Protein Arginine Methyltransferase 5 Inhibition Research Articles

Towards the Targeted Protein Degradation of PRMT1.

Targeting the protein arginine methyltransferase 1 (PRMT1) has emerged as a promising therapeutic strategy in cancer treatment. The phase 1 clinical trial for GSK3368715, the first PRMT1 inhibitor to enter the clinic, was terminated early due to a lack of clinical efficacy, extensive treatment-emergent effects, and dose-limiting toxicities. The incidence of the latter two events may be associated with inhibition-driven pharmacology as a high and sustained concentration of inhibitor is required for therapeutic effect. The degradation of PRMT1 using a proteolysis targeting chimera (PROTAC) may be superior to inhibition as proceeds via event-driven pharmacology where a PROTAC acts catalytically at a low dose. PROTACs containing the same pharmacophore as GSK3368715, combined with a motif that recruits the VHL or CRBN E3-ligase, were synthesised. Suitable cell permeability and target engagement were shown for selected candidates by the detection of downstream effects of PRMT1 inhibition and by a NanoBRET assay for E3-ligase binding, however the candidates did not induce PRMT1 degradation. This paper is the first reported investigation of PRMT1 for targeted protein degradation and provides hypotheses and insights to assist the design of PROTACs for PRMT1 and other novel target proteins.

PRMT1 promotes epigenetic reprogramming associated with acquired chemoresistance in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis due to therapeutic resistance. We show that PDAC cells undergo global epigenetic reprogramming to acquire chemoresistance, a process that is driven at least in part by protein arginine methyltransferase 1 (PRMT1). Genetic or pharmacological PRMT1 inhibition impairs adaptive epigenetic reprogramming and delays acquired resistance to gemcitabine and other common chemo drugs. Mechanistically, gemcitabine treatment induces translocation of PRMT1 into the nucleus, where its enzymatic activity limits the assembly of chromatin-bound MAFF/BACH1 transcriptional complexes. Cut&Tag chromatin profiling of H3K27Ac, MAFF, and BACH1 suggests a pivotal role for MAFF/BACH1 in global epigenetic response to gemcitabine, which is confirmed by genetically silencing MAFF. PRMT1 and MAFF/BACH1 signature genes identified by Cut&Tag analysis distinguish gemcitabine-resistant from gemcitabine-sensitive patient-derived xenografts of PDAC, supporting the PRMT1-MAFF/BACH1 epigenetic regulatory axis as a potential therapeutic avenue for improving the efficacy and durability of chemotherapies in patients of PDAC.

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 7595: PRMT5 inhibitor synergizes with PARP inhibitors in triple-negative breast cancer cells

Abstract Background: Treating triple-negative breast cancer (TNBC) patients, an aggressive subtype of breast cancer, is an ongoing clinical challenge. Small-molecule inhibitors targeting the poly ADP-ribose polymerase (PARP) DNA repair enzymes, have been approved for treating only a subset of TNBC patients with a specific genetic landscape (BRCA1 mutation). However, TNBC patients with wildtype BRCA1 (BRCA1WT) are still lacking targeted therapy and chemotherapy remains their main treatment strategy. High expression of protein arginine methyltransferase 5 (PRMT5) is linked to poor prognosis in breast cancer and PRMT5 alone or via its interacting proteins plays an oncogenic role in breast cancer. Recent evidence suggests that PRMT5 is a key player in regulating the repair of DNA double strand breaks (DSBs) either through homologous recombination (HR) or non-homologous end joining (NHEJ) depending on the PRMT5 expression level within the cell. This dependency of cancer cells on PRMT5 expression levels in deciding the repair pathway of choice can be exploited for developing therapies. We hypothesize that in BRCA1 wildtype TNBC cells, inhibiting PRMT5 activity will force the cells to rely on NHEJ rather than HR to repair DNA DSBs, and introducing PARP inhibitors in this setting will inactivate NHEJ as well, causing synthetic lethality and cell death. In this study, we evaluated the pre-clinical benefit of combining PARP inhibitors with a PRMT5 inhibitor in TNBC cells. Methods: Human TNBC cell lines with wildtype BRCA1 (MDA-MB-468, HCC1806) and mutant BRCA1 (MDA-MB-436, SUM149-PT) were treated with varying doses of two PARP inhibitors (Olaparib, Talazoparib) and a PRMT5 inhibitor (GSK3326595) as single agents or in combination. MTT-based cell viability assay was used to determine cytotoxicity of the treatments. mRNA expression was determined by RT-qPCR after drug treatment. Results and Conclusion: We report that PRMT5 inhibition synergizes with both Olaparib and Talazoparib, to potentiate cell death in two BRCA1WT but not in BRCA1mut TNBC cell lines. Furthermore, we show that inhibiting PRMT5 in BRCA1WT cells upregulates the mRNA expression of RAD50, a DNA damage sensing gene in the HR pathway. Our ongoing study is to delineate the molecular mechanism of action for the observed synergy and whether PRMT5 inhibition can overcome resistance to PARP inhibitors. Through this study, we will identify a novel treatment strategy for the majority of TNBC patients (BRCA1WT) who currently lack targeted therapies. Citation Format: Samyuktha Suresh, Lisa A. McPherson, James M. Ford. PRMT5 inhibitor synergizes with PARP inhibitors in triple-negative breast cancer cells [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 7595.

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 1775: The profiling of MTAP homozygous deletion (MTAPhd) in a Chinese pan-cancer cohort

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that catalyzes the transfer of methyl groups from S-adenosylmethionine (SAM) to the arginine residues on histones and other proteins. The dysregulation of this methylation is critical in the development of certain cancers. MTAPhd can cause anti-tumor effect of PRMT5 inhibitor via a synthetic lethality mechanism. Methods: 9,202 cases were collected and subjected to NGS for genomic alteration detection. The testing was carried out by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory, Shanghai, China. The hybrid capture panel covered 450 genes, heterozygous snps were used for MTAPhd identification, the detection rate of MTAPhd was measured and fisher’s exact test was used for analyzing the correlation with age, stage, subtype and gender. Results: In total, 6.8% (626/9202) cases had MTAPhd. In 626 dataset, 220 was female and 406 was male. Age was from 3 to 91 with median 63. 25, 43, 74 and 119 was in clinical stage I, II, III, IV, respectively. Patients with age < 60 had lower odds (OR 0.60; 95% CI 0.51-0.71; p < 0.001) of occurring MTAPhd compared to age >= 60. Clinical stage I and II had lower odds (OR 0.59; 95% CI 0.44-0.79; p < 0.001) compared to stage III and IV. Adenocarcinoma had lower odds (OR 0.17; 95% CI 0.14-0.21; p < 0.001) compared to squamous carcinoma. Female had lower odds (OR 0.70; 95% CI 0.59-0.83; p < 0.001). MTAPhd occurred in multiple cancers, among which it was more likely to occur in esophageal carcinoma (72/298; 24.2%), brain tumor (14/66; 21.2%) and bladder urothelial carcinoma (67/348, 19.3%). The detection rates of the top three cancer types in the sample size were lung cancer (208/2743, 7.6%), pancreatic cancer (48/794, 6.0%) and colorectal carcinoma (7/756, 0.9%), respectively. Lung squamous cell carcinoma (LUSC) (13.5% vs 19.3%, p = 0.008) and bladder urothelial carcinoma (BLCA) (19.3% vs 25.5%, p = 0.045) of Chinese cohort had lower rate of MTAPhd compared to TCGA cohort. 53 out of 626 had no driver mutations associated with recommended drug use currently. One lung adenocarcinoma patient received crizotinib (MET amplification) in 2021, then changed to osimertinib (EGFR exon19del), underwent chemotherapy in 2023, developed hip metastases, and was currently undergoing radiotherapy. Conclusion: This study, for the first time, revealed that MTAPhd were found in approximately 6.8% of Chinese patients across different tumors. Patients with age >= 60, stage III and IV, squamous carcinoma and male, were more likely to occur MTAPhd. Esophageal carcinoma and brain tumor had a higher detection rate. Chinese cohort had lower detection rate of MTAPhd for LUSC and BLCA compared to TCGA cohort. Our results indicated that PRMT5 inhibitors would be a potential therapeutic strategy to be considered for Chinese patients with MTAPhd in the near future. Citation Format: Shuirong Zhang, Wenjin Liu, Mingmin Wang, Caiping Chen, Xiaowei Dong, Aodi Wang, Kai Wang. The profiling of MTAP homozygous deletion (MTAPhd) in a Chinese pan-cancer cohort [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 1775.

Abstract 4596: Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497

Abstract It remains largely challenging to treat cancer patients with chromosome 9p21-deletion which occurs in approximately 15% of human cancers. Recently, specific targeting of protein arginine methyltransferase 5 (PRMT5), a key member of the type-II PRMT family and a master epigenetic modulator of arginine methylome essential for cancer progression, has emerged as a promising therapy in various hematological and solid tumors with MTAP deletion serving as a precision biomarker. To optimize the potential of targeting PRMT5, we developed CTS3497, a potent, brain-penetrable, orally bioavailable MTA-cooperative PRMT5 inhibitor, using our EpigenPLUSTM platform. The overall excellent drug-like properties of CTS3497 support fast track IND application, and it is anticipated to enter Phase 1 trials in 2024. CTS3497 demonstrated potent PRMT5 inhibitory activity in cellular pharmacodynamics (PD) assays, evidenced by the suppression of symmetric dimethylarginine (SDMA). Additionally, it exhibited profound cell growth inhibition with a low single-digit nM IC50 and 171-fold high selectivity for MTAPnull over isogenic MTAPwt cell lines. Moreover, CTS3497 displayed strong inhibitory effects on cell growth across diverse panels of MTAPnull cancer cell lines and patient-derived xenograft organoids (PDXOs) spanning various lineages. In vivo, oral administration of CTS3497 resulted in significant tumor regression in multiple MTAP-deleted xenograft models, notably MTAPnull brain tumors. The observed anti-tumor activity correlated with PRMT5 modulation, as indicated by reduced SDMA levels. Impressively, sustained tumor growth inhibition led to the complete tumor regression, with no regrowth even after discontinuation of CTS3497 treatment. The brain penetration property of CTS3497 underscores its therapeutic potential for both primary brain tumors, and brain metastases arising from specific cancers like lung cancer. At the molecular level, PRMT5 inhibition by CTS3497 induced substantial and distinctive RNA splicing defects and differentially expressed gene signatures, impacting key cellular activities downstream of arginine and epigenetic reprogramming in MTAPnull cancer cells. Furthermore, a synergistic anti-tumor effect was observed in multiple MTAP-deleted tumors when combining PRMT5 inhibition with CTS2190, an investigational type-I PRMT inhibitor currently in phase I/II clinical trials. In summary, CTS3497, a PRMT5 inhibitor selectively targeting MTAPnull tumor cells, demonstrated robust and durable anti-tumor responses both in vitro and in vivo across diverse lineages of MTAP-deleted tumors. Its superior ADME properties and favorable safety profiles observed in preclinical studies mark a breakthrough in the next generation epigenetic therapy. CTS3497 emerges as a promising therapeutic option for patients with MTAP-deleted malignancies. Citation Format: Yilin Liu, Long Wang, Hui Shi, Yuanyuan Liu, Qiugeng Ouyang, Xingnian Fu, Meng Wang, Jiannan Guo, Yiqin Wang, Youzhen Wang, Guoliang Xu, Yuan Mi, Haiping Wu. Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497 [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 4596.

PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.

Inhibition of PRMT1 Suppresses the Growth of U87MG-Derived Glioblastoma Stem Cells by Blocking the STAT3 Signaling Pathway.

Glioblastoma stem cells (GSCs) play a pivotal role in the initiation, progression, resistance to treatment, and relapse of glioblastoma multiforme (GBM). Thus, identifying potential therapeutic targets and drugs that interfere with the growth of GSCs may contribute to improved treatment outcomes for GBM. In this study, we first demonstrated the functional role of protein arginine methyltransferase 1 (PRMT1) in GSC growth. Furamidine, a PRMT1 inhibitor, effectively inhibited the proliferation and tumorsphere formation of U87MG-derived GSCs by inducing cell cycle arrest at the G0/G1 phase and promoting the intrinsic apoptotic pathway. Moreover, furamidine potently suppressed the in vivo tumor growth of U87MG GSCs in a chick embryo chorioallantoic membrane model. In particular, the inhibitory effect of furamidine on U87MG GSC growth was associated with the downregulation of signal transducer and activator of transcription 3 (STAT3) and key GSC markers, including CD133, Sox2, Oct4, Nanog, aldehyde dehydrogenase 1, and integrin α6. Our results also showed that the knockdown of PRMT1 by small interfering RNA significantly inhibited the proliferation of U87MG GSCs in vitro and in vivo through a molecular mechanism similar to furamidine. In addition, combined treatment with furamidine and berbamine, a calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) inhibitor, inhibited the growth of U87MG GSCs more strongly than single-compound treatment. The increased antiproliferative effect of combining the two compounds resulted from a stronger downregulation of STAT3-mediated downstream GBM stemness regulators through dual PRMT1 and CaMKIIγ function blockade. In conclusion, these findings suggest that PRMT1 and its inhibitor, furamidine, are potential novel therapeutic targets and drug candidates for effectively suppressing GSC growth.

Abstract C076: New insights into the role of FOXA1- HNF4 axis in pancreatic cancer

Abstract Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late-stage disease and have limited treatment options. Therefore, there is an immediate need for the generation of new and innovative therapeutic targets. Little is known about the role of pioneer factors such as FOXA1 and their interaction partners in a PDAC context. Inspired by literature reports implicating FOXA1 and GATA5/GATA6 in regulating pancreatic cancer resistance and metastasis, our study hypothesised the possibility of an undiscovered nuclear receptor that works with FOXA1 (similar to Estrogen receptor in breast cancer). Using innovative ‘omic’ based approaches (RIME) developed in our laboratory 1 we discovered a nuclear receptor (NR) complex involving HNF4A and HNF4G in the classical sub-type of pancreatic cancer. Subsequently the interaction was independently validated in Whipple surgical biopsies from PDAC patients using ChIP-sequencing studies. Across multiple patient tumors (n=7) a binding overlap specifically between FOXA1 and HNF4G (3461 sites) was established. To investigate the therapeutic potential of HNF4G in the classical subtype of PDAC, we generated CRISPR deletions (KO) of HNF4G in the HPAF-II cells. HNF4G-KO cells were orthotopically implanted into the pancreas of NSG mice. A significant survival advantage of 12 days (<0.001) and reduced growth (<0.02) was observed in these mice compared to the controls. To better understand the impact of HNF4G-KO on gene expression, the orthotopic tumors were subjected to RNA-seq analyses. Gene set enrichment and pathway analysis revealed significantly down-regulated pathways to include EMT transition. TCGA data highlighted HNF4G amplification in 9% of PDAC patients with concomitant decreased progression free survival. These data point towards HNF4G being a therapeutically viable target for further exploration. Protein Arginine Methyl Transferase 1 (PRMT1) is a common interactor of both FOXA1 and HNF4G. Our study reveals a unique dependency of PRMT1 on the HNF4G-FOXA1 complex in PDAC biopsies. HNF4G-KO drastically reduces the chromatin binding of PRMT1, implicating them as functionally dependent. Treatment of HNF4G-KO cells with GSK3368715 (PRMT1 inhibitor) further sensitizes these cells and results in a significant survival advantage (10 days <0.02). We propose a model of HNF4G inhibition in combination with PRMT1 as a novel therapeutic opportunity to treat the classical sub-type of PDAC. Further, our study reveals a unique reliance of primary classical tumors on HNF4G. Although HNF4G appears to dominate FOXA1 functionality in the primary disease, FOXA1 remains instrumental in priming metastasis and enhancer reprograming. 1. Papachristou EK, Kishore K, Holding AN, Harvey K, Roumeliotis TI, Chilamakuri CSR, et al. A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes. Nature Communications. 2018;9(1):2311. Citation Format: Shalini V. Rao, Lisa Young, Danya Cheeseman, Stephanie Mack, Jill Temple, Chandra Shekar Reddy Chilamakuri, Evangelia Papachristou, Catherina Pelicano, Amy Smith, Dominique-Laurent Couturier, Michael Gill, Duncan Jodrell Jodrell, Alasdair Russell, Igor Chernukhin, Jason Carroll. New insights into the role of FOXA1- HNF4 axis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C076.

PRMT5 as a Potential Therapeutic Target in MYC-Amplified Medulloblastoma.

MYC amplification or overexpression is most common in Group 3 medulloblastomas and is positively associated with poor clinical outcomes. Recently, protein arginine methyltransferase 5 (PRMT5) overexpression has been shown to be associated with tumorigenic MYC functions in cancers, particularly in brain cancers such as glioblastoma and medulloblastoma. PRMT5 regulates oncogenes, including MYC, that are often deregulated in medulloblastomas. However, the role of PRMT5-mediated post-translational modification in the stabilization of these oncoproteins remains poorly understood. The potential impact of PRMT5 inhibition on MYC makes it an attractive target in various cancers. PRMT5 inhibitors are a promising class of anti-cancer drugs demonstrating preclinical and preliminary clinical efficacies. Here, we review the publicly available preclinical and clinical studies on PRMT5 targeting using small molecule inhibitors and discuss the prospects of using them in medulloblastoma therapy.

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.

Abstract C089: PRMT5 as a novel druggable vulnerability for EWSR1-ATF1-driven clear cell sarcoma

Abstract Clear cell sarcoma of soft tissue (CCSST) is a rare and aggressive soft tissue sarcoma. The 5-year survival rate is only 20% for metastatic cases of CCSST. CCSST is insensitive to existing chemotherapies or radiotherapy. New target therapies will have tremendous impact on CCSST patients. The hallmark of CCSST is a chromosome translocation, which generates an oncogene EWSR1-ATF1, where Ewing’s sarcoma gene (EWSR1) is fused with activating transcription factor 1 (ATF1). EWSR1-ATF1 is a constitutively active form of ATF1 and is independent of extracellular cues to drive ATF1-dependent gene transcription. We recently discovered that protein arginine methyltransferase 5 (PRMT5) is involved in EWSR1-ATF1-mediated gene transcription. Genetic silencing of PRMT5 in CCSST cells results in severely inhibited cell growth. We further identified a PRMT5 inhibitor JNJ-64619178 to potently and efficaciously inhibit CCSST cell growth in vitro and in vivo. This becomes clinically very relevant as JNJ-64619178 finished phase I clinical trial (NCT03573310) in advanced solid tumors and non-Hodgkin lymphoma patients and it demonstrated manageable toxicity. Our results presented here provide a strong mechanistic basis for further clinical evaluation of JNJ-64619178 to treat CCSST patients. Citation Format: Bingbing X Li, Larry L David, Lara E Davis, Xiangshu Xiao. PRMT5 as a novel druggable vulnerability for EWSR1-ATF1-driven clear cell sarcoma [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 C089.

PRMT5 participates in B cell overactivation in patients with primary Sjogren's syndrome (pSS) through RSAD2-mediated NF-κB signaling.

There are new evidences that protein arginine methyltransferase 5 (PRMT5) is widely involved in the progression of various diseases, but its effect is unclear on Primary Sjogren's syndrome (pSS). The main purpose of this study is to explore the regulatory effect of PRMT5 on pSS and its potential mechanisms. CD40L treated CD19 + B cells to construct a cell model of pSS. CCK-8 assay and Annexin V-FITC/PI kits were used to measure cell proliferation and apoptosis. ELISA assay was used to determine the contents of IL-6 and TNF-α in CD19 + B cells. And commercial kits were used to detect the levels of immunoglobins (IgG, IgM, and IgA) in CD40L-treated CD19 + B cells. And successfully constructed a pSS mouse model. The results revealed an increase in the expression of PRMT5 in CD19 + B cells from patients with pSS. After CD40L treatment, the knockdown of PRMT5 prominently decreased cell viability, the production level of immunoglobulins (IgG, IgM, and IgA), and the content of IL-10, increased the content of IL-6 and IL-8, and promoted the apoptosis of pSS CD19 + B cells. Mechanistically, PRMT5 negatively regulated the RSAD2 and nuclear factor kappa-B (NF-κB) signaling pathway. Furthermore, overexpression of RSAD2 and p65 significantly rescued the effect of PRMT5 knockdown on proliferation, immunoglobin production and secreting cytokines in CD40L-treated CD19 + B cells. More importantly, inhibition of PRMT5 significantly inhibited the symptoms of pSS mice. Low-expression of PRMT5 through inactivation of RSAD2/NF-κB signalling pathway alleviates the hyperactivity of B cells, which may provide theoretical basis and potential therapeutic targets for clinical treatment of pSS.

PRMT1 Promotes Chronic Lymphocytic Leukemia Progression Via Modulating Methylation of MAST1

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

PRMT1 Inhibition Activates the Interferon Pathway to Potentiate Antitumor Immunity and Enhance Checkpoint Blockade Efficacy in Melanoma.

Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.

Loss of the methylarginine reader function of SND1 confers resistance to hepatocellular carcinoma.

Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.

EXTH-74. TUMOR PHARMACOKINETICS AND PHARMACODYNAMICS IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE PRMT5 INHIBITOR SKL27969

Abstract Epigenetic regulation plays a key role in the development of several cancers, including glioblastoma (GBM). Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising therapeutic target for GBM. PRMT5 is the primary type II methyltransferase that catalyzes symmetric arginine dimethylation in multiple proteins. In this study, we evaluated the pharmacokinetics, pharmacodynamics, and radiation sensitization properties of SKL27969, a highly selective PRMT5 inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 50 mg/kg (oral) SKL27969 vs. placebo for two cycles either alone or in combination with radiation. SKL27969 levels in plasma and contrast-enhancing tumor tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unbound fractions were determined by equilibrium dialysis. Immunohistochemistry was performed to assess levels of SDMA, phospho-Histone H3, pH2AX, and cleaved caspase 3 (CC3). For survival studies, mice with intracranial tumors were randomized into four cohorts – vehicle, radiotherapy, SKL27969 monotherapy, and SKL27969 plus radiotherapy. The median brain-to-plasma and tumor-to-plasma partition coefficients of unbound SKL27969 were 1.8 and 2.9, respectively – indicating favorable brain and tumor penetration ability of SKL27969. A decrease in SDMA (+) cells (54% vs. 0.1%, p < 0.001) indicated PRMT5 inhibition and was accompanied by an increase in CC3 (+) cells (0.7% vs. 1.2%, p = 0.01). SKL27969 is well tolerated, achieves pharmacologically relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Ongoing studies are evaluating in vivo survival benefit from combining PRMT5 inhibition with radiation therapy.

PRMT5 and CDK4/6 inhibition result in distinctive patterns of alternative splicing in melanoma.

Drugs targeting cyclin-dependent kinases 4 and 6 (CDK4/6) are promising new treatments for melanoma and other solid malignancies. In studies on CDK4/6 inhibitor resistance, protein arginine methyltransferase 5 (PRMT5) regulation of alternative splicing was shown to be an important downstream component of the CDK4/6 pathway. However, the full effects of inhibition of CDK4/6 on splicing events in melanoma and the extent to which they are dependent on PRMT5 has not been established. We performed full-length mRNA sequencing on CHL1 and A375 melanoma cell lines treated with the CDK4/6 inhibitor palbociclib and the PRMT5 inhibitor GSK3326595 and analysed data for differential gene expression and differential pre-mRNA splicing induced by these agents. Changes in gene expression and RNA splicing were more extensive under PRMT5 inhibition than under CDK4/6 inhibition. Although PRMT5 inhibition and CDK4/6 inhibition induced common RNA splicing events and gene expression profiles, the majority of events induced by CDK4/6 inhibition were distinct. Our findings indicate CDK4/6 has the ability to regulate alternative splicing in a manner that is distinct from PRMT5 inhibition, resulting in divergent changes in gene expression under each therapy.