Methylthioadenosine Phosphorylase Research Articles

Loss of methylthioadenosine phosphorylase immunoreactivity correlates with poor prognosis and elevated uptake of 11C-methionine in IDH-mutant astrocytoma.

The proximate localization of MTAP, which encodes methylthioadenosine phosphorylase, and CDKN2A/B on Chromosome 9q21 has allowed the loss of MTAP expression as a surrogate for homozygous deletion of CDKN2A/B. This study aimed to determine whether MTAP status correlates with clinical outcomes and 11C-methionine uptake in astrocytomas with IDH mutations. We conducted immunohistochemistry for MTAP in 30 patients with astrocytoma, IDH-mutant who underwent 11C-methionine positron emission tomography scans prior to surgical resection. The tumor-to-normal (T/N) ratio of 11C-methionine uptake was calculated using the mean standardized uptake value (SUV) for tumor and normal brain tissues. Cox regression analysis was used for multivariate survival analysis. Among IDH-mutant astrocytomas, 26.7% (8/30) exhibited the loss of cytoplasmic MTAP expression, whereas 73.3% (22/30) tumors retained MTAP expression. The median progression-free survival (PFS) was significantly shorter in patients with MTAP loss than those with MTAP retention (1.88years vs. 6.80years, p = 0.003). The median overall survival (OS) was also shorter in patients with MTAP loss than in MTAP-retaining counterparts (5.23years vs. 10.69years, p = 0.019). Multivariate analysis identified MTAP status (hazard ratio (HR), 0.081) and extent of resection (HR, 0.104) as independent prognostic factors for PFS. Astrocytomas lacking cytoplasmic MTAP expression showed a significantly higher median T/N ratio for 11C-methionine uptake than tumors retaining MTAP (2.12 vs. 1.65, p = 0.012). Our study revealed that the loss of MTAP expression correlates with poor prognosis and an elevated T/N ratio of 11C-methionine uptake in astrocytoma, IDH-mutant.

Abstract 4536: MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma

Abstract 5’-methylthioadenosine phosphorylase (MTAP), is deleted in 15% of all cancers (estimated 100,000 patients per year in the United States), including 12-15% of cholangiocarcinoma (CCA) cases. MTAP deficiency accumulates 5’-methylthioadenosine (MTA) that subsequently binds protein arginine methyl transferase 5 (PRMT5) and inhibits its activity. This renders selective targeting of MTAP null tumors with agents (MRTX1719, AMG193, TNG462, TNG908) targeting MTA bound PRMT5 (PRMT5:MTA), while sparing surrounding normal MTAP wild-type (WT) tissue. Early signs of clinical activity was seen with MRTX1719, including objective responses in patients with MTAP loss tumors, including biliary tract cancer from the phase I/II study (NCT05245500). Despite these advances, prolonged targeted therapy poses the risk of development of acquired resistance demanding the need for better therapeutic strategies. To address this pressing need, we co-treated CCA cell lines with MRTX1719 and PARP inhibitor olaparib. We hypothesize that since PRMT5 is a critical regulator of splicing and DNA damage pathways, inhibition of PRMT5 disrupts these downstream processes creating an additional vulnerability to DNA damage inducing agents such as PARP inhibitors. Our preliminary data combining MRTX1719 with olaparib in three MTAP loss CCA cell lines (RBE, YSCCC, TFK1) show synergy (combination index; CI<1), and significant reduction in cell viability as well as colony formation ability when compared to monotherapy in vitro. To further gain mechanistic insights into the biology of MTAP loss, we stably expressed MTAP in MTAP null CCA cell line, RBE and demonstrated that MTAP loss drives tumorigenesis in vivo highlighting its tumor suppressor function in CCA. Mass spectrometry-based profiling of histone modifications from the isogenic RBE cell lines showed heightened methylation and reduced acetylation marks in the absence of MTAP. We also noted substantial differences in the distribution of splicing events, with increased intron retention, 3’ alternative splicing and decreased exon skipping as a result of MTAP loss. Similar differences in proportion of splicing events were also observed in MTAP loss CCA patient derived xenografts as compared to WT models. Collectively, these data implicate MTAP to be a crucial player in modulation of the chromatin and splicing events that may drive therapeutic response to PRMT5 inhibition. Citation Format: Pooja A. Shah, Ajay Kumar Saw, William Padron, Ming Zhao, Argun Akcakanat, Kurt Evans, Funda Meric-Bernstam, Milind Javle, Jordi Rodon, Kunal Rai. MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4536.

Abstract 4591: PH020-2: an MTA-cooperative PRMT5 inhibitor with excellent selectivity and brain-penetration capability that targets MTAP-deleted tumors

Abstract Background: Methylthioadenosine phosphorylase (MTAP) gene deletion is highly prevalent across many cancer types such as brain, pancreatic and lung cancer. Accumulated MTA in MTAP loss cells competes with S-adenosylmethionine (SAM, the methyl donor) and leads to partial inhibition on protein arginine methyltransferase 5 (PRMT5), resulting in the sensitivity of these cells to PRMT5 inhibition. On this basis, MTA cooperative PRMT5 inhibitors (such as MRTX1719 and TNG908) have been designed to stabilize the binding of PRMT5 to MTA, but not SAM, resulting in further improvement of selectivity window. The clinical data showed that MRTX1719 has the capability to inhibit MTAP-loss tumor growth while avoid hematology toxicity observed with first generation PRMT5 inhibitors. Methods: A pair of HCT116 isogenic cell lines (MTAP-/- and MTAP+/+) were used to determine the effects of PH020-2 on cell proliferation and intracellular symmetric dimethylarginine (SDMA) content. Human CD34+ hematopoietic stem cells (HSCs) were collected to evaluate hematological toxicity. Intravenous injection of PH020-2 in rats were performed to assess the brain-penetration capability. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with tumor harboring MTAP gene deletion. The in vitro and in vivo pharmacokinetic (PK) and safety properties were assessed with corresponding assay methods. Results: PH020-2 dramatically decreased SDMA content and inhibited proliferation in HCT116 MTAP-/- cells (IC50, SDMA, 0.31 nM; proliferation, 5.9 nM), but had very weak effect on HCT116 WT cells (IC50, SDMA, 296.2 nM; proliferation, 1381 nM), suggesting an excellent selectivity (proliferation, 253×). In comparison, IC50 of MRTX1719 and TNG908 on MTAP-/- cell viability was 8.38 and 52.75 nM, respectively, generating a selectivity of 118× and 28×, respectively. PH020-2 mildly inhibited CD34+ HSC (IC50=807.1 nM), compared to the test on MRTX1719 (IC50=481.9 nM). The tests on a panel of cancer cells confirmed activity and selectivity of PH020-2. PH020-2 possessed excellent PK profiles in all species with absolute bioavailability of 98% in dogs. At the same time, PH020-2 showed outstanding brain penetration in rats (Kp, 0.67). In three CDX models (H838, H2170, Hup-T4), PH020-2 at a dose of 25 mg/kg BID potently inhibited tumor growth, and tumor regression was observed at higher doses in all tested models. Methyltransferase screening (44 targets) showed that PH020-2 potently inhibits PRMT5, but not others. Safety panel assays covering 47 targets demonstrated excellent safety profiles of PH020-2. All other in vitro and in vivo tests showed favorable results and supported for preclinical development. Conclusion: The data suggest that PH020-2 is an MTA-cooperative PRMT5 inhibitor with excellent brain-penetration that selectively targets MTAP-deleted tumors. Citation Format: Feng Gao, Bin Liu, Jing Wang, Liandong Jing, Yongyong Wu, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH020-2: an MTA-cooperative PRMT5 inhibitor with excellent selectivity and brain-penetration capability that targets MTAP-deleted tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4591.

Abstract 4598: Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers

Abstract Homozygous deletions of the p16/CDKN2a (cyclin dependent kinase inhibitor 2A) locus, which is responsible for regulating the cell cycle, are commonly found in cancer and often involve the deletion of adjacent genes. One such adjacent gene is methylthioadenosine phosphorylase (MTAP), involved in metabolism, located on chromosome 9p21 in close proximity to the p16/CDKN2A tumor-suppressor locus. Co-deletion of MTAP is observed in approximately 80-90% of tumors with homozygous deletion of CDKN2A, representing 10-15% of all human tumors. These tumor types, including non-small cell lung cancer, pancreatic adenocarcinoma, glioblastoma, and mesothelioma, have a poor prognosis, highlighting the significant unmet medical need in this area. Deletion of MTAP leads to a significant accumulation of methylthioadenosine (MTA) in cells. MTA, at high concentrations, selectively inhibits the PRMT5 methyltransferase enzyme, competing with the substrate S-adenosylmethionine (SAM) required for methylation reactions. As a result, the overall level of symmetric arginine dimethylation throughout the proteome is reduced. This heightened sensitivity to modulation of methylosome activity makes cells with MTAP deletion more susceptible to therapeutic targeting of PRMT5. Hence, selective targeting of PRMT5 in cancers with homozygous MTAP deletion represents a promising strategy for specifically eliminating cancer cells with this genetic alteration. Ryvu has developed MTA-cooperative PRMT5 inhibitors characterized by good drug-like physicochemical properties and inhibition of methyltransferase activity with IC50 values in the low nanomolar range. A structure-based lead optimization delivered compounds coming from two independent series with high selective potency in MTAP-deleted cell lines and DMPK profiles allowing an oral administration. The antitumor activities were compared in vitro and in vivo to MRTX1719 and AMG193 in MTAP null tumors such as HCT116 MTAP KO, DoHH-2 and Lu99. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies. Taken together, these studies confirm that MTA cooperative PRMT5 inhibitors exert strong synthetic lethal phenotype in MTAP deleted cancers and offer an exciting therapeutic opportunity for a large patient population. Citation Format: Adam Radzimierski, Aneta Bobowska, Agata Stachowicz, Kamil Kuś, Kamila Kozłowska-Tomczyk, Agnieszka Ludwig-Słomczyńska, Paulina Podkalicka-Gołda, Aniela Gołas, Monika Żukowska, Pavlo Lebed, Przemysław Wyrębek, Daria Szukiel, Matylda Stefaniak, Oleksandr Popika, Julia Krzywik, Sujit Sasmal, Paulina Niedziejko-Ćwiertnia, Klara Korta-Piątek, Karolina Fijołkowska, Aleksandra Więckowska, Marta Olszak-Płachta, Swapnil Nipunge, Mateusz Świrski, Marek Wronowski, Henryk Pawlik, Quỳnh Vũ, Katarzyna Łagosz-Ćwik, Mateusz Stoszko, Szymon Woroszyło, Igor Tomczyk, Ewelina Gabor-Worwa, Nilesh Gaud, Anna Kowal-Chwast, Dawid Gogola, Magdalena Miodek, Róża Starczak, Agata Dudek, Jacek Faber, Bożena Winnik, Sanja Novak-Ratajczak, Agnieszka Świrska, Karolina Gluza, Paweł Guzik, Katarzyna Banaszak, Nicolas Boutard, Grzegorz Ćwiertnia, Krzysztof Brzózka, Mateusz Nowak, Anna Bartosik, Didier Pez. Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4598.

Abstract 539: LN18-MRTX1719R cell line, a useful cell model for the discovery of the new PRMT5 inhibitors

Abstract In recent years, the PRMT5-MTA complex has emerged as a "synthetic lethal" novel target for the treatment of MTAP (methylthioadenosine phosphorylase)-deficient cancers. Kryukov et al. first reported the "synthetic lethal" effect of inhibiting PRMT5 in MTAP-deficient tumors, and they identified MTAP as the synthetic lethal partner of PRMT5 in 2016. MTAP is a tumor suppressor gene frequently lost in various cancers, including pancreatic cancer, non-small cell lung cancer, and mesothelioma. Furthermore, the loss of MTAP leads to the accumulation of MTA in cancer cells, which competes with PRMT5's co-factor SAM for binding and forms the PRMT5-MTA complex. Cancer cells lacking MTAP show enhanced sensitivity to PRMT5 loss. Therefore, this complex has emerged as a potential therapeutic target, particularly for the treatment of MTAP-deficient cancers. MRTX1719 is a highly effective selective inhibitor of the PRMT5-MTA complex, capable of selectively inhibiting PRMT5 activity in MTAP-deficient cells. As the most promising candidate drug, MRTX1719 is undergoing Phase I/II clinical evaluation for patients with MTAP-deficient cancers. Drug resistance is an inevitable problem with small molecule inhibitors. Currently, the mechanism of resistance to MRTX1719 is not clear, as MTAP/CDKN2A loss is an early driver event in most cancers. To further explore the resistance mechanism of MRTX1719, our group used the MTAP-deficient LN-18 cell line and induced mutagenesis using the mutagen ENU. We then used a drug low-concentration gradient method to establish the LN-18-MRTX1719R-resistant cell line. Specifically, LN-18 cells were treated with increasing doses of MRTX1719 for 6 months, and mixed clones that tolerated MRTX1719 were selected. Subsequently, single clones were selected, and a stable MRTX1719-resistant clone was obtained, named LN-18-MRTX1719R. Cell proliferation inhibition assay showed that the IC50 of LN-18-MRTX1719R was over 1000nM, which is 100-fold more resistant than the parental LN-18 cells (9.87 nM). We explored the resistance mechanisms using RNA-Seq and WES methods. The resistance to MRTX1719 of the LN-18-MRTX1719R cell line was highly stable after 20 cell passages. The LN-18-MRTX1719R cell line can be a useful cell line mode in the development of future PRMT5·MTA inhibitors. Citation Format: Yao Tang, Liping Wang, Guoqian Wang, Jinying Ning, Feng Hao. LN18-MRTX1719R cell line, a useful cell model for the discovery of the new PRMT5 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 539.

Abstract 1808: Discovery of highly selective novel MTA-cooperative PRMT5 inhibitors for the treatment of cancers

Abstract Protein arginine methyltransferase 5 (PRMT5), a type II PRMT catalyzing the formation of symmetric dimethylation of arginine residues on histone and non-histone proteins, regulates many biological pathways in mammalian cells, including cell growth and differentiation. Methylthioadenosine phosphorylase (MTAP) is required for the methionine salvage pathway, deletion of MTAP leads to the accumulation of inhibitory PRMT5 cofactor methylthioadenosine (MTA). MTAP gene is adjacent to and frequently co-deleted with CDKN2A gene, the most commonly deleted tumor suppressor gene in human cancers. The increase in MTA significantly reduced PRMT5 activity in MTAP-deficient cancer cells, rendering them more vulnerable to PRMT5 inhibition than normal cells. Selective Inhibitors of PRMT5-MTA complex (MTA-cooperative) are supposed to exhibit an increased therapeutic index compared to first generation PRMT5 inhibitors for the treatment of MTAP-deleted (MTAP-del) cancer patients. Guided by computation-aided drug design (CADD), binding mode analysis of known PRMT5 inhibitors has led to the design and synthesis of a novel series of MTA-cooperative PRMT5 inhibitors. Many compounds within this series were found to inhibit PRMT5/MEP50 complex with single digit nM of IC50 in enzymatic assay. Cell based activity of these compounds was assessed by measuring the symmetric demethylarginine (SDMA) and 10-day cell proliferation assays. Several compounds exhibited low double-digit nM of cellular potency in HCT116 MTAP-del cells, the inhibition of SDMA and anti-proliferation activities in HCT116 MTAP-del cells over HCT116 MTAP-WT cells have excellent selectivity (80 - 400 fold). Further optimization of in vitro and in vivo pharmacokinetic properties has yielded candidate compounds suitable for evaluation of pharmacodynamic effect in the LU99 MTAP-del NSCLC xenograft model. Tumor growth inhibition (TGI) was observed in a dose-dependent manner, accompanied by a reduction of PRMT5-mediated SDMA levels in both the tumor and bone marrow. In summary, we have identified compounds with novel scaffolds that are potent and highly selective MTA-cooperative PRMT5 inhibitors. Further development of the second-generation PRMT5 inhibitors for treatment of cancers is also planned. Citation Format: Wen-Lian Wu, Taishan Hu, Zhilin Deng, Honghai Li, Quanrong Shen, Lei Zhang, Xiaochu Ma, Peihua Sun, Cindy Cheng, Fang Liu, Xin Chen, Ye Hua, Bryan Huang. Discovery of highly selective novel MTA-cooperative PRMT5 inhibitors for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1808.

Abstract 7603: Targeting MTAP-deleted tumor via synthetic lethality with a combination of a PRMT5i and an MAT2Ai

Abstract MTAP (methylthioadenosine phosphorylase) is located adjacent to the tumor-suppressor gene CDKN2A (cyclin-dependent kinase inhibitor 2A) and sometimes co-deleted in 10-15% of all cancers, especially in glioblastoma, mesothelioma, and pancreatic cancers. Recent studies shown that MTAP deletions in cancer create vulnerability to synthetic lethal with the MAT2A/PRMT5/RIOK1 axis. Herein, we report the discovery of a PRMT5 inhibitor GH56, and an MAT2A inhibitor GH31, both showed promising single agent efficacy and synergy in MTAP loss tumor models. GH56 is an MTA-Cooperative PRMT5 inhibitor. In vitro, GH56 showed potent SDMA inhibition in MTAP-deleted cancer cells with an IC50 from 0.8‒3 nM. It also showed excellent selectivity with more than 100-folds in MTAP-WT cells. In a cell panel with more than 100 cell-lines, GH56 selectively inhibited the growth of cells with MTAP-deletion while separated the cells with MTAP-WT. In the safety panel and kinase panel, GH56 all showed good selectivity. In addition, GH56 had an IC50 > 10 μM for hERG. In vivo, GH56 showed dose-dependent efficacy in a series MTAP-deleted models and were well tolerated. In U87MG glioblastoma CDX model, NCI-H838 NSCLC CDX model, BXPC-3/HupT4 PDAC CDX models and RT112-84 BLCA model, TGI for GH56 at high dose were all >100%. On the other hand, GH31 is a potent and selective MAT2A inhibitor. GH31 also showed excellent selectivity with MTAP-deleted and MTAP-WT cell lines. The combination of GH56 and GH31 showed synergy both in vitro and in vivo. The low dose GH31 combination with low-dose GH56 achieved the similar efficacy as high dose GH56 and a more rapid tumor regression. In view of the delayed onset of the tumor response and high dose needed by PRMT5 inhibitor (reported by clinic stage compounds), the combination of PRMT5i and MAT2Ai will be advantageous. Furthermore, both GH56 and GH31 are brain penetrant, especially for GH31, with a Kpuu > 0.6 and B/P > 2. The combination of GH56 and GH31 in a brain metastasis model and a glioblastoma in situ model all showed strong synergy, demonstrated the potential for the treatment of glioblastoma and other cancer with brain metastasis. In summary, we discovered potent, selective and brain penetrant PRMT5 inhibitor GH56 and MAT2A inhibitor GH31. Both compounds are currently under evaluation in IND-enabling studies and will provide more options for MTAP-deleted cancers. Citation Format: Guiping Zhang, Jiapeng Li, Jie Jack Li. Targeting MTAP-deleted tumor via synthetic lethality with a combination of a PRMT5i and an MAT2Ai [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7603.

Abstract 3020: The tumor suppressive role of methylthioadenosine phosphorylase in melanoma

Abstract Methylthioadenosine phosphorylase (MTAP) is an enzyme that is expressed in virtually all tissues but lost in many cancers. MTAP deficiency can be due to either deletion of the MTAP gene or methylation of the MTAP promoter. In normal cells, MTAP catalyzes the conversion of methylthioadenosine (MTA), produced during polyamine biosynthesis, to adenine and 5-methylthioribose-1-phosphate, which is subsequently converted to methionine. MTAP deficiency results in intracellular accumulation of MTA. Although recent genome wide association studies (GWAS) have associated the MTAP locus with melanoma risk, the molecular mechanisms linking MTAP loss to increased tumorigenesis are not yet fully understood. In this study, we hypothesized that loss of MTAP and the resulting accumulation of MTA would dysregulate microphthalmia transcription factor (MITF), a master transcriptional regulator of melanocytes that is also an amplified or mutated oncogene in melanoma. We identify a novel signaling mechanism in which MTA accumulation from MTAP deficiency aberrantly induces MITF expression via inhibition of the cAMP phosphodiesterase PDE4D3 in melanocytes. Inhibition of PKA abolishes this MITF induction, suggesting that the PKA pathway is hyperactivated upon MTAP deficiency. Downregulation of MTAP leads to increased proliferation of melanoma cells in vitro and increased melanoma tumor growth in vivo. We also show that MTAP deficient melanoma cells are more resistant to the BRAF inhibitor vemurafenib and observe MTAP downregulation in BRAF inhibitor resistant melanoma clinical specimens. Furthermore, MTAP downregulation via decreased expression and/or genomic loss corresponds to significantly worse outcomes in early-stage cutaneous melanomas from multiple datasets, thereby identifying a clinically important sub-category of “thin-lethal” tumors. Citation Format: Jennifer J. Hsiao, Pedro Andreu-Perez, Lajos V. Kemény, Miroslav Hejna, Jérémie Nsengimana, Jennifer Allouche, Abdullah Al Emran, D. Timothy Bishop, Göran B. Jönsson, Julia Newton-Bishop, Jun Song, David E. Fisher. The tumor suppressive role of methylthioadenosine phosphorylase in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3020.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

Polyamine Inhibitor SAM486A Augments Cytarabine Cytotoxicity in Methylthioadenosine Phosphorylase-deficient Leukemia Cells.

Methionine metabolism contributes to supplying sulfur-containing amino acids, controlling the methyl group transfer reaction, and producing polyamines in cancer cells. Polyamines play important roles in various cellular functions. Methylthioadenosine phosphorylase (MTAP), the key enzyme of the methionine salvage pathway, is reported to be deficient in 15-62% of cases of hematological malignancies. MTAP-deficient cancer cells accumulate polyamines, resulting in enhanced cell proliferation. The aim of this study was to investigate the combined effects of the polyamine synthesis inhibitor SAM486A and the anticancer antimetabolite cytarabine in MTAP-deficient leukemic cells in vitro. The leukemia cell line U937 and the subline, U937/MTAP(-), in which MTAP was knocked down by shRNA, were used. The experiments were performed in media supplemented with 20% methionine (low methionine), which was the minimum concentration for maintaining cellular viability. The knockdown efficiency test confirmed a 70% suppression of the expression of the MTAP gene in U937/MTAP(-) cells. Even in the media with low methionine, the intracellular methionine concentration was not reduced in U937/MTAP(-) cells, suggesting that the minimum supply of methionine was sufficient to maintain intracellular levels of methionine. Both U937/MTAP(+) and U937/MTAP(-) cells were comparably sensitive to anticancer drugs (cytarabine, methotrexate, clofarabine and 6-thioguanine). The combination of SAM486A and cytarabine was demonstrated to have synergistic cytotoxicity in U937/MTAP(-) cells with regard to cell growth inhibition and apoptosis induction, but not in U937/MTAP(+) cells. Mechanistically, SAM486A altered the intracellular polyamine concentrations and reduced the antiapoptotic proteins. Methionine metabolism and polyamine synthesis can be attractive therapeutic targets in leukemia.

Methylthioadenosine Phosphorylase Genomic Loss in Advanced Gastrointestinal Cancers

Abstract Background One of the most common sporadic homozygous deletions in cancers is 9p21 loss, which includes the genes methylthioadenosine phosphorylase (MTAP), CDKN2A, and CDKN2B, and has been correlated with worsened outcomes and immunotherapy resistance. MTAP-loss is a developing drug target through synthetic lethality with MAT2A and PMRT5 inhibitors. The purpose of this study is to investigate the prevalence and genomic landscape of MTAP-loss in advanced gastrointestinal (GI) tumors and investigate its role as a prognostic biomarker. Materials and Methods We performed next-generation sequencing and comparative genomic and clinical analysis on an extensive cohort of 64 860 tumors comprising 5 GI cancers. We compared the clinical outcomes of patients with GI cancer harboring MTAP-loss and MTAP-intact tumors in a retrospective study. Results The prevalence of MTAP-loss in GI cancers is 8.30%. MTAP-loss was most prevalent in pancreatic ductal adenocarcinoma (PDAC) at 21.7% and least in colorectal carcinoma (CRC) at 1.1%. MTAP-loss tumors were more prevalent in East Asian patients with PDAC (4.4% vs 3.2%, P = .005) or intrahepatic cholangiocarcinoma (IHCC; 6.4% vs 4.3%, P = .036). Significant differences in the prevalence of potentially targetable genomic alterations (ATM, BRAF, BRCA2, ERBB2, IDH1, PIK3CA, and PTEN) were observed in MTAP-loss tumors and varied according to tumor type. MTAP-loss PDAC, IHCC, and CRC had a lower prevalence of microsatellite instability or elevated tumor mutational burden. Positive PD-L1 tumor cell expression was less frequent among MTAP-loss versus MTAP-intact IHCC tumors (23.2% vs 31.2%, P = .017). Conclusion In GI cancers, MTAP-loss occurs as part of 9p21 loss and has an overall prevalence of 8%. MTAP-loss occurs in 22% of PDAC, 15% of IHCC, 8.7% of gastroesophageal adenocarcinoma, 2.4% of hepatocellular carcinoma, and 1.1% of CRC and is not mutually exclusive with other targetable mutations.

Reliability assessment of methylthioadenosine phosphorylase immunohistochemistry as a surrogate biomarker for CDKN2A homozygous deletion in adult-type IDH-mutant diffuse gliomas.

According to the 2021 World Health Organization classification of brain tumors, astrocytomas containing a CDKN2A/B homozygous deletion (HD) are designated as grade 4 even when no microvascular proliferation and/or necrosis is present. In this study, we aimed to investigate the relationship between CDKN2A HD and loss of methylthioadenosine phosphorylase (MTAP) expression in adult-type IDH-mutant gliomas and to assess the sensitivity and specificity of MTAP immunohistochemistry (IHC) along with interobserver agreement as a surrogate biomarker for CDKN2A HD. Eighty-eight astrocytomas and 71 oligodendrogliomas cases that were diagnosed between 2014 and 2021 at Hacettepe University were selected and tissue microarrays were conducted to perform CDKN2A fluorescence in situ hybridization and MTAP IHC. Twenty-five (15.7%) cases harbored CDKN2A HD. MTAP loss was detected in 28 (15.7%) cases by the first observer and 27 (17%) cases by the second observer. The sensitivity and specificity of MTAP were calculated as 88% and 95.52%-96.27% for 2 observers. A very good/perfect agreement was noted between the observers (Cohen kappa coefficient = 0.938). Intratumoral heterogeneity was observed in 4 cases. MTAP IHC was found to be a reliable surrogate biomarker as a possible alternative to CDKN2A HD identification with a high sensitivity and specificity along with high interobserver agreement.

SERS analysis of cancer cell-secreted purines reveals a unique paracrine crosstalk in MTAP-deficient tumors.

The tumor microenvironment (TME) is a dynamic pseudoorgan that shapes the development and progression of cancers. It is a complex ecosystem shaped by interactions between tumor and stromal cells. Although the traditional focus has been on the paracrine communication mediated by protein messengers, recent attention has turned to the metabolic secretome in tumors. Metabolic enzymes, together with exchanged substrates and products, have emerged as potential biomarkers and therapeutic targets. However, traditional techniques for profiling secreted metabolites in complex cellular contexts are limited. Surface-enhanced Raman scattering (SERS) has emerged as a promising alternative due to its nontargeted nature and simplicity of operation. Although SERS has demonstrated its potential for detecting metabolites in biological settings, its application in deciphering metabolic interactions within multicellular systems like the TME remains underexplored. In this study, we introduce a SERS-based strategy to investigate the secreted purine metabolites of tumor cells lacking methylthioadenosine phosphorylase (MTAP), a common genetic event associated with poor prognosis in various cancers. Our SERS analysis reveals that MTAP-deficient cancer cells selectively produce methylthioadenosine (MTA), which is taken up and metabolized by fibroblasts. Fibroblasts exposed to MTA exhibit: i) molecular reprogramming compatible with cancer aggressiveness, ii) a significant production of purine derivatives that could be readily recycled by cancer cells, and iii) the capacity to secrete purine derivatives that induce macrophage polarization. Our study supports the potential of SERS for cancer metabolism research and reveals an unprecedented paracrine crosstalk that explains TME reprogramming in MTAP-deleted cancers.

The diagnostic utility of methylthioadenosine phosphorylase immunohistochemistry for pancreatic ductal adenocarcinoma in FNA and small biopsy specimens.

Accurate diagnosis of pancreatic lesions by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or fine-needle biopsy can be challenging. Although surrogate immunohistochemical markers for genetic alterations associated with pancreatic ductal adenocarcinoma (PDAC) have been identified, they have modest sensitivity. Biallelic loss of CDKN2A occurs in up to 46% of PDACs, and methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) has been identified as a reliable surrogate marker for this alteration. The current study evaluates the utility of MTAP IHC for the diagnosis of PDAC. In total, 136 cases of EUS-FNA cell block or core biopsy targeting solid pancreatic masses were identified. MTAP IHC was performed and evaluated for complete loss of expression in neoplastic cells. These results were correlated with available clinical next-generation sequencing that was performed on a subset of cases. Complete loss of MTAP expression was identified in 23 of 80 (29%) PDACs. A subset of cases classified as suspicious (4 of 21) and atypical (4 of 22) showed MTAP loss. All morphologically indeterminate cases with MTAP loss were confirmed as PDAC on resection/additional sampling. No benign samples (n=13) showed loss of MTAP. In samples that had available clinical next-generation sequencing data (n=13), copy number loss of CDKN2A was detected in all cases that had loss of MTAP expression (n=4). Loss of MTAP was identified in approximately 30% of PDAC small biopsy specimens. As loss of MTAP expression is not expected in nonneoplastic cells, and these findings suggest that MTAP IHC can support a diagnosis of PDAC in small biopsy samples.

Discovery of Potent and Oral Bioavailable MAT2A Inhibitors for the Treatment of MTAP-Deleted Tumors.

Inhibition of methionine adenosyltransferase 2A (MAT2A) has received significant interest because of its implication as a synthetic lethal target in methylthioadenosine phosphorylase (MTAP)-deleted cancers. Here, we report the discovery of a series of 3H-pyrido[1,2-c]pyrimidin-3-one derivatives as novel MAT2A inhibitors. The selected compound 30 exhibited high potency for MAT2A inhibition and a favorable pharmacokinetic profile. Furthermore, in an HCT-116 MTAP-deleted xenograft model, compound 30 showed better in vivo potency than current clinical compound AG-270.

Abstract C135: Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers

Abstract Homozygous deletions of the p16/CDKN2a (cyclin dependent kinase inhibitor 2A) locus, which is responsible for regulating the cell cycle, are commonly found in cancer and often involve the deletion of adjacent genes. One such adjacent gene is methylthioadenosine phosphorylase (MTAP), involved in metabolism, located on chromosome 9p21 in close proximity to the p16/CDKN2A tumor-suppressor locus. Co-deletion of MTAP is observed in approximately 80-90% of tumors with homozygous deletion of CDKN2A, representing 10-15% of all human tumors. These tumor types, including non-small cell lung cancer, pancreatic adenocarcinoma, glioblastoma, and mesothelioma, have a poor prognosis, highlighting the significant unmet medical need in this area. Deletion of MTAP leads to a significant accumulation of methylthioadenosine (MTA) in cells. MTA, at high concentrations, selectively inhibits the PRMT5 methyltransferase enzyme, competing with the substrate S-adenosylmethionine (SAM) required for methylation reactions. As a result, the overall level of symmetric arginine dimethylation throughout the proteome is reduced. This heightened sensitivity to modulation of methylosome activity makes cells with MTAP deletion more susceptible to therapeutic targeting of PRMT5. Hence, selective targeting of PRMT5 in cancers with homozygous MTAP deletion represents a promising strategy for specifically eliminating cancer cells with this genetic alteration. Ryvu is developing two independent series of PRMT5 inhibitors characterized by good drug-like physicochemical properties and inhibition of methyltransferase activity with IC50 values in the low nanomolar range. Structure-based lead optimization enabled rapid expansion and delivery of a compound library with novel intellectual property (IP), high target engagement in cells and selective potency in MTAP-deleted cell lines. In long-term three-dimensional (3D) cell culture, Ryvu compounds selectively inhibit the growth of MTAP-deficient cancer cells, which correlate strongly with the inhibition of PRMT5-dependent symmetric protein dimethylation  (SDMA) in these cells. The synthesized library allowed the identification of compounds with more than 350-fold selectivity between the effects observed in MTAP-deficient and WT cells for both SDMA and growth inhibition. The DMPK profile of these compounds allows for oral administration, which enables testing of antitumor activity in MTAP null tumor xenograft bearing mice. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies. Taken together, these studies provide a rationale for the further development of our chemical series of MTA cooperative PRMT5 inhibitors toward a clinical candidate. Citation Format: Adam Radzimierski, Aneta Bobowska, Agata Stachowicz, Kamil Kus, Kamila Kozłowska-Tomczyk, Agnieszka Ludwig-Słomczyńska, Aniela Gołas, Paulina Podkalicka, Andrzej Gondela, Nicolas Boutard, Grzegorz Ćwiertnia, Krzysztof Brzózka, Anna Bartosik, Mateusz Nowak, Didier Pez. Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C135.

Abstract B177: The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers

Abstract Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene occurs in approximately 15% of all cancers due to its proximity to the commonly deleted tumor suppressor gene CDKN2A. Elevated methylthioadenosine (MTA) levels, driven by loss of MTAP, compete with the methyl donor S-adenosylmethionine (SAM) for binding to protein arginine N-methyltransferase 5 (PRMT5), placing the methyltransferase in a hypomorphic state, and vulnerable to further PRMT5 inhibition. The screening of a DNA-encoded library, designed to identify small molecules that preferentially bind to PRMT5 in the presence of MTA, resulted in the identification of AM-9934 as a promising hit. Using structure-based drug design, potency and cooperativity were enhanced to deliver AM-9747, an MTA-cooperative PRMT5 inhibitor suitable for in vivo proof-of-concept studies. Further optimization led to AMG 193, an orally bioavailable and MTA-cooperative PRMT5 inhibitor with potent biochemical and cellular activity, and an improved pharmacokinetic profile. Insights into the binding mode of AMG 193 were elucidated by the crystal structure of AMG 193 bound to the MTA-PRMT5 complex. AMG 193 activity is enriched in MTAP-null cells in vitro, as illustrated in the HCT116 isogenic pair; AMG 193 is ~40X selective for MTAP-null cells (IC50 = 0.1 mM) over MTAP WT cells (IC50 > 4 mM) in viability assays and >100X selective in an assay evaluating symmetric dimethylarginine (SDMA) levels.  AMG 193 was profiled in a panel of >850 cancer cell lines, and response demonstrated preferential sensitivity in MTAP-null cells compared to WT across multiple cancer lineages, including pancreatic, lung, biliary tract, and lymphoma. Response strongly correlated with both MTAP and CDKN2A copy number loss, and cell lines that were dependent on PRMT5, as shown by RNAi and CRISPR knockdown, were most sensitive to AMG 193 treatment. In vitro mechanism-of-action studies demonstrated that PRMT5 inhibition induced DNA damage, cell cycle arrest in G2/M, and an increase in alternative mRNA splicing in MTAP-null cells. In mice oral, once-daily administration of AMG 193 selectively inhibited SDMA in tumor versus normal tissue and was well tolerated with no hematologic perturbations. Importantly, AMG 193 demonstrated robust anti-tumor activity across a broad range of MTAP-null CDX and PDX tumor models derived from NSCLC, pancreatic, and esophageal cancers. In vitro synergy was observed with the combination of AMG 193 and several chemotherapeutic agents, including carboplatin and paclitaxel, or the KRASG12C inhibitor, sotorasib. In vivo, these combinations led to enhanced anti-tumor activity relative to single agent in MTAP-null NSCLC tumor models. In February 2022, AMG 193 became the first MTA-cooperative PRMT5 inhibitor to enter clinical development and is currently being evaluated in subjects with advanced MTAP-null solid tumors in the ongoing FIH study (NCT05094336). Citation Format: Brian Belmontes, Katherine Slemmons, Siyuan Liu, Antonia Policheni, Jodi Moriguchi, Hong Tan, Fang Xie, Famke Aeffner, Matthew G Rees, Melissa M Ronan, Jennifer A Roth, Mikkel Vestergaard, Sanne Cowland, Jan Andersson, Ian Sarvary, Patricia Lopez, Nuria Tamayo, Liping H Pettus, Susmith Mukund, Jennifer R Allen, Sanne Glad, Matthew P Bourbeau. The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B177.

Abstract PR006: Initial results from first-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in biomarker-selected solid tumors

Abstract Background: AMG 193 is a second-generation protein arginine methyltransferase 5 (PRMT5) inhibitor that targets the MTA-bound state of PRMT5 in methylthioadenosine phosphorylase (MTAP)-null tumors. PRMT5 is responsible for methylation and gene silencing of cell-essential proteins dysregulated in cancer and is partially inhibited in tumors harboring MTAP deletion, which occurs in ~15% of solid tumors. First generation PRMT5 inhibitors were intolerable due to indiscriminate inhibition of PRMT5 leading to dose-limiting myelosuppression. In preclinical studies, AMG 193 demonstrated selective antitumor activity in MTAP-null models by further suppressing PRMT5 function while sparing normal function, thereby improving upon first generation molecules. We report the initial clinical results from dose-escalation in the ongoing first-in-human (FIH) study. Methods: AMG193 was orally administered in continuous 28-day cycles to patients (pts) with advanced MTAP-null solid tumors. Dose escalation proceeded via a BLRM method. The primary objectives include safety, tolerability, and identification of the maximum tolerated dose (MTD). Secondary objectives include preliminary antitumor activity by investigator-assessed RECIST, pharmacokinetics (PK) and pharmacodynamic (PD) effects. Results: As of August 8, 2023, 47 pts with MTAP-null cancer (PDAC n = 10; NSCLC n = 6; CCA = 5; MESO n = 3; others n = 23) were enrolled in seven escalating cohorts. Five pts had DLTs, and exploration continues per protocol to identify the MTD. The most common TRAEs were nausea (45%), fatigue (26%), decreased appetite (17%), and vomiting (17%). Preliminary PK analyses showed dose-proportional systemic exposure with a half-life of 7–11 hrs. Among 31 pts who had at least one postbaseline scan, there were 5 with confirmed PRs [PDAC (–100%), ovarian Sertoli-Leydig (–59%), RCC (–58%), esophageal (–46%), and gallbladder cancer (–63%), 1 each], 14 with stable disease (including 9 with some degree of tumor shrinkage), and 12 with disease progression. All PRs were ongoing at the data cutoff with treatment durations of 140–275 days. PD effects demonstrated dose-dependent reduction in serum total SDMA levels and complete PRMT5 inhibition was confirmed in five pts with on-treatment biopsies spanning multiple dose levels. Exploratory analysis of changes in variant allele frequency by ctDNA demonstrated rapid treatment effects that was predictive and correlated with response. Conclusion: AMG 193 is an MTA-cooperative PRMT5 inhibitor designed to induce synthetic lethality in MTAP-null solid tumors while sparing hematologic toxicity. The initial results of the FIH study demonstrate proof-of-concept with encouraging signs of preliminary clinical activity without evidence of myelosuppression. Dose escalation continues to proceed to establish the MTD. AMG 193 has demonstrated promise as a potential new therapeutic for pts with tumors that have MTAP loss. Citation Format: Jordi Rodon, Noboru Yamamoto, Toshihiko Doi, François Ghiringhelli, Maria-Elisabeth Goebeler, Yutaka Fujuwara, Miguel Villalona-Calero, Nicolas Penel, Amita Patnaik, Jean-Pascal Machiels, Alfredo Addeo, James Abbruzzese, Sophie Postel-Vinay, Sylvie Rottey, Chunxu Liu, Chen-Hua Chuang, William Kormany, Paul Hughes, Kiana Keyvanjah, Bert O'Neil. Initial results from first-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in biomarker-selected solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR006.

Primary pericardial angiosarcoma with pleural metastasis: an unusual clinical presentation

Abstract Introduction/Objective Primary pericardial angiosarcoma is an uncommon, aggressive cardiac neoplasm that arises from the endothelial cells of blood vessels. It typically affects middle-aged men and has a poor prognosis due to early metastasis. Unfortunately, there are currently no established guidelines or effective treatments for this rare malignancy. The purpose of this case study is to emphasize the significance of recognizing the unspecific presentations of pericardial angiosarcoma and ensuring timely diagnosis and appropriate treatment. Methods/Case Report A 56-year-old male presented with recurrent pericardial effusion and exudative pleural effusion. The radiologic studies showed a thickened pericardium with right-sided pericardial effusion and a large right- sided pleural effusion. Resected partial pericardium and parietal pleura showed the presence of epithelioid to spindled cells that formed solid sheets and vascular structures filled with numerous red blood cells. The tumor cells were cytologically malignant, with angulated shapes and hyperchromatic nuclei, and showed extensive invasion into the surrounding connective tissue. Mitotic figures were variably visualized, and there was abundant hemorrhage. Immunohistochemical analysis of the lesional cells showed positivity for ERG, CD31, Factor VIII, and retention of Methylthioadenosine phosphorylase (MTAP) and BRCA1-associated protein 1 (BAP1), while they were negative for WT1, calretinin, claudin-4, and p40. Based on the morphology and immunoprofile, a diagnosis of high grade angiosarcoma was rendered, and the patient underwent chemotherapy. The overall survival of the patient was 8 months to date. Results (if a Case Study enter NA) NA Conclusion Prompt diagnosis of pericardial angiosarcoma is crucial, as nonspecific symptoms may lead to misdiagnosis, such as in this case where the patient was initially diagnosed with pericarditis. Video-assisted thoracic surgery and histologic analysis are necessary to reach a correct diagnosis, which ensures appropriate treatment, as a delayed diagnosis can worsen the prognosis and even lead to death.

Epigenetic Silencing of MTAP in Hodgkin's Lymphoma Renders It Sensitive to a 2 nd Generation PRMT5 Inhibitor

Background: PRMT5 is type II arginine methyltransferase that catalyses symmetric dimethylation of arginine residues (SDMA) and plays an important role in cancer biology. By methylating a number of substrates, PRMT5 can regulate important processes such as DNA repair, RNA splicing, and cellular proliferation. In addition, PRMT5 is overexpressed in various cancer types and has been identified as a candidate for therapeutic intervention through the development of small molecules that inhibit PRMT5 methyltransferase activity. However, first generation PRMT5 inhibitors have shown limited clinical benefit mainly due to development of on-target toxicity, primarily in the bone marrow. AstraZeneca has developed a second generation PRMT5 inhibitor (AZ-PRMT5i), that selectively inhibits PRMT5 in MTAP deficient tumours while sparing MTAP proficient normal cells. MTAP (methylthioadenosine phosphorylase) is a metabolic enzyme involved in methionine salvage pathway. MTAP deficiency results in accumulation of the metabolite methylothioadenosine (MTA) in tumor cells that induces partial inhibition of PRMT5, rendering these tumors sensitive to PRMT5 inhibition. Homozygous deletion of the MTAP gene, that results in the loss of MTAP protein, has been found in approximately 15% of advanced solid tumors. Here, for the first time, we describe epigenetic silencing of the MTAP gene in Hodgkin's Lymphoma (HL) cell lines. This silencing results in the loss of MTAP protein expression thus increasing sensitivity to PRMT5 inhibition. Importantly, MTAP protein loss was also observed in primary cHL samples, opening a novel opportunity for the treatment of HL. Methods: Bioinformatic data mining of the Cancer Cell Line Encyclopedia (CCLE) dataset has been used to overlay levels of expression of MTAP mRNA with MTAP copy number and MTAP promoter DNA methylation. MTAP protein expression and activity of AZ-PRMT5i were assessed in 5 HL cell lines in vitro; efficacy of AZ-PRMT5i and levels of target engagement were tested in vivo in the L540 xenograft model. MTAP expression levels were determined by IHC analysis using 55 primary samples from cHL patients. Results: Using an unbiased analysis of the CCLE dataset, we have identified a subset of cell lines that in the absence of MTAP genetic loss were showing low levels of MTAP mRNA expression, equivalent to levels of MTAP mRNA in MTAP homozygous deleted cell lines. Interestingly, this was predominantly the case in HL cell lines where it was detected in 67% of cases (4 out of 6 cell lines). MTAP methylation profile analysis of HL cell lines revealed elevated MTAP DNA promoter methylation in cell lines with low MTAP mRNA expression. In addition, MTAP protein was not detected by Western Blot. Accordingly, lack of MTAP protein expression resulted in increased sensitivity of these cells to AZ-PRMT5i compared to a HL cell line that expressed MTAP protein. Utilizing RNA-seq for transcriptional profiling of AZ-PRMT5i on a MTAP-deficient HL cell line has identified robust changes in genes that regulate the cell cycle, DNA replication and TP53 pathways thus confirming on target activity. In addition, AZ-PRMT5i demonstrated strong dose dependent efficacy in a MTAP silenced L540 xenograft model where greater than 90% of tumour growth inhibition was detected, with no significant body weight loss. Corresponding dose-dependent inhibition of SDMA is observed in the treated tumours. Finally, IHC analysis of MTAP expression in 55 primary cHL samples has demonstrated that 46 of 55 cHL samples (84%) had absent nuclear MTAP expression. Conclusion: Here we are presenting a novel finding showing MTAP protein expression loss in the majority of tested primary cHL samples, which could provide a collateral vulnerability and novel therapeutic opportunity to 2 nd generation PRMT5 inhibitors as demonstrated in our pre-clinical models.