MTA-synergistic PRMT5 inhibitors for treatment of cancer

Abstract

The present specification relates to methods of treating wild-type MTAP gene cancers comprising administering to a patient in need thereof an MTA synergistic PRMT5 inhibitor.

Description

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

[0002] The present description relates to methods for treating cancer, which methods include administering an MTA-synergistic PRMT5 inhibitor to a patient in need, wherein the patient has a tumor in which the wild-type MTAP gene is silenced, i.e., a tumor that carries the wild-type MTAP gene but still accumulates methylthioadenosine (MTA). One type of cancer in which this situation has been found to be prevalent is Hodgkin's lymphoma (HL). The present description also relates to methods for identifying cancer patients who will benefit from treatment with an MTA-synergistic PRMT5 inhibitor.

[0003] Protein arginine methyltransferase 5 (PRMT5) is a member of the PRMT family of arginine methyltransferases that catalyze the addition of methyl groups to the guanidine motif of arginine residues using S-adenosyl-L-methionine (SAM) as a methyl donor. PRMT5 is a type II arginine methyltransferase that symmetrically dimethylates the guanidine groups of arginine residues, thereby converting the guanidine NH 2 Conversion of groups to NMe 2 PRMT5 methylates many different substrates, including histone and non-histone proteins, thereby regulating processes such as RNA splicing, cell proliferation, and DNA repair. Notably, PRMT5 is overexpressed in multiple cancer types and has been identified as a candidate for therapeutic intervention by developing small molecules that inhibit PRMT5 methyltransferase activity (see, e.g., Kim et al., (2020) Cell Stress 4(8)199-2151).

[0004] Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a tumor suppressor gene that is homozygously deleted in approximately 15% of cancers. The loss of the 9p21 chromosome locus (where CDKN2A is located) leads to the co-deletion of additional genes, including the gene MTAP encoding methylthioadenosine phosphorylase (MTAP). MTAP is a metabolic enzyme involved in methionine salvage. The loss of MTAP leads to an increase in the concentration of MTAP substrate methylthioadenosine (MTA) in cancer cells that lack CDKN2A / MTAP. MTA itself acts as a weak PRMT5 inhibitor and therefore the accumulation of MTA in cancer cell lines that lack CDKN2A / MTAP leads to partial inhibition of PRMT5 activity. Impaired PRMT5 activity makes cancer cells that lack CDKN2A / MTAP susceptible to further targeting of PRMT5 (e.g., using short hairpin RNA (shRNA)). “Collateral vulnerability” has been identified in cancer, where CDKN2A / MTAP-deficient tumors can be selectively targeted by PRMT5 inhibition (see Marjon et al., (2016) Cell Reports 15, 574-587; Mavrakis et al., (2016) Science 11; 351(6278): 1208-13; Kryukov et al., (2016) Science 11; 351(6278): 1214-8).

[0005] Recently, there have been reports of MTA-synergistic PRMT5 inhibitors, i.e., PRMT5 inhibitors that preferentially bind to PRMT5 in the presence of MTA (see, e.g., WO 2022 / 026892A1, WO 2022 / 115377, WO 2021 / 163344, WO 2021 / 050915, WO 2022 / 192745, WO 2023 / 278564, WO 2022 / 132914, WO 2022 / 14619948, WO 2023 / 036974, WO 2023 / 081367, CN 202310191381, CN 116462676, CN 116462677, WO 2023 / 098439, and WO 2021 / 086879). These MTA-synergistic PRMT5 inhibitors are designed to exploit the “collateral vulnerability” caused by CDKN2A / MTAP gene loss described in the literature. Notably, MTA-synergistic PRMT5 inhibitors exert greater inhibition of PRMT5 in the presence of relatively high concentrations of MTA (e.g., as found in CDKN2A / MTAP-deficient tumor cells) rather than in healthy tissues (where inhibition of PRMT5 would otherwise result in toxic side effects). Thus, MTA-synergistic PRMT5 inhibitors should have a high therapeutic index (and low off-target toxicity) because their antiproliferative activity will be selectively manifested in tumor cells in the targeted, MTA-rich, CDKN2A / MTAP-deficient environment.

[0006] Hodgkin lymphoma (HL) is a B-cell lymphoma that accounts for approximately 15% of all lymphomas. Although HL has a low incidence in the general population, with 2-3 cases per 100,000 individuals in people of European descent (see, e.g., JM Connors et al., Nature Rev Disease Primers, 6, Art.: 61 (2020)), it is one of the most common types of cancer in young adults. HL is also seen in older individuals, but less frequently.

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

[0008] HL is characterized by the presence of a small number of malignant cells surrounded by a large number of immune effector cells in the tumor microenvironment. HL malignant cells are large mononuclear or multinuclear cells with a unique morphology and are derived from B cells. The malignant cells in cHL are called Hodgkin and Reed-Sternberg (HRS) cells, while the malignant cells 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 (HRS or LP) only account for approximately 1% of the cell composition of HL tumors. The remainder of HL tumors is mainly composed of noncancerous immune cells and stromal cells. The low frequency of malignant cells in HL tumors makes it very challenging to analyze the genomic alterations of HRS and LP cells. Recently, methods such as laser capture microdissection and fluorescence-correlated cell sorting have enabled multiple research groups to effectively enrich and genomic profile HRS and LP cells. In addition, methods based on ctDNA capture have also been successfully used to analyze the genomic landscape of HL.

[0010] Radiation therapy and multi-agent chemotherapy as first-line treatment for HL have a high chance of cure. For relapsed or refractory (R / R) disease, high-dose chemotherapy and autologous hematopoietic stem cell transplantation are usually used. Immunotherapy approaches using immune checkpoint inhibitors and antibody-drug conjugates have brought promising results for the treatment of patients with R / R HL. Nevertheless, there is still a need for alternative and improved methods to treat HL.

[0011] As described herein, it has been found that in certain tumor types that express wild-type MTAP genes and do not delete MTAP genes, hypermethylation at or around the MTAP gene can reduce or eliminate the mRNA level of MTAP, and thus cause the tumor to accumulate MTA due to the absence of MTAP protein. Therefore, an opportunity for treating certain MTAP wild-type tumor patients that has not been realized so far is revealed. Thus, the purpose of this specification is to provide a new method for treating tumors that carry wild-type MTAP genes but still accumulate MTA. In addition, this specification also provides a method for identifying patients suitable for treatment with MTA-synergistic PRMT5 inhibitors, the method comprising identifying from a sample obtained from a patient that the patient suffers from a tumor that carries a wild-type MTAP gene but still accumulates MTA in tumor cells.

[0012] According to a first aspect of the present specification, a method of treatment is provided, the method 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. Tumors in which the wild-type MTAP gene is silenced are those that carry a complete wild-type MTAP gene but nonetheless have reduced or zero expression of MTAP mRNA or MTAP protein. Due to the silencing of their wild-type MTAP gene, the ability of such tumor cells to phosphorylate MTA is reduced or zero and thus accumulates MTA. Because the MTA-synergistic PRMT5 inhibitor binds to and inhibits PRMT5 that synergizes with MTA, this in turn is enhanced in an MTA-rich environment, thereby presenting new "collateral vulnerability" opportunities.

[0013] In another aspect of the present disclosure, 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 carrying a wild-type MTAP gene and characteristically accumulating MTA.

[0014] In another aspect of the present specification, 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 carrying a wild-type MTAP gene and characteristically accumulating MTA due to MTAP gene silencing.

[0015] In another aspect of the present specification, a treatment method is provided, which comprises administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has been identified as having a tumor carrying a wild-type MTAP gene and characteristically accumulating MTA due to MTAP gene silencing mediated by hypermethylation of MTAP.

[0016] In another aspect, the disclosure provides 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 tumor carrying a wild-type MTAP gene and accumulating MTA due to downregulation of MTAP at the protein level.

[0017] In another aspect, the disclosure 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 carries a wild-type MTAP gene but accumulates MTA due to downregulated MTAP protein expression.

[0018] In another aspect, the disclosure provides a method of treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but characteristically accumulates MTA due to epigenetic downregulation of MTAP mRNA.

[0019] In another aspect, the disclosure provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but accumulates MTA due to partial or complete silencing of MTAP protein expression.

[0020] In another aspect, the present disclosure provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but accumulates MTA due to partial or complete silencing of MTAP protein expression caused by epigenetic modification of the MTAP gene.

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

[0022] In another aspect, the disclosure provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but accumulates MTA due to downregulation of MTAP mRNA.

[0023] In another aspect, the present disclosure provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but accumulates MTA due to downregulation of MTAP mRNA caused by hypermethylation at or around the MTAP gene.

[0024] In another aspect, the present disclosure provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries 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 (e.g., CDKN2A) or any other genomic location.

[0025] In another aspect, the present disclosure provides a method for identifying patients who would benefit from treatment with an MTA-synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient to confirm that the tumor carries a wild-type MTAP gene but is still susceptible to accumulating MTA, optionally wherein the identification is performed via an immunohistochemical assay demonstrating that the relevant cell population lacks MTAP protein.

[0026] In the above aspects, the accumulation of MTA may be in both the nucleus and cytoplasm of the relevant cells, or may be localized in the nucleus of the relevant cells.

[0027] In the above aspects, the accumulation of MTA can be determined by immunochemical staining of MTAP in a sample obtained from the patient.

[0028] In another aspect, the disclosure provides a method for identifying a patient who would benefit from treatment with an MTA-synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient and identifying that the associated tumor cells are wild-type MTAP gene silenced.

[0029] In another aspect, the disclosure provides a method for identifying a patient who would benefit from treatment with an MTA-synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient and determining reduced MTAP protein or mRNA expression levels in relevant tumor cells, optionally as identified by immunohistochemistry.

[0030] In another aspect, the disclosure provides a method of identifying a patient who would benefit from treatment with an MTA-synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient to confirm that the tumor i) carries a wild-type MTAP gene and ii) is MTAP-deficient or MTAP-null.

[0031] In another aspect, the disclosure provides a method of identifying a patient who would benefit from treatment with an MTA-synergistic PRMT5 inhibitor, the method comprising analyzing a sample obtained from the patient to confirm that the tumor i) carries a wild-type MTAP gene and ii) is null or deficient in MTAP mRNA.

[0032] In another aspect, the disclosure provides a method for identifying a patient who would benefit from treatment with an MTA-synergistic PRMT5 inhibitor, the method comprising performing an immunohistochemical assay for MTAP on a tumor sample obtained from the patient and identifying the associated tumor cells as MTAP null or deficient.

[0033] In another aspect, the disclosure provides a method of treating cancer comprising the steps of: i) identifying a patient as having a tumor that accumulates MTA, such as by performing an immunohistochemical assay for MTAP, and ii) administering to the patient an MTA-synergistic PRMT5 inhibitor.

[0034] In another aspect, the disclosure provides an MTA-synergistic PRMT5 inhibitor for use in treating cancer, wherein the cancer carries a wild-type MTAP gene and accumulates MTA.

[0035] In another aspect, the disclosure provides an MTA-synergistic PRMT5 inhibitor for use in treating cancer, wherein the tumor carries a wild-type MTAP gene but remains MTAP null or deficient at the protein level.

[0036] In another aspect, the disclosure provides a kit comprising an MTA-synergistic PRMT5 inhibitor and instructions for use in treating a cancer that carries a wild-type MTAP gene but is still MTAP null or deficient at the protein level.

[0037] In order to more easily understand the present description, the following drawings are referred to herein:

[0038] Figure 1 :Cancer Cell Line Encyclopedia (CCLE, https: / / sites.broadinstitute.org / ccle / ) is a graph showing MTAP mRNA expression in tumor cells compared to MTAP copy number. Figure 1 A presents a complete diagram of tumor cells in CCLE. Samples within the boxed area have at least one copy of the wild-type MTAP gene. Figure 1 B is Figure 1 The region of the graph of A comprises cell lines that carry the wild-type MTAP gene but whose MTAP gene expression is still reduced (as reflected by low MTAP mRNA expression). Figure 1 C is Figure 1 A diagram of the seven Hodgkin lymphoma cell lines presented in .

[0039] Figure 2 : Figure 2 A Graph of MTAP DNA methylation (y axis) compared to MTAP mRNA expression in seven HL cell lines in CCLE; Figure 2 B: Methylation of all MTAP gene transcription sites. The X-axis shows the chromosomal location of the promoter methylation CpG cluster of the MTAP promoter region by reduced representation bisulfite sequencing (RRBS) (data obtained from Broad Institute https: / / data.broadinstitute.org / ccle / ); Figure 2 C Western blot of MTAP and GAPDH of four HL cell lines (L540, L1236, KMH2, HDLM2) carrying wild-type MTAP gene but with its gene silenced, along with the non-MTAP silenced HL cell line L428. The HCT116 colorectal cell line was included as a positive (MTAP wild-type) and negative (MTAPKO) control.

[0040] Figure 3: This graph illustrates that 84% of HL histopathology tumor samples were stained for MTAP protein in both the nucleus and cytoplasm. Of the 46 HL samples lacking nuclear MTAP, 14 also lacked MTAP in the cytoplasm, 27 showed faint MTAP staining in the cytoplasm, and 5 had more than +1 MTAP staining in the cytoplasm.

[0041] Figure 4 :Normal tonsil tissue( Figure 4 A) and NSCLC ( Figure 4 B) Histopathological sections. Figure 4 MTAP staining was visible in all cells in A. In contrast, Figure 4 In B, MTAP staining was limited to tumor-infiltrating lymphocytes (right, darkly stained areas), whereas NSCLC cells extending from the upper left to the lower right corner of the figure showed no staining for MTAP.

[0042] Figure 5 : Histopathological image taken from Küppers, R. and Hansmann, M.-L., Int J Biochem & Cell Biol., 37(3), 2005 p511-17, showing the characteristic tumor clone of Hodgkin lymphoma, Hodgkin Reed / Sternberg (HRS) cells, stained with CD30 within a larger lymphoma cell population.

[0043] Figure 6 : Using MTAP antibody and diluent containing casein ( Figure 6 A, 2 μg / mL mAb) or standard casein-free diluent ( Figure 6 B, Histopathological image of sample No. 243969-LN-1 (HL subtype: MC interfollicular) obtained with 0.5 μg / mL of MTAP. There was no MTAP staining in both the nuclear and cytoplasmic compartments of HRS cells.

[0044] Figure 7 : Using MTAP antibody and diluent containing casein ( Figure 7 A, 2 μg / mL mAb) or standard casein-free diluent ( Figure 7 B, Histopathological images of sample No. 243957-LN-1 (HL subtype: NS) obtained with 0.5 μg / mL of MTAP. For MTAP, a complete absence of nuclear staining was observed in HRS cells, but some faint cytoplasmic staining.

[0045] Figure 8 : Using MTAP antibody and diluent containing casein ( Figure 8 A, 2 μg / mL mAb) or standard casein-free diluent ( Figure 8 B, Histopathological images of sample No. 243958-LN-1 (HL subtype: NS syncytia) obtained with 0.5 μg / mL of MTAP. For MTAP, a complete absence of nuclear staining was observed in HRS cells, but some faint cytoplasmic staining.

[0046] Fig. 9 : Using MTAP antibody and diluent containing casein ( Fig. 9 A, 2 μg / mL mAb) or standard casein-free diluent ( Fig. 9 B, Histopathological images of sample No. 243965-LN-1 (HL subtype: MC) obtained with 0.5 μg / mL of MTAP. For MTAP, a complete absence of nuclear staining was observed in HRS cells, but some faint cytoplasmic staining.

[0047] Fig.10 : Histopathological images obtained for sample number 243970-LN-1 (HL subtype: NS syncytium) in which HRS cells are easily discerned as "light" areas, in which case cells are designated as exhibiting a complete absence of nuclear staining and 1+ cytoplasmic staining in HRS cells. Fig.10 A, 2 μg / mL mAb) or standard casein-free diluent ( Fig.10 B, 0.5 μg / mL) for staining.

[0048] Fig.11 : Histopathological images obtained for sample number 243963-LN-1 (HL subtype: LRHL) in which HRS cells are easily discerned as "light" areas, in which case cells are designated as showing a complete absence of nuclear staining and 1+ cytoplasmic staining in HRS cells. Fig.11 A, 2 μg / mL mAb) or standard casein-free diluent ( Fig.11 B, 0.5 μg / mL) for staining.

[0049] Fig.12 : Histopathological images of sample No. 243959-LN-1 (HL subtype: NS) obtained, in which HRS cells were stained for MTAP in the nucleus and cytoplasm of HRS cells. MTAP antibody and diluent containing casein ( Fig.12 A, 2 μg / mL mAb) or standard casein-free diluent ( Fig.12 B, 0.5 μg / mL) for staining.

[0050] Fig.13: Histopathological images of sample No. 243961-LN-1 (HL subtype: NLPHL) obtained, in which HRS cells were stained for MTAP in the nucleus and cytoplasm of LP (lymphocyte-predominant) cells. Fig.13 A, 2 μg / mL mAb) or standard casein-free diluent ( Fig.13 B, 0.5 μg / mL) for staining.

[0051] Fig.14 : This graph illustrates the effect of Compound C treatment on relative tumor volume in the L540 HL xenograft model.

[0052] Fig.15 : This graph illustrates the effect of Compound C treatment on mouse body weight in the L540 HL xenograft model.

[0053] Fig.16 : This graph illustrates the effect of Compound C treatment on SDMA protein levels in the L540 HL xenograft model.

[0054] The application opportunity of MTA synergistic PRMT5 inhibitors in the treatment of tumors that do not have CDKN2A / MTAP gene deletion is realized from the following new observations: some tumors carry wild-type MTAP genes, but still accumulate MTA due to complete or partial silencing of MTAP genes. Based on the research described in this article, this specific phenotype seems to be derived from hypermethylation at or around the MTAP gene in some tumors, which in turn leads to significant or complete silencing of the MTAP gene or downregulation of MTAP gene expression, resulting in reduced intratumoral concentrations of MTAP protein. Functionally, hypermethylation at or around the MTAP gene reduces MTAP gene expression, wherein MTAP mRNA levels are reduced to low levels or eliminated, i.e., the wild-type MTAP gene is silenced. This in turn leads to a reduction or absence of MTAP protein in tumor cells. MTAP is an enzyme that plays a major role in polyamine metabolism and is important for the salvage of adenine and methionine. In the context of cancer treatment strategies, the loss of MTAP protein removes the clearance mechanism of methylthioadenosine (MTA) and leads to the accumulation of MTA. Tumor cells or tumors containing a population of related cells carrying a wild-type MTAP gene but still accumulating MTA are identified herein as targets that are susceptible to treatment with MTA-synergistic PRTM5 inhibitors (PRMT5 inhibitors that bind PRMT5 in combination with MTA). In more detail, since MTA-synergistic PRMT5 inhibitors express their optimal activity only in cells with high concentrations of MTA, selective cytotoxic effects can be used to avoid or significantly reduce off-target toxicity associated with non-MTA-selective PRMT5 inhibitors, a phenomenon that has been observed in the clinic.

[0055] The opportunity to selectively target certain tumors carrying the wild-type MTAP gene with MTA-synergistic PRMT5 inhibitors was realized from the analysis of the Cancer Cell Line Encyclopedia (CCLE, https: / / sites.broadinstitute.org / ccle / ). In more detail, CCLE was searched for tumor cell lines with low MTAP mRNA levels, a feature that was previously associated with cells with homozygous deletion of CDKN2A / MTAP in the work described herein. Therefore, MTAP mRNA levels (determined by RNAseq) were plotted against MTAP (gene) copy number (see Figure 1 A) As expected, the search results revealed that Figure 1 A Cluster of MTAP gene-deficient cell lines in the lower left corner (note that Figure 1 The copy number ratio on the x-axis of A, 1B, and 1C is the log of the copy number 2 –1, so log 2 Cells with a copy number ≥ -1 expressed at least one wild-type MTAP gene copy, while log 2 Cells with a copy number of -1≤-2 are MTAP null, i.e., they do not express the MTAP gene. Figure 1 The cells carrying the wild-type MTAP gene in the upper right corner of A (those with log 2 Unexpectedly, a group of cells in CCLE were found to carry the wild-type MTAP gene and were therefore placed on the right side of the graph, but nonetheless displayed MTAP mRNA levels comparable to those of tumor cells with CDKN2A / MTAP gene deletion (see Figure 1 A shows a cluster of cells in the lower right box (MTAP copy number ≥ -1, MTAP mRNA ≤ 0). We refer to such cells that carry the wild-type MTAP gene but still exhibit reduced MTAP mRNA expression as wild-type MTAP gene-silenced cells, and by extension, tumors containing clonal tumor cells of this phenotype are referred to herein as wild-type MTAP gene-silenced tumors.

[0056] Figure 1 B presents a magnified view of the MTAP gene-silenced tumor cell population, i.e., those tumor cells that carry the wild-type MTAP gene but also exhibit low levels or complete absence of MTAP mRNA. Table 1 provides a table correlating the MTAP gene-silenced tumor cell types compared to the total number of models, broken down by tissue of origin or tumor type, and categorized by the incidence of the MTAP gene-silenced phenotype for each tissue of origin. Figure 1B and inspection of Table 1 show that 23 tumor cell lines from CCLE were identified as carrying both wild-type MTAP gene and MTAP silenced. Although many tumor cells are outliers in this figure in terms of their tissue of origin or tumor type incidence, a large proportion of Hodgkin lymphomas (4 / 7) and non-Hodgkin lymphomas (5 / 27) exhibited this MTAP silenced feature. Figure 1 Data for all seven Hodgkin lymphoma cell lines in the CCLE are presented in C.

[0057] Table 1: Tumor cell models in CCLE that carry the wild-type MTAP gene but are MTAP-silenced (as evidenced by their low MTAP mRNA levels) and the incidence of this feature based on tissue of origin or tumor type

[0058]

[0059] This observation inspired further exploration of the origins of MTAP gene silencing, aimed at determining whether the ‘collateral vulnerability’ opportunity provided by the availability of MTA-synergistic PRMT5 inhibitors could be exploited outside of non-CDKN2a / MTAP-deficient tumors.

[0060] To understand the origin of MTAP gene silencing in tumor cell lines carrying wild-type MTAP genes, we searched for unifying features present in 4 / 7 Hodgkin lymphoma cell lines that carry wild-type MTAP genes but are MTAP gene-silenced (relative to the remaining 3 non-MTAP gene-silenced HL cell lines). It was determined that 4 of the 7 HL cell lines with gene silencing, HDLM2, L540, KMH2, and L1236 (see Figure 2 All MTAP gene transcription sites in A are also methylated ( Figure 2 B), i.e., the MTAP gene is hypermethylated. In contrast, the three HL cell lines in which the MTAP gene is not methylated express normal levels of MTAP mRNA and should therefore express MTAP at the protein level. Western blots of MTAP protein are shown in Figure 2 C, and confirmed that MTAP protein is only present in the L428 cell line expressing MTAP mRNA, while the HDLM2, L540, KMH2, and L1236 cell lines in which the MTAP gene is hypermethylated are MTAP protein null. Therefore, the data indicate that hypermethylation of the MTAP gene leads to MTAP gene silencing in many tumors.

[0061] Having identified the possible epigenetic origin of MTAP gene silencing in HL cell lines, an experiment was performed to confirm that an MTA-synergistic PRMT5 inhibitor would be able to inhibit the growth of MTAP gene-silenced HL cell lines and should accumulate MTA accordingly. Therefore, the activity of 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 gene silenced) and L428 (intact MTAP expression), as well as wild-type and MTAP knockout HCT116 cells. The activity of the non-selective PRMT5 inhibitor Compound B (GSK3326595, as described in WO 2015 / 198229A1, commercially available from, for example, medchemexpress.com - catalog number HY-101563) in all the same cells was evaluated in parallel. The results obtained from these experiments are presented in Table 2.

[0062] Table 2: In vitro activity of MTA-synergistic (Compounds A and C) and non-selective (Compound B) PRMT5 inhibitors on MTAP gene silencing and proliferation of MTAP-expressing Hodgkin lymphoma cells as well as wild-type (wt) and MTAP knockout (KO) HCT116 cells.

[0063]

[0064] *Average from two or more experiments.

[0065] As can be seen from Table 2, all cell lines are sensitive to treatment with non-selective PRMT5 inhibitor compound B. In contrast, MTA synergistic PRMT5 inhibitor compound A mainly expresses its activity in MTAP gene silencing cell lines (HDLM2, L540 and L1236) and MTAP knockout HCT116 cell lines (wherein MTAP mRNA levels are low or absent, and MTA is accumulated accordingly due to the absence or reduction of MTAP protein). Similarly, compound C shows preferential activity in the same cell lines treated with compound A (i.e., those cell lines that accumulate MTA due to the absence or reduction of MTAP protein). Therefore, the potential of targeting tumors carrying wild-type MTAP genes but still accumulating MTA using MTA synergistic PRMT5 inhibitors is demonstrated in vitro.

[0066] To investigate whether the prevalence of the MTAP wild-type / MTAP-silenced phenotype in CCLE is representative of HL in the clinic, we obtained a panel of Hodgkin lymphoma samples and set out to assess whether a significant proportion of clinical Hodgkin lymphoma samples lack MTAP protein expression and, therefore, accumulate MTA. To perform this analysis, an immunohistochemical (IHC) method for detecting MTAP at the protein level was developed and applied to the analysis of clinical samples. The results obtained from these experiments are summarized in Figure 3 and Table 3a and Table 3b.

[0067] Table 3a: Immunohistochemical analysis of MTAP protein in 15 Hodgkin's lymphoma clinical samples

[0068]

[0069]

[0070]

[0071] * = age at diagnosis. Note that no 2+ or 3+ staining scores were obtained for nuclear or cytoplasmic MTAP in any of the 15 samples.

[0072] Table 3b: Immunohistochemical analysis of MTAP protein in 40 additional Hodgkin's lymphoma clinical samples

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] As can be seen in Tables 3a and 3b, IHC analysis revealed that 46 of the 55 primary HL samples (84%) had nuclear MTAP loss, in other words, according to the judgment of an expert histopathologist, MTAP protein was completely absent from the nuclei of Hodgkin and Reed / Sternberg cells in these 46 Hodgkin lymphoma samples. Interestingly, 32 of the 46 samples with MTAP loss expressed some degree of MTAP in the cytoplasm, and the cytoplasmic MTAP score was +1 or faint. Based on this sample set, it is clear that a large proportion of Hodgkin lymphomas have reduced MTAP expression, a finding consistent with the finding that MTAP expression is reduced in Hodgkin lymphomas. Figure 1 The data presented in the above CCLE are well correlated.

[0080] To illustrate the IHC studies performed to obtain the above data, Figure 4 Shown is a normal tonsil tissue sample stained with MTAP antibody, showing staining across the entire panel ( Figure 4 A), while staining of NSCLC samples ( Figure 4 B) shows darkly stained areas (attributed to tumor infiltrating lymphocytes) and essentially no MTAP-stained tumor cells, and faint blue and light-colored areas revealing the nucleus and cytoplasm in the color image.

[0081] To understand Figure 5 The importance of the histopathological images presented in the next, first of all, it is important to remember that Hodgkin lymphoma tissue contains relatively few cloned tumor cells in a wider group of normal cells (see above). In more detail, Hodgkin and Reed / Sternberg cells (herein referred to as HRS cells) are the hallmark cells of Hodgkin lymphoma, and are large, usually multinuclear cells with characteristic and unique morphology and unusual immunophenotypes, which are not similar to any normal cells in the body (see, for example, Küppers, R. and Hansmann, M.-L., Int J Biochem & Cell Biol. [International Journal of Biochemistry and Cell Biology], 37 (3), 2005p 511-17). Hodgkin cells are typical mononuclear cells, while Reed / Sternberg cells are multinuclear cells. Although HRS cells are rare in HL tissues, they are cloned tumor cells of HL. HRS cells in almost all HL cases are derived from B cells, and only a few are derived from T cells. Notably, the somatic mutation pattern of their rearranged immunoglobulin V genes suggests that they are derived from pre-apoptotic germinal center B cells. The pathogenesis of HL remains largely unresolved, but it is now clear that aberrant activation of several signaling pathways (e.g., the NFκB pathway) is essential for HRS cell survival. HRS cells or HRS-like cells are also present in several other diseases, for example, rare hybrid cells are present in some non-Hodgkin lymphomas and infectious mononucleosis. To illustrate the incidence of HRS cells in HL, images of HL samples stained with CD30 from the literature are provided here, as shown in Figure 5 A and Figure 5 The enlarged image in B.

[0082] Go to the data of a single IHC analysis, Figure 6 The results show that the MTAP antibody and casein-containing diluent ( Figure 6 A, 2 μg / mL mAb) or standard casein-free diluent ( Figure 6B, histopathological image of sample No. 243969-LN-1 (HL subtype: MC interfollicular) obtained with 0.5 μg / mL. Figure 6 All IHC images mentioned in the table were obtained under the same conditions, using MTAP antibody and casein-containing diluent ( Figure X a, where X = 7 to 13) or the same concentration of standard casein-free diluent ( Figure X b) In this image, HRS cells are the light-colored areas of a stained histopathology slide and IHC analysis revealed the complete absence of MTAP from both the nuclear and cytoplasmic compartments of HRS cells.

[0083] Figure 7 Data for sample number 243957-LN-1 (HL subtype: NS) are presented. Likewise, no nuclear staining of MTAP was observed in the nuclei of HRS cells, but faint MTAP staining was observed in the cytoplasmic compartment of HRS cells. Figure 8 (Sample number: 243958-LN-1, HL subtype: NS syncytial) and Fig. 9 (Sample number: 243965-LN-1, HL subtype: MC) also revealed a complete absence of nuclear staining in HRS cells, but some faint cytoplasmic staining.

[0084] Fig.10 Data are presented for sample number 243970-LN-1 (HL subtype: NS syncytia), in which HRS cells were readily discernible as "light" areas, in which case cells were designated as exhibiting a complete absence of nuclear staining in HRS cells and 1+ cytoplasmic staining. LRHL subtype sample number: 243963-LN-1 (see Fig.11 ) were similarly found in HRS cells, where the cells showed a complete absence of nuclear staining and 1+ cytoplasmic staining.

[0085] In contrast, Fig.12 and Fig.13 IHC slides of sample number: 243959-LN-1, HL subtype: NS and sample number: 243961-LN-1, HL subtype: NLPHL are shown in FIG, respectively, where MTAP staining was observed in HRS cells in each case.

[0086] Thus, based on the 55 clinical samples analyzed and in accordance with the observations obtained from the CCLE, we identified that a large proportion of Hodgkin lymphomas accumulate MTA and that this MTA accumulation is due to MTAP gene silencing. The observation that loss of MTAP protein expression was very common in the nucleus (as seen in 46 of the 55 HL samples), but residual levels of MTAP protein expression were present in 32 of the 46 samples that did 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, the “Protein Subcellular Localization Prediction Tool” (PSORTII) analysis revealed no nuclear localization signal for MTAP protein and predicted that MTAP protein was proportionally distributed in the cytoplasm (78.3%), nucleus (17.4%), and endoplasmic reticulum (4.3%) (see https: / / psort.hgc.jp / form2.html ). The interpolation obtained from this prediction suggests that when MTAP protein levels are low (as determined by partial MTAP gene silencing), it is possible that MTAP protein may still be detected in the cytoplasm when MTAP protein levels in the nucleus are reduced to below the detection limit. In other words, a uniform % reduction in MTAP protein throughout the cell is more likely to result in MTAP protein levels in the nucleus relative to the cytoplasm being below the detection limit.

[0087] Reports in the wider scientific literature indicate that if MTAP protein is expressed in a cell, then depending on the expression level, the MTAP protein is expected to be in one or both of the cytoplasm and the nucleus (when the expression level is high) or only in the cytoplasm (when the expression level is low) but is unlikely to be observed only in the nucleus.

[0088] In the context of the data reported here, we conclude that loss of MTAP protein from the nucleus in HL samples, as detected by IHC, is a strong indicator of overall MTAP protein deficiency, which would lead to intracellular MTA accumulation and could be used as a surrogate marker of sensitivity to MTA-synergistic PRMT5 inhibitors.

[0089] Thus, the IHC experiments reported herein demonstrate that a very large proportion of HL clinical tumor samples lack MTAP. Since these tumors will inevitably accumulate elevated concentrations of MTA, and given the availability of MTA-synergistic PRMT5 inhibitors, a new approach to treating HL, where HL characteristically accumulates MTA, is revealed for the first time. This new "collateral vulnerability" opportunity should provide an effective option for the treatment of HL, characterized by a favorable side effect profile, since significant targeting of PRMT5 in healthy cells expressing the MTAP protein should be minimal. Furthermore, this opportunity should be equally applicable to other tumor types, such as those selected from those disclosed in Table 1 in which MTAP gene silencing is present, which are characterized by carrying a wild-type MTAP gene, but still having the MTAP gene silencing phenotype described for the first time herein.

[0090] 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 L540 HL xenograft model (see Biological Example 1).

[0091] As described above, in a first embodiment, the present specification provides a method of treating cancer, the method 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 a wild-type MTAP gene has been silenced.

[0092] In an embodiment, MTAP gene silencing causes partial or complete loss of MTAP gene expression protein in relevant tumor cells. Partial or complete loss of MTAP gene expression can be determined by immunohistochemical analysis or any other appropriate technique (e.g., RT-qPCR that allows quantification of MTAP protein or mRNA in relevant cells or cell compartments).

[0093] In embodiments, MTAP gene silencing results in partial or complete loss of MTAP protein in the nuclei of relevant tumor cells.

[0094] In an embodiment, MTAP gene silencing results in partial or complete loss of MTAP mRNA in relevant tumor cells. Partial or complete loss of MTAP mRNA can be determined by RNA-Seq, in situ hybridization or any other appropriate technique.

[0095] In embodiments, MTAP gene silencing results in reduced MTAP protein expression in the nuclei of tumor cells. In embodiments, MTAP gene silencing results in reduced MTAP protein expression in the nuclei of clonal tumor cells. In embodiments, the tumor cells are Hodgkin's Reed / Sternberg cells and the cancer is Hodgkin's lymphoma.

[0096] In an embodiment, the disclosure 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 tumor carrying a wild-type MTAP gene and characteristically accumulating MTA.

[0097] In an embodiment, the present description 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 tumor carrying a wild-type MTAP gene and characteristically accumulating MTA due to MTAP gene silencing.

[0098] In an embodiment, the present description provides 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 tumor carrying a wild-type MTAP gene and characteristically accumulating MTA in associated tumor cells due to MTAP gene silencing mediated by hypermethylation of MTAP.

[0099] In an embodiment, the present description provides 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 tumor carrying a wild-type MTAP gene and characteristically accumulating MTA in associated tumor cells due to epigenetic modification of the MTAP gene.

[0100] In an embodiment, the present description provides a method of treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient has a tumor carrying a wild-type MTAP gene but accumulating MTA in associated tumor cells due to downregulation of MTAP at the protein level.

[0101] In an embodiment, the present description 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 carrying a wild-type MTAP gene but accumulating MTA in associated tumor cells due to downregulated MTAP protein expression.

[0102] In an embodiment, the present description provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but characteristically accumulates MTA in associated tumor cells due to epigenetic downregulation of MTAP mRNA.

[0103] In an embodiment, the present description provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but accumulates MTA in associated tumor cells due to partial or complete silencing of MTAP protein expression.

[0104] In an embodiment, 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 carries a wild-type MTAP gene but accumulates MTA in associated tumor cells due to partial or complete silencing of MTAP protein expression caused by epigenetic modification of the MTAP gene.

[0105] In an embodiment, 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 carries a wild-type MTAP gene but accumulates MTA in associated tumor cells due to partial or complete silencing of MTAP protein expression caused by hypermethylation of the MTAP gene.

[0106] In an embodiment, the present description provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein the patient's tumor carries a wild-type MTAP gene but accumulates MTA in associated tumor cells due to downregulation of MTAP mRNA.

[0107] In an embodiment, 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 carries a wild-type MTAP gene but accumulates MTA in associated tumor cells due to downregulation of MTAP mRNA caused by hypermethylation at or around the MTAP gene.

[0108] In an embodiment, 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 carries 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 (e.g., CDKN2A).

[0109] In an embodiment, the present specification provides a method for treating cancer, the method comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, the method comprising analyzing a sample obtained from the patient to confirm that the tumor carries a wild-type MTAP gene but is still susceptible to accumulating MTA (based on an immunohistochemical assay that indicates that the relevant tumor cell population lacks MTAP protein).

[0110] In an embodiment, the present specification provides a method for treating cancer, comprising administering an MTA-synergistic PRMT5 inhibitor to a patient in need thereof, wherein MTA can accumulate in both the nucleus and cytoplasm of relevant tumor cells, or can be localized in the nucleus of relevant cells.

[0111] In embodiments, accumulation of MTA may be in both the nucleus and cytoplasm of the relevant cell, or may be localized or substantially localized to the nucleus of the relevant cell.

[0112] In an embodiment, identification of a tumor's propensity to accumulate MTA can be determined by immunochemical staining for MTAP on a sample obtained from a patient.

[0113] In an embodiment, use of an MTA-synergistic PRMT5 inhibitor for treating cancers in which the wild-type MTAP gene is silenced is provided.

[0114] In an embodiment, a use of an MTA-synergistic PRMT5 inhibitor for treating cancer in which the wild-type MTAP gene is silenced is provided, wherein the MTAP gene is silenced

[0115] (a) causing partial or complete loss of MTAP protein in relevant tumor cells; or

[0116] (b) causing partial or complete loss of MTAP protein in the nucleus of the relevant tumor cells; or

[0117] (c) leading to decreased expression of MTAP protein in the nucleus of tumor cells; or

[0118] (d) resulting in decreased MTAP protein expression in the nuclei of cloned tumor cells; or

[0119] (e) characteristically results in the accumulation of MTA in the relevant tumor cells; or

[0120] (f) MTAP gene silencing mediated by MTAP hypermethylation characteristically leads to the accumulation of MTA in the relevant tumor cells; or

[0121] (g) characteristically results in the accumulation of MTA in the relevant tumor cells due to epigenetic modification of the MTAP gene; or

[0122] (h) characteristically leads to accumulation of MTA in the relevant tumor cells due to downregulation of MTAP at the protein level; or

[0123] (i) due to down-regulation of MTAP protein expression, characteristically leading to the accumulation of MTA in relevant tumor cells; or

[0124] (j) leading to epigenetically driven downregulation of MTAP mRNA; or

[0125] (k) accumulation of MTA in the relevant tumor cells due to partial or complete silencing of MTAP protein expression; or

[0126] (l) partial or complete silencing of MTAP protein expression due to epigenetic modification of the MTAP gene, leading to accumulation of MTA in the relevant tumor cells; or

[0127] (m) causing the tumor to characteristically accumulate MTA due to partial or complete silencing of MTAP protein expression caused by hypermethylation of the MTAP gene; or

[0128] (n) causing tumors to characteristically accumulate MTA due to downregulation of MTAP mRNA caused by hypermethylation at or around the MTAP gene; or

[0129] (o) Tumors characteristically accumulate MTA due to downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or neighboring genes (e.g., CDKN2A).

[0130] In an embodiment, there is provided a use of an MTA-synergistic PRMT5 inhibitor for the manufacture of a medicament for treating a cancer characterized by silencing of a wild-type MTAP gene.

[0131] In an embodiment, a use of an MTA-synergistic PRMT5 inhibitor for the manufacture of a medicament for treating a cancer in which a wild-type MTAP gene is silenced is provided, wherein the MTAP gene is silenced

[0132] (a) causing partial or complete loss of MTAP protein in relevant tumor cells; or

[0133] (b) causing partial or complete loss of MTAP protein in the nucleus of the relevant tumor cells; or

[0134] (c) leading to decreased expression of MTAP protein in the nucleus of tumor cells; or

[0135] (d) resulting in decreased MTAP protein expression in the nuclei of cloned tumor cells; or

[0136] (e) characteristically results in the accumulation of MTA in the relevant tumor cells; or

[0137] (f) MTAP gene silencing mediated by MTAP hypermethylation characteristically leads to the accumulation of MTA in the relevant tumor cells; or

[0138] (g) characteristically results in the accumulation of MTA in the relevant tumor cells due to epigenetic modification of the MTAP gene; or

[0139] (h) characteristically leads to accumulation of MTA in the relevant tumor cells due to downregulation of MTAP at the protein level; or

[0140] (i) due to down-regulation of MTAP protein expression, characteristically leading to the accumulation of MTA in relevant tumor cells; or

[0141] (j) leading to epigenetically driven downregulation of MTAP mRNA; or

[0142] (k) accumulation of MTA in the relevant tumor cells due to partial or complete silencing of MTAP protein expression; or

[0143] (l) partial or complete silencing of MTAP protein expression due to epigenetic modification of the MTAP gene, leading to accumulation of MTA in the relevant tumor cells; or

[0144] (m) causing the tumor to characteristically accumulate MTA due to partial or complete silencing of MTAP protein expression caused by hypermethylation of the MTAP gene; or

[0145] (n) causing tumors to characteristically accumulate MTA due to downregulation of MTAP mRNA caused by hypermethylation at or around the MTAP gene; or

[0146] (o) Tumors characteristically accumulate MTA due to downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or neighboring genes (e.g., CDKN2A).

[0147] As used herein and above, the term "relevant tumor cells" is used to refer to those cells that drive tumor growth and maintain tumor survival. An example of a relevant tumor cell in each embodiment referring to a "relevant tumor cell" is a Hodgkin's Reed / Sternberg cell as found in Hodgkin's lymphoma.

[0148] In an embodiment, a method for identifying patients who will benefit from treatment with an MTA-synergistic PRMT5 inhibitor is provided, the method comprising the following steps: identifying relevant tumor cells in the sample that exhibit a reduction in MTAP protein in their nuclei and, optionally, also in their cytoplasm by performing an immunohistochemical assay for MTAP protein on a sample obtained from the patient.

[0149] In an embodiment, a method for identifying a patient with Hodgkin lymphoma who will benefit from treatment with an MTA-synergistic PRMT5 inhibitor is provided, the method comprising the steps of analyzing MTAP expression in a tumor sample obtained from the patient and identifying the associated tumor cells as being wild-type MTAP gene silenced. In such an embodiment, identification of the wild-type MTAP gene silenced state is performed by an immunohistochemical assay that shows reduced or absent MTAP protein expression in the nucleus and / or cytoplasm of Hodgkin / Schmidt cells.

[0150] In embodiments involving the above-described methods of treatment, the methods of treatment may further include the step of analyzing a sample obtained from a patient suffering from cancer, the patient suffering from a cancer in which the wild-type MTAP gene is silenced. In such embodiments, the silenced wild-type MTAP gene may be determined based on an immunochemical assay for the MTAP protein, which reveals, for example, in the case of Hodgkin's lymphoma in Hodgkin's Reed / Sternberg cells, reduced or absent MTAP protein expression in the nucleus and / or cytoplasm of the relevant tumor cells.

[0151] In embodiments involving the use of an MTA-synergistic PRMT5 inhibitor for use in treating cancer, the use may be indicated based on results obtained from analysis of a sample obtained from a patient in need of treatment that indicates that the patient suffers from a cancer in which the wild-type MTAP gene is silenced. In such embodiments, the silenced wild-type MTAP gene may be determined based on an immunochemical assay for the MTAP protein that reveals, for example, in the case of Hodgkin's lymphoma in Hodgkin's Reed / Sternberg cells, reduced or absent MTAP protein expression in the nucleus and / or cytoplasm of the relevant tumor cells.

[0152] In embodiments involving the use of an MTA-synergistic PRMT5 inhibitor for the manufacture of a medicament for the treatment of cancer, the use of the resulting medicament can be indicated based on the identification of the patient as having a tumor in which the wild-type MTAP gene has been silenced following analysis of a sample obtained from the patient. In such embodiments, the silenced wild-type MTAP gene can be determined based on an immunochemical assay for the MTAP protein that reveals, for example, in the case of Hodgkin's lymphoma in Hodgkin's Reed / Sternberg cells, reduced or absent MTAP protein expression in the nucleus and / or cytoplasm of the relevant tumor cells.

[0153] In an embodiment, a kit is provided, the kit comprising an MTA-synergistic PRMT5 inhibitor and instructions for use in treating a cancer in which the wild-type MTAP gene is silenced. In such an embodiment, the instructions can characterize a cancer in which the wild-type MTAP gene is silenced based on:

[0154] (a) causing partial or complete loss of MTAP protein in relevant tumor cells; or

[0155] (b) causing partial or complete loss of MTAP protein in the nucleus of the relevant tumor cells; or

[0156] (c) leading to decreased expression of MTAP protein in the nucleus of tumor cells; or

[0157] (d) resulting in decreased MTAP protein expression in the nuclei of cloned tumor cells; or

[0158] (e) characteristically results in the accumulation of MTA in the relevant tumor cells; or

[0159] (f) MTAP gene silencing mediated by MTAP hypermethylation characteristically leads to the accumulation of MTA in the relevant tumor cells; or

[0160] (g) characteristically results in the accumulation of MTA in the relevant tumor cells due to epigenetic modification of the MTAP gene; or

[0161] (h) characteristically leads to accumulation of MTA in the relevant tumor cells due to downregulation of MTAP at the protein level; or

[0162] (i) due to down-regulation of MTAP protein expression, characteristically leading to the accumulation of MTA in relevant tumor cells; or

[0163] (j) leading to epigenetically driven downregulation of MTAP mRNA; or

[0164] (k) accumulation of MTA in the relevant tumor cells due to partial or complete silencing of MTAP protein expression; or

[0165] (l) partial or complete silencing of MTAP protein expression due to epigenetic modification of the MTAP gene, leading to accumulation of MTA in the relevant tumor cells; or

[0166] (m) causing the tumor to characteristically accumulate MTA due to partial or complete silencing of MTAP protein expression caused by hypermethylation of the MTAP gene; or

[0167] (n) causing tumors to characteristically accumulate MTA due to downregulation of MTAP mRNA caused by hypermethylation at or around the MTAP gene; or

[0168] (o) Tumors characteristically accumulate MTA due to downregulation of MTAP mRNA caused by hypermethylation of the MTAP gene and / or neighboring genes (e.g., CDKN2A).

[0169] Thus, for illustration, the kit may provide instructions for use of an MTA-synergistic PRMT5 inhibitor in treating a wild-type MTAP gene-silenced cancer that characteristically accumulates MTA due to MTAP gene silencing mediated by hypermethylation of the MTAP gene, as specified in use (f).

[0170] In an embodiment, the cancer in which the wild-type MTAP gene is silenced is a cancer of the lymphatic system, for example, 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) and may be classical HL (cHL) (classified as nodular sclerosis (NSHL), mixed cell type (MCHL), lymphocyte-rich type (LRHL) and lymphocyte-depleted type (LDHL)) or may be nodular lymphocyte-based HL. In an embodiment, non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0171] 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.

[0172] In embodiments, the cancer in which the wild-type MTAP gene is silenced is Hodgkin's lymphoma.

[0173] In an embodiment, the wild-type MTAP gene-silenced cancer is Hodgkin lymphoma, and determination of the gene-silencing status is based on an immunohistochemical assay for MTAP protein that demonstrates reduced or ineffective MTAP protein levels in the nuclei of Hodgkin's Reed / Sternberg (HRS) cells, as assessed relative to normal cells (e.g., non-HRS cells in the sample).

[0174] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor described in WO 2021 / 163344. In such an embodiment, the inhibitor has the general formula I

[0175]

[0176] its tautomer, its stereoisomer or a pharmaceutically acceptable salt of any one of the foregoing,

[0177] in represents a single bond or a double bond;

[0178] X 1 and X 2 is independently N or C at each occurrence; wherein if X 1 is C, which may be optionally halogenated or C 1-6 Alkyl substitution;

[0179] Ar is a six-membered aromatic ring having 0-2 N atoms, each of which may be independently replaced by 0-2 R a Group substitution;

[0180] Where R a is independently selected at each occurrence from cyano, halo, optionally substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl, OR\NR c R d 、-C(O)NR c R d , =S, -SO 2 、-SO 2 C 1-6 Alkyl, -C(O)H, -C(O)C 1-6 Alkyl, C(O)OC 1-6 alkyl, difluoro-pyrrolidinyl and 4 to 6 membered heterocyclic rings having 0-2 heteroatoms independently selected from O and N, and the heterocyclic rings may be further independently substituted with 0-2 halogen, C 1-6 Alkyl, -C(O)H, -C(O)C 1-6 alkyl or optionally substituted cycloalkoxy;

[0181] Each R b is independently selected at each occurrence from H, optionally substituted C 1-6 Alkyl, wherein the substituents may be selected from halo, or oxetanyl;

[0182] Each R e and R d Independently selected from H, C 1-3 Alkyl, C 1-3 Haloalkyl or -CO;

[0183] Where R e In each case selected from H or C 1-6 alkyl;

[0184] Where R f and R g is independently selected from H and C 1-6 alkyl;

[0185] wherein R is H or methyl;

[0186] Where R 1 and R 2 is independently selected at each occurrence from H, optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkynyl, -C(OR e), optionally substituted monocyclic and bicyclic groups having 0-3 N, S or O atoms; wherein the substituents are selected from halo, optionally substituted C 1-6 Alkyl, -C(O)NR f R g , OH and an optionally substituted 5-membered ring having 0-3 N atoms;

[0187] or R 1 and R 2 and the carbon atoms to which they are attached may together form an optionally substituted mono- or bi-carbocyclic or heterocyclic ring, which may be saturated, partially saturated or aromatic, and further wherein the heterocyclic ring comprises 1, 2 or 3 heteroatoms independently selected from N, O and S;

[0188] Wherein the substituent is selected from the following group: optionally substituted C1-6 alkyl, halo, CN, OR e and-C(OR e ),

[0189] The condition is R 1 and R 2 Not at the same time H;

[0190] And where R 3 and R 4 is independently selected at each occurrence from H, halogen, alkynyl, cyano and C 1-6 Alkyl, optionally substituted by halogen or deuterium.

[0191] In such an embodiment, the compound can be selected from the list of compounds presented in claim 19 of WO 2021 / 163344, as presented on pages 267 to 305 of the International Publication.

[0192] In an embodiment, the compound may be a compound having the following formula II as described in claim 1 of WO 2022 / 026892A1, and as presented on pages 2309 to 2311 of the international publication. In an embodiment, the compound may be selected from the compounds presented in Table 1 of WO 2022 / 026892A1, as presented on pages 122 to 470 of the international publication.

[0193]

[0194] Within the scope of 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:

[0195]

[0196] or a pharmaceutically acceptable salt thereof.

[0197] Within the scope of 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:

[0198]

[0199] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt of N-(6-amino-5-methylpyridin-3-yl)-2-((2R,5S)-2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxoacetamide:

[0200]

[0201] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor described in WO 2022 / 115377A1.

[0202] In such embodiments, the MTA-synergistic PRMT5 inhibitor may be a compound having the following formula III

[0203]

[0204] Its tautomer, its stereoisomer, or a pharmaceutically acceptable salt of any one of the foregoing, wherein:

[0205] R is independently selected from tricyclic rings of formula IA and IB:

[0206]

[0207] in is a single bond or a double bond,

[0208] X 1 , X 2 , X 6 and X 7 is N or C in each case, where X 1 and X 2 Both cannot be N at the same time, and if X 1 is C, which may be optionally substituted by halogen;

[0209] X 3 , X 4 and X 5is independently selected at each occurrence from optionally substituted C, O, N and S; wherein the substituents are independently selected from C 1-3 Alkyl, C 1-3 Alkyl (OH), wherein the alkyl group may be optionally substituted by halogen;

[0210] R 3 is independently selected from H or C 1-3 alkyl;

[0211] Ar 1 is a six-membered optionally substituted aryl or heteroaryl independently selected from:

[0212]

[0213] Wherein these substituents are independently selected from C 1-3 Alkyl, -OC 1-3 Alkyl or halo;

[0214] R 1 is independently selected at each occurrence from H, halo, optionally substituted C 1-3 Alkyl, wherein the substituents are selected from halo, -CN, optionally substituted -OC 1-3 Alkyl, wherein the substituents are selected from halo, -C(O)OC 1-3 Alkyl, where C 1-3 The alkyl group may be optionally substituted with halo and morpholinyl; and

[0215] R 2 is independently selected at each occurrence from optionally substituted C 1-8 Alkyl, wherein the substituents are selected from halo, hydroxy, amino, -OC 1-3 alkyl or -CN, a 5- or 6-membered ring or heterocycle, an optionally substituted hydroxyl, an amino, an optionally substituted C 1-6 Alkyl, wherein the substituents are selected from halo, optionally substituted C 1-6 Alkyl-OC 1-3 alkyl, wherein the substituents are selected from halo, 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridinyl, C 1-3 Alkyl-heterocyclyl, wherein the heterocyclyl is selected from optionally substituted 3,4-dihydro-2H-pyrano[2,3-c]pyridinyl, pyridazinyl, triazolyl, pyrimidinyl, tetrahydrofuranyl, 1H-pyrrolo[2,3-b]pyridinyl, cyclohexyl; wherein the substituents are selected from C 1-3 Alkyl, -CN and halo, or optionally substituted C 1-6 Alkyl-0-C 1-3alkyl, wherein the substituents are selected from halo, optionally substituted phenyl, wherein the substituents are selected from halo or C 1-3 alkyl.

[0216] In an embodiment, the compound may be selected from the compounds presented in claim 20 of WO2022 / 115377A1, as presented on pages 331 to 378 of the international publication. In such an embodiment, in one embodiment, the MTA-synergistic PRMT5 inhibitor is selected from those presented in claim 21 of WO 2022 / 115377, as presented on pages 377 and 378 of the international publication.

[0217] In an embodiment, 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-(cyclopropyloxy)-3-fluoro-benzonitrile (as described in Smith et al., https: / / doi.org / 10.1016 / j.bmc.2022.116947 ):

[0218]

[0219] In an embodiment, 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-(cyclopropyloxy)-3-fluoro-benzonitrile:

[0220]

[0221] or a pharmaceutically acceptable salt thereof.

[0222] In an embodiment, 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-(cyclopropyloxy)-3-fluoro-benzonitrile:

[0223]

[0224] In an embodiment, 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-(cyclopropyloxy)-3-fluoro-benzonitrile hydrochloride:

[0225]

[0226] In an embodiment, the MTA-synergistic PRMT5 inhibitor is a compound of formula (IV) as described in WO 2023 / 036974 or a pharmaceutically acceptable salt thereof:

[0227]

[0228] in:

[0229] The ring containing X and Y is pyrrole, and X is NH and Y is CH, or X is CH and Y is NH;

[0230] Z is selected from CH, CF, CCl, or, when Q is not N, is also selected from N;

[0231] Q is selected from CH, CF, CCl, or, when Z is not N, is also selected from N;

[0232] m is 0, 1, or 2;

[0233] n is 0, 1, or 2;

[0234] p is 1 or 2;

[0235] R 1 independently selected at each occurrence from F, Cl, CN, Me, CF 3 , C 1 -C 3 Alkyl, cyclopropyl, C 1 -C 3 Fluoroalkyl, OMe or C 1 -C 3 Alkoxy;

[0236] R 2 is independently selected at each occurrence from F, Cl, Me, MeO and CF 3 ;

[0237] R 3 It is H, Me, C 1 -C 3 Alkyl or C 1 -C 3 Fluoroalkyl;

[0238] R 4 is H, Me or C 1 -C 3 alkyl;

[0239] R 5 It is H, Me, C 1 -C 3 Alkyl, C 1 -C 3 Fluoroalkyl, CH 2 OMe、CH 2OCHF 2 , CH 2 OCF 3 , CH 2 O(C 1 -C 3 alkyl), CH 2 O(C 1 -C 3 Fluoroalkyl), C(CH 2 CH 2 )R 6 、CCR 7 , CH 2 R 8 , R 9 or CH 2 R 10 ;

[0240] R 6 It is H, Me, CH 2 F. CHF 2 CF 3 , CH 2 OH or CH 2 OMe;

[0241] R 7 is H, Me, cyclopropyl, C 1 -C 3 Alkyl, C 1 -C 3 Fluoroalkyl, C 3 -C 6 Cycloalkyl or optionally Me, C 1 -C 3 an alkyl, F or Cl substituted 5-membered heteroaryl group;

[0242] R 8 is optionally Me, C 1 -C 3 Alkyl, F or Cl substituted 5-membered heteroaryl;

[0243] R 9 is an optionally substituted phenyl, 5-membered or 6-membered heteroaryl or bicyclic heteroaryl group; and R 10 is an optionally substituted phenyl, 5-membered or 6-membered heteroaryl or bicyclic heteroaryl group.

[0244] In an embodiment, 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:

[0245]

[0246] or a pharmaceutically acceptable salt thereof.

[0247] In an embodiment, 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:

[0248]

[0249] In an embodiment, the MTA-synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt 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:

[0250]

[0251] In an embodiment, 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:

[0252]

[0253] or a pharmaceutically acceptable salt thereof.

[0254] In an embodiment, 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:

[0255]

[0256] In an embodiment, the MTA-synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt 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:

[0257]

[0258] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula V as described in claim 1 of WO 2021 / 050915, as presented on pages 321 and 322 of the International Publication:

[0259]

[0260] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 88 of WO 2021 / 050915, as presented on pages 331 to 349 of that international publication.

[0261] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 89 of WO 2021 / 050915, as presented on pages 349 and 350 of the International Publication.

[0262] In an embodiment, 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 presented on pages 512 and 513 of the International Publication:

[0263]

[0264]

[0265] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 25 of WO2022 / 192745, as presented on pages 523 to 536 of that international publication.

[0266] Within the scope of 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-1 as described in claim 26 of WO 2022 / 192745, and as presented on pages 536 and 537 of the International Publication:

[0267]

[0268]

[0269] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 27 of WO 2022 / 192745, as presented on page 538 of that International Publication.

[0270] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula VII as described in claim 1 of WO 2023 / 278564, as presented on pages 145 to 147 of the International Publication:

[0271]

[0272] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 19 of WO 2023 / 278564, as presented on pages 149 to 154 of that international publication.

[0273] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula VIII as described in claim 1 of WO 2022 / 132914, as presented on pages 188 and 189 of the International Publication:

[0274]

[0275] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 19 of WO 2022 / 132914, as presented on pages 192 and 193 of the International Publication.

[0276] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 20 of WO2022 / 132914, as presented on pages 194 and 195 of that international publication.

[0277] Within the scope of 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:

[0278]

[0279] or a pharmaceutically acceptable salt thereof.

[0280] Within the scope of 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:

[0281]

[0282] Within such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt of (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl]methanone:

[0283]

[0284] Within 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:

[0285]

[0286] or a pharmaceutically acceptable salt thereof.

[0287] Within 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:

[0288]

[0289] Within such embodiments, in embodiments, the MTA synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt of (R)-(4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)(2-(4-(trifluoromethyl)phenyl)piperidin-1-yl)methanone:

[0290]

[0291] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula IX as described in claim 1 of WO 2022 / 169948, as presented on pages 240 and 241 of the International Publication:

[0292]

[0293] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 23 of WO 2022 / 169948, as presented on pages 243 and 244 of the International Publication.

[0294] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor having Formula X as described in Claim 1 of WO 2023 / 081367, as presented on pages 161 and 162 of the International Publication:

[0295]

[0296] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is an inhibitor having formula XA as described in claim 6 of WO 2023 / 081367, as presented on pages 164 and 165 of the International Publication:

[0297]

[0298] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 17 of WO 2023 / 081367, as presented on pages 168 to 181 of the International Publication.

[0299] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 18 of WO 2023 / 081367, as presented on pages 181 to 185 of that international publication.

[0300] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 19 of WO 2023 / 081367, as presented on pages 185 to 188 of that international publication.

[0301] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 20 of WO 2023 / 081367, as presented on pages 188 to 189 of that International Publication.

[0302] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 21 of WO 2023 / 081367, as presented on pages 189 to 190 of the International Publication.

[0303] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor having formula XI as described in claim 1 of CN 116178347, as presented on page 2 of the A publication:

[0304]

[0305] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 10 of CN116178347, as presented on pages 6 and 7 of that A publication.

[0306] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor having Formula XII as described in claim 1 of WO 2023 / 098439, as presented on pages 55 to 59 of the international publication:

[0307]

[0308] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 11 of WO 2023 / 098439, as presented on pages 68 and 69 of that International Publication.

[0309] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula XIII as described in claim 1 of WO 2021 / 086879, as presented on pages 497 and 498 of the International Publication:

[0310]

[0311] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is an inhibitor having Formula XIII-a as described in Claim 5 of WO 2021 / 086879, as presented on pages 499 and 500 of the International Publication:

[0312]

[0313] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula XIII-b as described in claim 63 of WO 2021 / 086879, as presented on pages 507 to 509 of the International Publication:

[0314]

[0315] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula XIII-c as described in claim 65 of WO 2021 / 086879, as presented on pages 509 and 510 of the International Publication:

[0316]

[0317] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those listed in Table 1 of WO 2021 / 086879, as presented on pages 103 to 114 of the International Publication.

[0318] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor of Formula XIV as described in Claim 1 of CN 116462676, as presented on pages 2 to 4 of the A publication:

[0319]

[0320] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 14 of CN116462676, as presented on pages 12 to 17 of that A publication.

[0321] In an embodiment, the MTA-synergistic PRMT5 inhibitor is an inhibitor having Formula XV as described in Claim 1 of CN 116462677, as presented on pages 2 to 5 of the A publication:

[0322]

[0323] Within the scope of such embodiments, in embodiments, the MTA-synergistic PRMT5 inhibitor is selected from those described in claim 18 of CN116462677, as presented on pages 17 to 22 of that A publication.

[0324] In an embodiment, the present specification provides a pharmaceutical composition comprising an MTA-synergistic PRMT5 inhibitor for use in treating cancer, wherein the cancer is characterized by silencing of the wild-type MTAP gene. In such an embodiment, the MTA-synergistic PRMT5 inhibitor can be selected from the list of inhibitors disclosed above.

[0325] 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 the present disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised edition, 2000, Oxford University Press provide a general dictionary reference to many of the terms used in the present disclosure to the skilled person.

[0326] Units, prefixes and symbols are expressed in their form accepted in the Système International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range.

[0327] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional components that are unacceptably toxic to the subject to which it is to be administered. Such compositions can be sterile. The pharmaceutical composition according to this specification will contain an MTA synergistic PRMT5 inhibitor and at least one pharmaceutically acceptable excipient. One or more pharmaceutically acceptable excipients can be selected from a group comprising fillers, binders, diluents, etc.

[0328] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "toalleviate" refer to (1) therapeutic measures that result in the diagnosis of a pathological condition or disorder being cured, slowed, relieved of symptoms, and / or stopped from progressing and (2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathological condition or disorder. Thus, those in need of treatment include those already suffering from the disorder; those prone to suffering from the disorder; and those in whom prevention of the disorder is desired.

[0329] The term "subject" refers to a person who will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to a human subject.

[0330] MTA synergistic PRMT5 inhibitors or pharmaceutically acceptable salts thereof are usually administered via the oral route in the form of pharmaceutical preparations comprising the active ingredient or a pharmaceutically acceptable salt or solvate thereof, 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, these compositions can be administered at different doses.

[0331] Pharmaceutical formulations of MTA-synergistic PRMT5 inhibitors may conveniently be administered in unit dosage form and may be prepared by any methods well known in the art of pharmacy, for example, as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pennsylvania, (1985).

[0332] 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 may be prepared with the following: binders; fillers; lubricants; and surfactants. Liquid compositions may contain conventional additives such as suspending agents; emulsifiers; and preservatives. Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form. Solid oral dosage forms include tablets, two-piece hard shell capsules, and soft elastic gelatin (SEG) capsules.

[0333] The following examples are provided to aid in understanding the therapeutic applicability of MTA-synergistic inhibitors for use in treating cancers in which the wild-type MTAP gene is silenced.

[0334] Examples

[0335] Proliferation assay

[0336] For Hodgkin lymphoma cell lines L428, L540, L1236, HDLM2 and KM-H2 (purchased from DSMZ, http: / / www.dsmz.de): Assay preparation plates were prepared by adding compounds to 384-well plates (Corning #3712) using an Echo liquid handler. On day 0, 1200 cells / well of L428, L540 or KMH2 cells in 60 μl of growth medium (RPMI1640 + 10% FBS + 1% L-Glu + 1% P / S) were dispensed into assay preparation plates with a final top concentration of 10 μM, diluted 1:3, for a total of 10 doses. The same volume was also dispensed into 1 empty plate as a day 0 control plate. 30 μl / well of CellTiter Glo reagent (Promega #7573) was added to the day 0 plate, incubated at room temperature in the dark for 20 minutes, and luminescence was measured on a plate reader Tecan M200 (using 100 ms integration time, 0 ms decay and stabilization time). The assay plates were incubated at 37°C, 90% humidity, 5% CO 2 The cells were incubated in an incubator at 4 °C for 7 days. Luminescence was measured as for the control plate on day 0. IC was calculated using GraphPad Prism 8 and nonlinear regression (curve fitting) analysis. 50 .

[0337] Alternatively, for HDLM2 (purchased from DSMZ): cells were grown as adherent cells in growth medium (RPMI without phenol red + 10% FCS + 2 mM glutamine). Cells were seeded at 1000 cells / well in 96-well clear bottom black tissue culture treated plates in 90 μl growth medium and placed at 37°C, 5% CO 2incubator. Obtain compounds from the liquid reservoir at a concentration of 10 mM in DMSO solution and then perform half-log serial dilutions in DMSO to prepare x1000-fold stock solutions. The concentration is then diluted 10 to 1 in DMSO and subsequently 10 to 1 in growth medium to obtain a final concentration of x10 in 10% DMSO in the addition plate. 10 μl of compound is then added to 90 μl of cells (diluted 1 to 10) to obtain a concentration range of 0.1 nM to 10 μM in 1% DMSO. CellTiterGlo readings are performed at the time of dosing and on day 6. Add CellTiterGlo reagent to the current volume of culture medium (100 μl) on the cells. Mix on a plate shaker at room temperature for 2 minutes 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 luminescent signal (cover with a silver seal plate). Luminescence was read on an EnvisionF (ultrasensitive luminescent 96-well format with 384-well aperture). IC was calculated using GraphPad Prism 8 and nonlinear regression (curve fitting) analysis. 50 .

[0338] For HCT116 isogenic cell line (parental model purchased from ATCC, MTAPKO clones generated in-house using CRISPR technology): cells were harvested to a density of 400 cells / well (McCoys 5A + 10% FCS + 1% Glutamax) and seeded into 384-well plates (Greiner, Kremsmunster, Austria; 781090) at 40 μl / well using a Multidrop Combi. For day 0 plates, 4 μl of Alamar Blue reagent (Thermo; DAL1100) was immediately added using a multidrop combi and incubated at 37°C, 5% CO 2 Incubate for 3 h at 4 °C. Use Envision plate reader to measure the day 0 cell plate, where the fluorescence excitation wavelength is 540-570 nm (peak excitation is 570 nm) and the fluorescence emission is 580-610 nm (peak emission is 585 nm). Add the test compound using Echo 555 and place it at 37 °C, 5% CO 2 On day 5, 4 μl of Alamar Blue reagent was added using a Multidrop Combi and incubated at 37°C, 5% CO 2Incubate for 3 h at 4 °C. The day 4 cell plates 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. The proliferation rate (IC) was determined by evaluating the total cell count of the Envision plate reader from the day 0 and day 4 plates using the Genedata screener software. 50 value).

[0339] Western blot experiments

[0340] The cell pellet was washed 2x with ice-cold PBS and lysed in 1x SDS lysis buffer (100 mM Tris-HCl buffer, pH 7.4, 10% glycerol and 1% SDS) and then frozen at -80°C. The samples were thawed and heated at 95°C for 5 minutes. After spinning at 14000 rpm for 10 minutes, the supernatant was transferred to a fresh tube. The supernatant was lysed using a Pierce TM Protein concentration was measured using the BCA protein assay kit (Pierce, catalog number 23225). 25 μg of protein from each cell line was loaded onto the NuPAGE TM 4%-12% Bis-Tris gel (Invitrogen, catalog number WG1403BOX10), run at 120V for 1.5 hours, and then transferred to nitrocellulose membrane using Bio Rad semi-dry transfer system (Bio-Rad, model Trans-Blot SD Cell). The membrane was blotted using the following primary and secondary antibodies: MTAP (Cell Signaling, 4158), GAPDH (Cell Signaling, 2118), HRP-linked anti-rabbit IgG (Cell Signaling, 7074).

[0341] Evaluation of MTAP protein expression in Hodgkin lymphoma tumor samples by immunohistochemistry

[0342] IHC analysis was performed on the Ventana Benchmark platform (Roche Diagnostics) using the Ventana human immunohistochemistry staining protocol as provided by the instrument supplier.

[0343] Formalin-fixed paraffin-embedded (FFPE) Hodgkin lymphoma samples were obtained from Tristar Technology Group LLC, Washington, DC, USA.

[0344] Antigen retrieval was performed at pH 8.55 at 100°C for 24 min.

[0345] Positive controls: HCT116 cells (human MTAP wild-type colorectal cancer cell line), human tonsil cells (tonsil FFPE block (ID 68282B2(4)-4), purchased from ProteoGenex, Inglewood, CA 90301, USA).

[0346] Negative controls: MCF7 cells (human MTAP-deficient breast metastatic adenocarcinoma cell line) and xenograft tumors based on MCF7 MTAP-null human breast cancer cells (FFPE blocks from the AZ Archival Research Repository).

[0347] The following equipment was used for IHC analysis:

[0348] Ventana Benchmark Ultra and Prep Kit dispenser (Roche Diagnostics); Leica XL automated stainer (ST5010) and Leica CV5030 coverslipper (Leica Biosystems (leicabiosystems.com)).

[0349] The following reagents were used:

[0350] Ventana bulk reagents: Benchmark Ultra LCS [Roche: 05424534001 (650-210)]; 10x EZ preparation solution [Roche: 05279771001 (950-102)]; reaction buffer 10x concentrate [Roche: 05353955001 (950-300)]; 10x SSC [Roche: 05353947001 (950-110)]; ULTRA cell regulator (ULTRA CC1) [Roche: 05424569001 (950-224)]

[0351] Ventana dispenser reagents: Optiview DAB IHC detection kit [Roche: 06396500001 (760-700)]; Hematoxylin [Roche: 05266726001 (760-2021)]; Bluing reagent [Roche: 05266769001 (760-2037)]; Primary antibody: MTAP (clone A8N9F) rabbit IgG monoclonal antibody, CST (Cell Signaling Technology) #62765S (www.cellsignal.com)

[0352] Staining kit: OptiView DAB IHC Detection Kit #760-700 (Roche)

[0353] Other reagents: deionized water, Fairy liquid (detergent, Proctor & Gamble).

[0354] program:

[0355] 1. Refer to the user manual of the BenchMark ULTRA IHC / ISH System (Roche) for instructions on how to operate the Ventana and fill bulk reagents, empty waste containers, and print slide labels. Note: Dilute the EZ Prep Solution and Reaction Buffer to 1X in deionized water before use.

[0356] 2. In a Ventana preparation kit dispenser, prepare MTAP (A8N9F) rabbit mAb [CST#62765S] in Ventana diluent [Roche: 05261899001 (251-018)] to a working concentration of 0.5 μg / ml or in Ventana diluent containing casein [Roche: 06440002001 (760-219)] to a working concentration of 2 μg / ml.

[0357] 3. In the Ventana Option dispenser, register and fill with Dako serum-free protein block [Agilent: X090930-2].

[0358] 4. Print slide labels (refer to the BenchMark ULTRA IHC / ISH System User Manual).

[0359] 5. Attach labels to slides and load slides and reagent dispensers onto the Ventana and run the machine.

[0360] 6. Once the Ventana run is complete; remove the slides from the slide tray and load them into the coverslip rack.

[0361] 7. Wash the slides in soapy water to remove the LCS oil, then rinse under running tap water.

[0362] 8. Repeat the wash and then load the rack into the Leica XL Autostainer and select program 3 "VentanaClearing" to pass the slides through running tap water, graded ethanol and xylene.

[0363] 9. Once completed, coverslip the slides using 24x50 mm cover slips on a CV5030 coverslipper.

[0364] 10. Scanning was performed at 40X using a Leica Aperio AT2 scanner (https: / / www.leicabiosystems.com / ) prior to manual pathology scoring. Manual scoring was performed by an experienced pathologist. The H-score was adjusted and used for nuclear MTAP staining in tumor cells. The presence and intensity of any cytoplasmic MTAP staining in tumor cells for each sample was also determined and annotated. For a description of the H-score and its application, see: DA Budwit-Novotny et al. Cancer Res. 1986; 46: 5419–5425, F. Aeffner et al. Archives of pathology & laboratory medicine 141(9), 1267-1275, DK Meyerholz et al., Laboratory Investigation 98(7): 844-855.

[0365] 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)

[0366] Compound A can be prepared according to the method disclosed in WO 2023 / 036974, for example, the method disclosed herein.

[0367] Methyl 2-(2-bromo-4-fluorophenyl)acetate

[0368]

[0369] 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 room temperature. The reaction mixture was stirred at 60 ° C for 4 hours, cooled and the solvent was removed in vacuo. The residue was dissolved in EtOAc (250 mL) and saturated NaHCO 3 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 provide the title compound as a colorless oil (105 g, 99%). 1H 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 + Not observed.

[0370] Methyl 5-fluoro-2-(2-methoxy-2-oxoethyl)benzoate

[0371]

[0372] 2-(2-bromo-4-fluorophenyl)acetic acid methyl ester (45.0g, 182.14mmol) and triethylamine (27.90mL, 200.35mmol) are placed in a steel pressure vessel with MeOH (300mL). [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (complexed with dichloromethane) (4.46g, 5.46mmol) is added and the container is sealed. The container is purged with carbon monoxide and then filled to 7 bar with carbon monoxide. The pressure vessel is heated to 100°C and stirred for 2 hours. The reaction mixture is cooled, vented and filtered to remove the catalyst. The solvent is removed in vacuo and the residue is dissolved in EtOAc (250mL), washed with water (2x200mL) and brine (100mL). The organic phase is passed through a phase separation filter paper and the solvent is removed in vacuo. The crude product is purified by flash silica chromatography (elution gradient is 0 to 50% EtOAc in heptane). Pure fractions were evaporated to dryness to afford the title compound as a pale yellow oil (38.40 g, 93%). 1 H 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.

[0373] Racemic-2-(1-bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoic acid methyl ester

[0374]

[0375] 5-fluoro-2-(2-methoxy-2-oxoethyl)benzoic acid methyl ester (47.0g, 207.8mmol) is dissolved in chloroform (450mL). 1-bromopyrrolidine-2,5-dione (55.5g, 311mmol) is added, followed by 2,2'-azobis(2-methylpropionitrile) (3.41g, 20.8mmol), and the reaction mixture is stirred at reflux for 72 hours. The reaction mixture is cooled, washed with water (2x250mL), brine (100mL), and the solvent is removed in vacuo by phase separation filter paper. The crude product is purified by flash silica chromatography (elution gradient is 0 to 40% EtOAc in heptane). The pure fraction is evaporated to dryness to provide the title compound (50.50g, 80%) as a colorless oil. 1 H NMR (400MHz, DMSO-d6, 30℃)3.71(3H,s),3.86(3H,s),6.51(1H,s),7.56(1H,td),7.66(1H,dd),7.81(1H,dd); m / zMH + Not observed.

[0376] rac-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylic acid methyl ester

[0377]

[0378] 4-methoxybenzylamine (23.5g, 171mmol) is placed in a flask with MeCN (300mL) and sodium bicarbonate (23.9g, 285mmol) is added. Racemic-2-(1-bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoic acid methyl ester (43.5g, 142mmol) dissolved in MeCN (100mL) is slowly added by a dropping funnel, and the reaction mixture is brought to 80°C at the same time. The reaction mixture is stirred at 80°C for 3 hours. The reaction mixture is cooled, most of the MeCN is removed in vacuo and the residue is distributed between EtOAc (400mL) and water (400mL). The aqueous phase is extracted again with EtOAc (100mL), and the organic matter is combined and washed with brine (50mL). The organic phase is passed through a phase separation filter paper and the solvent is removed in vacuo. The crude product is purified by flash silica chromatography (elution gradient is 0 to 50% EtOAc in heptane). Pure fractions were evaporated to dryness to afford the title compound as a pale yellow oil (45.3 g, 96%). 1H NMR (400MHz, DMSO-d6, 30℃)3.69(3H,s),3.73(3H,s),4.31(1H,d),5.04(1H,d),5.18(1H, s),6.87–6.94(2H,m),7.17–7.24(2H,m),7.50(1H,ddd),7.57(1H,dd),7.62(1H,dd); m / z MH + 330.

[0379] rac-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylic acid methyl ester

[0380]

[0381] At 5 ° C, under nitrogen, rac-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylic acid methyl ester (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-(diphenylphosphinyl)benzamide) (2.52 g, 3.64 mmol) were stirred in THF (400 mL). 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. 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 separation filter paper. The solvent was removed in vacuo to afford an orange oil. 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 (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 MH + 370.

[0382] (S)-1-Allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylic acid methyl ester

[0383]

[0384] Racemic-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylic acid methyl ester (ca. 70:30, favoring the desired (S) enantiomer) (25.8 g, 69.7 mmol) was purified by SFC chromatography (column: Phenomenex C1, 30 x 250 mm, 5 micron, mobile phase: 10% IPA + 0.1% DEA / 90% scCO 2 , flow rate: 90 ml / min, BPR: 120 bar, column temperature: 40°C, UV max 210 nm). Pure fractions were evaporated to dryness to afford 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 MH + 370.

[0385] (S)-5-Fluoro-2-(4-methoxybenzyl)-3-oxo-1-(2-oxoethyl)isoindoline-1-carboxylic acid methyl ester

[0386]

[0387] To a solution of (S)-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylic acid methyl ester (60.0 g, 162 mmol) in 1,4-dioxane (800 mL) and water (200 mL) was added osmium (VIII) oxide (4% aqueous solution) (5.16 mL, 0.81 mmol), sodium periodate (87.0 g, 406 mmol) and 2,6-lutidine (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 rinsed thoroughly with dichloromethane (DCM, 500 mL). The filtrate was placed in a separatory funnel filled with water (500 mL) and partitioned. The aqueous phase was re-extracted with DCM (300 mL), the organic phases were combined, 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 (50.1 g, 83%) as a white crystalline solid. 1H 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 MH + 372.

[0388] (S)-5-Fluoro-1'-(4-fluorobenzyl)-2-(4-methoxybenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione

[0389]

[0390] (S)-5-fluoro-2-(4-methoxybenzyl)-3-oxo-1-(2-oxoethyl)isoindoline-1-carboxylic acid methyl ester (45g, 121.2mmol) and 4-fluorobenzylamine (22.75g, 181.8mmol) were placed in a flask with 1,2-dichloroethane (600mL) and stirred for 1 hour. The reaction mixture was placed in an ice bath and acetic acid (13.87mL, 242.4mmol) was added, followed by sodium triacetoxyborohydride (51.4g, 242.4mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was neutralized with 2MNaOH, diluted with water (200mL), and extracted with DCM (2x200mL). The combined organic phases were passed through a phase separation filter paper and the solvent was removed in vacuo to provide the title compound as a light yellow oil. The crude product was used in the next reaction, assuming 100% yield. m / z MH + 449.

[0391] Step 2: (S)-5-fluoro-1'-(4-fluorobenzyl)spiro[isoindoline-1,3'-pyrrolidine]-2',3-dione

[0392]

[0393] (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). Cerium (IV) ammonium nitrate (199.0 g, 363.2 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. 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 paper and the solvent was removed in vacuo. The crude product was purified by flash silica chromatography (elution gradient of 0 to 100% (10% MeOH in EtOAc) in heptane). The pure fractions were evaporated to dryness to provide the title compound (30.50 g, 77%) as a paste solid. 1 H NMR (400MHz, DMSO-d6, 30℃)2.37–2.44(1H,m),2.45–2.49(1H,m),3.48(1H,ddd),3.60(1 H,dt),4.49(2H,s),7.19–7.25(2H,m),7.32–7.37(2H,m),7.46(3H,d),9.11(1H,s); m / z MH + 329.

[0394] 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

[0395]

[0396] (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 with anhydrous 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 paper and the solvent removed in vacuo. The residue was triturated with diethyl ether (200 mL) and the resulting solid filtered, washed with ether and dried to afford the title compound (42.20 g, 80%) as a cream solid. 1 H 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 MH + 641.

[0397] (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

[0398]

[0399] (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), diphenylformanimine (14.18 g, 78.23 mmol) and sodium 2-methylpropan-2-ol (12.53 g, 130.4 mmol) were placed in a flask 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 stirred at 65 ° C for 30 minutes. The reaction mixture was cooled and distributed between EtOAc (600 mL) and water (600 mL). The organic phase was washed with brine (200 mL), the solvent was removed in vacuo by phase separation filter paper. The crude product was purified by flash silica chromatography (elution gradient was 0 to 100% EtOAc in heptane). The pure fraction was evaporated to dryness to provide 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 MH + 786.

[0400] (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

[0401]

[0402] (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 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 hours. 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 hours and then at room temperature overnight. The resulting solid was filtered and washed with MeCN (100 mL) to give about 20 g of the desired compound. The filtrate was reduced to about 200 mL and purified by reverse phase chromatography (Interchim C18-HP flash column, 2x415 g, 100 mL loading solution / run) using water (containing 1% NH4OH by volume) 4 OH (28%-30% H 2 (0 solution)) and a decreasing polarity mixture of MeCN as eluent (30%-60% gradient). The fractions containing the desired compound are combined and the previously obtained solid (about 20 g) is added. The slurry is stirred for 1 hour, and then the MeCN is removed in vacuo, resulting in the formation of a light yellow precipitate. The solid is filtered out and dried in vacuo for 2 hours. The solid is then suspended in MeCN (150 mL), and the slurry is gently refluxed for 2 hours, and then allowed to cool overnight. The solid is filtered out and dried in vacuo to provide the title compound (19.54 g, 63%) as a paste-like crystalline solid. 1 H 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 MH + 492.

[0403] 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)

[0404]

[0405] Compound C was prepared according to the method disclosed in WO 2023 / 036974.

[0406] Biological Example 1: Evaluation of Compound C Treatment on In Vivo Tumor Growth Inhibition in the L540 HL Xenograft Model Effect of target binding

[0407] This study was conducted to evaluate tumor growth inhibition and pharmacodynamic changes after treatment with MTA-synergistic PRMT5 inhibitor compound C in MTAP-silenced L540 HL xenograft models. Three doses of compound C (dose level 1, dose level 2, and dose level 3) were tested. The pharmacodynamic changes of SDMA (target engagement marker) reduction after compound C treatment were evaluated by Western blotting.

[0408] Table 4: Test animals used in the study

[0409]

[0410] Table 5: Cell lines used

[0411]

[0412]

[0413] RPMI = Roswell Park Memorial Institute-1640 medium; FBS = fetal bovine serum

[0414] Transplantation of xenografts

[0415] By suspending 5 × 10 e6 Xenografts were established by subcutaneous (SC) injection of 10 cells into the right anterior flank of 6- to 8-week-old animals. Tumors were allowed to reach 100-200 mm before randomization. 3 Tumors were measured by caliper and tumor volume was calculated using the following formula: Volume (mm3) = (tumor length) x (tumor width) x (tumor width) / 2

[0416] Tumor length (longest tumor dimension); Tumor width (longest tumor dimension perpendicular to the length).

[0417] Randomization

[0418] Animals were randomly divided into groups based on tumor size. Randomization was performed based on the "matched distribution" method (StudyDirector™ software, version 3.1.399.19). The randomization date was designated as day 0. There was no animal replacement.

[0419] Group Name and Dose Level

[0420] Control animals were dosed PO with vehicle (5% v / v DMSO / 20% v / v Kolliphor HS15 / 75% v / v purified water (pH 3.0–3.2)), and animals treated with vehicle and Compound C were dosed according to Table 6. Dosing began 1 day after selection and randomization. On dosing day 21, all animals in each group received the last dose in the morning. Mice were necropsied 6 hours later and snap-frozen tumors were collected for PD analysis.

[0421] Table 6: Summary of treatment groups

[0422]

[0423]

[0424] F = female; ROA = route of administration; PO = oral administration

[0425] Tumor measurements and body weight

[0426] Tumors were measured twice weekly with calipers and tumor volume was calculated using the following formula: Volume (mm 3 ) = (tumor length) x (tumor width) x (tumor width) / 2

[0427] The relative tumor volume (RTV) was calculated using the following formula: RTV on day X = (tumor volume on day X) / (tumor volume on day 0)

[0428] 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:

[0429] TGI percentage of treatment group on day X = (((vehicle RTV geometric mean on day X) - (treatment group RTV geometric mean on day X)) / ((vehicle RTV geometric mean on day X) - 1) x 100

[0430] Animals were weighed daily during the dosing phase and twice weekly during all other phases. The percent body weight change was calculated using the following formula: Percent body weight change on day X = (((body weight on day X) - (body weight on selected day) x 100))

[0431] Determination

[0432] Vehicle preparation

[0433] Kolliphor HS15 was melted in hot water (approximately 40°C) and vortexed to ensure a homogeneous solution. Subsequently, DMSO (5% of the final vehicle volume) was added to the glass bottle. Next, Kolliphor HS15 (20% of the final vehicle volume) was added to the glass bottle and vortexed to mix well. Prepare up to 80% of the final vehicle volume with purified water and vortex to mix well, and adjust the pH to 3.0–3.2 with hydrochloric acid (1 M) and vortex to mix well. Prepare up to 100% of the final vehicle volume with purified water and vortex to mix well.

[0434] Compound C preparation

[0435] Kolliphor HS15 was melted in hot water (approximately 40°C) and vortexed to ensure a homogeneous solution. Appropriate amounts of compound were weighed into glass vials. Subsequently, DMSO (5% of the final vehicle volume) was added to the glass vial and vortexed to completely dissolve the compound. Next, Kolliphor HS15 (20% of the final vehicle volume) was added to the glass vial and vortexed to mix. Up to 80% of the final vehicle volume was prepared with purified water and vortexed to mix, and the pH was adjusted to 3.0–3.2 with hydrochloric acid (1 M) and vortexed to mix. Up to 100% of the final vehicle volume was prepared with purified water and vortexed to mix.

[0436] Pharmacodynamic analysis

[0437] Western blotting

[0438] To determine the level of target protein in tumor samples, quick-frozen tumor fragments at the end of the PD study were used, and proteins were extracted by adding 600-1000ul lysis buffer for small and large tumors, respectively. The lysis buffer included: RIPA buffer (Thermo, #89901), complete protease inhibitor tablets (Roche, #58880600, 2 tablets / 50ml), phosphatase inhibitor mixture 2 and 3 (Sigma, #P5726, #P0044), and benzonase nuclease (Sigma, #E1014). The sample was homogenized three times at 6.5m / s in a fast preparation machine for 30 seconds each time. The lysate was then subjected to a highly ultrasonic treatment for 30 seconds in a cooling Diagenode Bioruptor for 1 cycle, and then cooled on ice for 30 minutes. The lysate was centrifuged twice at 13000rpm and 4 degrees for 10 minutes each time, and the test tube was replaced between each run to discard the fragments. The lysates were transferred to deep well plates and the protein in the supernatant was measured using the BCA standardized method (Thermofisher, #23225). The supernatant was washed with 4X sample buffer (Invitrogen, #NP007), 10X reducing agent (Invitrogen, #NP0009) and HO. 2O dilution, the protein concentration was standardized to 45ug. The samples were then boiled at 95 degrees for 5 minutes. After separating the proteins on a 4%-12% bis tris gel, the proteins were transferred to a nitrocellulose membrane (Thermo Fisher Scientific #IB21001) using Iblot2. The primary antibody that recognizes SDMA or focal adhesion protein was diluted in 0.05% Tween (TBST) + 5% Marvel and incubated overnight at 4 degrees Celsius. The membrane was washed three times in 20mL TBST for 15 minutes each. The second rabbit (CST#7074) or mouse (CST#7076) horseradish peroxidase (HRP)-linked antibody was diluted 1:2000 in TBST + 5% Marvel and incubated at room temperature for 1 hour. The membrane was washed three times in 20mL TBST for 15 minutes each, and the signal was detected using the chemiluminescent SuperSignal West Dura extended duration substrate (Thermo Fisher Scientific, #34075), and quantified using Syngene software. The 30 kDa molecular weight band of the PRMT5 substrate SDMA was quantified using Syngene software. The 110 kDa molecular weight band of vinculin was also quantified. Statistical analysis was performed using ordinary one-way ANOVA on values ​​normalized to vinculin compared to vehicle control. SDMA (SDMA #13222 1:1000 dilution obtained from CST); vinculin (#V913 1 1:10,000 dilution) obtained from Sigma.

[0439] Statistical methods

[0440] in vivo

[0441] Tumor volumes were plotted as geometric means using SEM. Percentage changes in body weight were plotted as means using SEM. Significant p values ​​for TGI on the last day of treatment relative to vehicle-treated controls (relative tumor volume) were obtained from Mann-Whitney one-tailed tests and calculated by GraphPad Prism 8.4.3

[0442] Pharmacodynamic analysis (PD)

[0443] Preliminary analysis was performed using Excel, where raw data were first normalized to focal adhesion protein, secondly normalized to the geometric mean of vehicle controls, and then multiplied by 100. Statistical analysis was performed using GraphPad Prism 8.4.3, where data were logarithmically transformed (Y=Log(Y)) and subjected to ordinary one-way ANOVA tests, adjusted for multiple comparisons (Dunnett). Mean differences were obtained from Prism and used to calculate percent inhibition, following the formula:

[0444] Mean difference values ​​were not converted to percentages = 1-((-1*mean difference)*10)*100. Significant p values ​​(if any) obtained from the analysis of variance (ANOVA) tests were quantified using GraphPad Prism 8.4.3.

[0445] Measurement of SDMA protein levels.

[0446] result

[0447] Compound C showed dose-dependent efficacy in inhibiting tumor growth in vivo ( Fig.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).

[0448] Table 7: Anticancer effect of compound C

[0449]

[0450] a The RTV was calculated using a one-tailed Mann-Whitney test on the last day of treatment.

[0451] Compound C was well tolerated at all doses tested, with no significant weight loss during treatment when compared to the vehicle-treated group ( Fig.15 ).

[0452] Pharmacodynamic changes in the reduction of SDMA (target engagement marker) following treatment with Compound C were evaluated by Western blotting. Administration of the compound at dose level 3, dose level 2, or dose level 1 resulted in a decrease in SDMA protein levels (99.1%, 97.6%, and 83.9%, respectively; Fig.16 and Table 8).

[0453] Table 8: Percent SDMA Inhibition with Compound C Treatment

[0454]

[0455] aOrdinary one-way ANOVA, adjusted for multiple comparisons (Dunnett)

[0456] in conclusion

[0457] Compound C demonstrated dose-dependent efficacy and target engagement in an in vivo MTAP-silenced subcutaneous Hodgkin lymphoma xenograft model without causing significant body weight loss compared to the vehicle group.

[0458] All references cited herein, including patents, patent applications, articles, textbooks, and the like, and references cited therein, to the extent not already incorporated herein, are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method of treating cancer, the method 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 a wild-type MTAP gene has been silenced.

2. An MTA-synergistic PRMT5 inhibitor for use in treating cancer, wherein the cancer is characterized by silencing of the wild-type MTAP gene.

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

4. A pharmaceutical composition comprising an MTA-synergistic PRMT5 inhibitor for use in treating cancer, wherein the cancer is characterized by silencing of the wild-type MTAP gene.

5. A kit comprising an MTA synergistic inhibitor and instructions for use in treating MTAP gene-silenced cancers.

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

7. 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 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 lymphoma, kidney cancer, leukemia, lung cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, sarcoma and skin cancer.

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

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

10. The method of treatment, inhibitor for use, composition for use or kit for use according to any one of claims 1 to 9, wherein the MTA-synergistic PRMT5 inhibitor is a compound of formula (IV) or a pharmaceutically acceptable salt thereof: in: The ring containing X and Y is pyrrole, and X is NH and Y is CH, or X is CH and Y is NH; Z is selected from CH, CF, CCl, or, when Q is not N, is also selected from N; Q is selected from CH, CF, CCl, or, when Z is not N, is also selected from N; m is 0, 1, or 2; n is 0, 1, or 2; p is 1 or 2; R 1 is independently selected at each occurrence from F, Cl, CN, Me, CF3, C1-C3 alkyl, cyclopropyl, C1-C3 fluoroalkyl, OMe or C1-C3 alkoxy; R 2 is independently selected at each occurrence from F, Cl, Me, MeO, and CF3; R 3 is H, Me, C1-C3 alkyl or C1-C3 fluoroalkyl; R 4 is H, Me or C1-C3 alkyl; R 5 is H, Me, C1-C3 alkyl, C1-C3 fluoroalkyl, CH2OMe, CH2OCHF2, CH2OCF3, CH2O(C1-C3 alkyl), CH2O(C1-C3 fluoroalkyl), C(CH2CH2)R 6 、CCR 7 、CH2R 8 , R 9 or CH2R 10 ; R 6 is H, Me, CH2F, CHF2, CF3, CH2OH or CH2OMe; R 7 is H, Me, cyclopropyl, C1-C3 alkyl, C1-C3 fluoroalkyl, C3-C6 cycloalkyl or a 5-membered heteroaryl group optionally substituted by Me, C1-C3 alkyl, F or Cl; R 8 is a 5-membered heteroaryl group optionally substituted by Me, C1-C3 alkyl, F or Cl; R 9 is an optionally substituted phenyl, 5-membered or 6-membered heteroaryl or bicyclic heteroaryl group; and R 10 is an optionally substituted phenyl, 5-membered or 6-membered heteroaryl or bicyclic heteroaryl group.

11. The method of treatment, inhibitor for use, composition for use or kit for use according to claim 10, wherein 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: or a pharmaceutically acceptable salt thereof.

12. The method of treatment, inhibitor for use, composition for use or kit for use according to claim 11, wherein 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:

13. The method of treatment, inhibitor for use, composition for use or kit for use according to claim 11, wherein the MTA-synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt 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:

14. The method of treatment, inhibitor for use, composition for use, or kit for use according to claim 11, wherein 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: or a pharmaceutically acceptable salt thereof.

15. The method of treatment, inhibitor for use, composition for use, or kit for use according to claim 11, wherein 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:

16. The method of treatment, inhibitor for use, composition for use, or kit for use according to claim 11, wherein the MTA-synergistic PRMT5 inhibitor is a pharmaceutically acceptable salt 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:

17. A method of identifying a patient who would benefit from treatment with an MTA-synergistic PRMT5 inhibitor, the method comprising the step of identifying a sample obtained from the patient as being wild-type MTAP gene silenced.

18. A method for identifying a tumor in which the wild-type MTAP gene is silenced, the method comprising the following steps: Relevant tumor cells in samples obtained from patients were identified by immunohistochemical assay for MTAP protein, which showed a decrease in MTAP protein in their nuclei and in their cytoplasm.

Images