMA protein levels were measured.

[0218] result Compound C demonstrated dose-dependent efficacy in tumor growth inhibition in vivo (Figure 14 and Table 7). Administration at dose level 3, dose level 2, or dose level 1 resulted in tumor growth inhibition (93%, 52%, and 22%, respectively).

[0219] [Table 9]

[0220] Compound C was well tolerated at all doses tested, and no significant weight loss was observed during the treatment period compared to the vehicle-treated group (Figure 15).

[0221] Pharmacodynamic changes were assessed by Western blot for the reduction of SDMA (a target binding marker) after treatment with Compound C. Administration of the compound at dose level 3, dose level 2, or dose level 1 reduced SDMA protein levels (99.1%, 97.6%, and 83.9%, respectively; Figure 16 and Table 8).

[0222] [Table 10]

[0223] conclusion Compound C demonstrated dose-dependent efficacy and target binding in an in vivo MTAP-silenced subcutaneous Hodgkin's lymphoma xenograft model without causing significant weight loss compared to the vehicle group.

[0224] All references cited herein, such as patents, patent applications, articles, textbooks, etc., and the references cited therein, to the extent not already incorporated, are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method for treating cancer, comprising administering an MTA synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a cancer in which the wild-type MTAP gene is silenced.

2. 1. An MTA synergistic PRMT5 inhibitor for use in the treatment of cancer, wherein the cancer is characterized as having a silenced wild-type MTAP gene.

3. 1. An MTA synergistic PRMT5 inhibitor for use in the manufacture of a medicament, wherein the medicament is for use in the treatment of a cancer in which the wild-type MTAP gene is silenced.

4. 1. A pharmaceutical composition comprising an MTA synergistic PRMT5 inhibitor for use in treating cancer, wherein the cancer is characterized as having a silenced wild-type MTAP gene.

5. A kit comprising an MTA synergistic inhibitor and instructions for its use in treating cancer in which the MTAP gene is silenced.

6. A method for treating cancer, comprising the steps of: i) analyzing a sample obtained from a patient in need of treatment, said patient having a cancer in which the wild-type MTAP gene is silenced; and ii) administering a therapeutically effective amount of an MTA synergistic PRMT5 inhibitor to said patient in need of treatment.

7. The method of treatment, inhibitor for use, composition for use, or kit for use according to any one of claims 1 to 6, wherein the cancer in which the wild-type MTAP gene is silenced is selected from bladder cancer, breast cancer, diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, kidney cancer, leukemia, lung cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, sarcoma, and skin cancer.

8. The method of treatment, inhibitor for use, composition for use, or kit for use according to claim 7, wherein the cancer in which the wild-type MTAP gene is silenced is Hodgkin's lymphoma.

9. 9. The method of treatment, inhibitor for use, composition for use, or kit for use according to claim 8, wherein the Hodgkin's lymphoma is classical Hodgkin's lymphoma, such as nodular sclerosing lymphoma (NSHL), mixed cellularity (MCHL), lymphocyte-rich (LRHL) and lymphocyte-depleted (LDHL) or nodular lymphocyte-predominant Hodgkin's lymphoma.

10. The MTA synergistic PRMT5 inhibitor is a compound of formula (IV), or a pharmaceutically acceptable salt thereof: 【Chemical 1】 (In the formula, the ring containing X and Y is pyrrole, where X is NH and Y is CH, or X is CH and Y is NH; Z is selected from CH, CF, CCl or, if Q is not N, N; Q is selected from CH, CF, CCl or, if Z is not N, N; m is 0, 1 or 2; n is 0, 1, or 2; p is 1 or 2; R １ is F, Cl, CN, Me, CF for each occurrence ３ , C １ ~C ３ Alkyl, cyclopropyl, C １ ~C ３ Fluoroalkyl, OMe or C １ ~C ３ independently selected from alkoxy; R ２ represents F, Cl, Me, MeO, and CF, respectively. ３ are independently selected from R ３ is H, Me, C １ ~C ３ Alkyl or C １ ~C ３ is fluoroalkyl; R ４ is H, M or C １ ~C ３ is alkyl; R ５ is H, Me, C １ ~C ３ Alkyl, C １ ~C ３ Fluoroalkyl, CH ２ OMe, CH ２ OCHF ２ , C.H. ２ OCF ３ , C.H. ２ O (C １ ~C ３ alkyl), CH ２ O (C １ ~C ３ fluoroalkyl), C(CH ２ CH ２ ) R ６ , CCR ７ , C.H. ２ R ８ , R ９ or CH ２ R １０ and R ６ is H, Me, CH ２ F, CHF ２ , C.F. ３ , C.H. ２ OH or CH ２ OMe; R ７ is H, Me, cyclopropyl, C １ ~C ３ Alkyl, C １ ~C ３ Fluoroalkyl, C ３ ~C ６ Cycloalkyl, or Me, C １ ~C ３ a 5-membered heteroaryl group optionally substituted with alkyl, F, or Cl; R ８ is Me, C １ ~C ３ 5-membered heteroaryl optionally substituted with alkyl, F or Cl; R ９ is an optionally substituted phenyl, 5- or 6-membered heteroaryl, or bicyclic heteroaryl group; R １０ is an optionally substituted phenyl, 5- or 6-membered heteroaryl, or bicyclic heteroaryl group. The method of treatment, the inhibitor for use, the composition for use, or the kit for use according to any one of claims 1 to 9, wherein

11. 1. The MTA synergistic PRMT5 inhibitor, wherein the compound is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1′-(4-fluorobenzyl)spiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

12. 1. The MTA synergistic PRMT5 inhibitor, wherein the compound is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1′-(4-fluorobenzyl)spiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: 【Chemistry 3】 12. The method of treatment, inhibitor for use, composition for use or kit for use according to claim 11, wherein

13. 1. The MTA synergistic PRMT5 inhibitor, wherein the compound is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-1′-(4-fluorobenzyl)spiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: 【Chemistry 4】 12. The method of treatment, inhibitor for use, composition for use or kit for use according to claim 11, which is a pharmaceutically acceptable salt of

14. 1. The MTA synergistic PRMT5 inhibitor, wherein the compound is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1′-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: 【Chemistry 5】 or a pharmaceutically acceptable salt thereof.

15. 1. The MTA synergistic PRMT5 inhibitor, wherein the compound is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1′-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: 【Chemistry 6】 12. The method of treatment, inhibitor for use, composition for use or kit for use according to claim 11, wherein

16. 1. The MTA synergistic PRMT5 inhibitor, wherein the compound is (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-1′-(but-2-yn-1-yl)-5-fluorospiro[isoindoline-1,3′-pyrrolidine]-2′,3-dione: 【Chemistry 7】 12. The method of treatment, inhibitor for use, composition for use or kit for use according to claim 11, which is a pharmaceutically acceptable salt of

17. A method for identifying a patient who will benefit from treatment with an MTA synergistic PRMT5 inhibitor, the method comprising identifying that the wild-type MTAP gene is silenced in a sample obtained from the patient.

18. A method for identifying a tumor in which the wild-type MTAP gene is silenced, the method comprising the step of identifying that relevant tumor cells in a sample obtained from a patient exhibit reduced MTAP protein in their nuclei and cytoplasm by performing an immunohistochemical assay of MTAP protein.

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