Protein Arginine Methyltransferase 5 Activity Research Articles

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.

Inhibition of PRMT5/MEP50 Arginine Methyltransferase Activity Causes Cancer Vulnerability in NDRG2low Adult T-Cell Leukemia/Lymphoma.

N-myc downstream-regulated gene 2 (NDRG2), which is a tumour suppressor, is frequently lost in many types of tumours, including adult T-cell leukaemia/lymphoma (ATL). The downregulation of NDRG2 expression is involved in tumour progression through the aberrant phosphorylation of several important signalling molecules. We observed that the downregulation of NDRG2 induced the translocation of protein arginine methyltransferase 5 (PRMT5) from the nucleus to the cytoplasm via the increased phosphorylation of PRMT5 at Serine 335. In NDRG2low ATL, cytoplasmic PRMT5 enhanced HSP90A chaperone activity via arginine methylation, leading to tumour progression and the maintenance of oncogenic client proteins. Therefore, we examined whether the inhibition of PRMT5 activity is a drug target in NDRG2low tumours. The knockdown of PRMT5 and binding partner methylsome protein 50 (MEP50) expression significantly demonstrated the suppression of cell proliferation via the degradation of AKT and NEMO in NDRG2low ATL cells, whereas NDRG2-expressing cells did not impair the stability of client proteins. We suggest that the relationship between PRMT5/MEP50 and the downregulation of NDRG2 may exhibit a novel vulnerability and a therapeutic target. Treatment with the PRMT5-specific inhibitors CMP5 and HLCL61 was more sensitive in NDRG2low cancer cells than in NDRG2-expressing cells via the inhibition of HSP90 arginine methylation, along with the degradation of client proteins. Thus, interference with PRMT5 activity has become a feasible and effective strategy for promoting cancer vulnerability in NDRG2low ATL.

PRMT5 regulates epigenetic changes in suppressive Th1-like iTregs in response to IL-12 treatment.

Induced regulatory T cells (iTregs) are a heterogeneous population of immunosuppressive T cells with therapeutic potential. Treg cells show a range of plasticity and can acquire T effector-like capacities, as is the case for T helper 1 (Th1)-like iTregs. Thus, it is important to distinguish between functional plasticity and lineage instability. Aplastic anemia (AA) is an autoimmune disorder characterized by immune-mediated destruction of hematopoietic stem and progenitor cells in the bone marrow (BM). Th1-like 1 iTregs can be potent suppressors of aberrant Th1-mediated immune responses such as those that drive AA disease progression. Here we investigated the function of the epigenetic enzyme, protein arginine methyltransferase 5 (PRMT5), its regulation of the iTreg-destabilizing deacetylase, sirtuin 1 (Sirt1) in suppressive Th1-like iTregs, and the potential for administering Th1-like iTregs as a cell-based therapy for AA. We generated Th1-like iTregs by culturing iTregs with IL-12, then assessed their suppressive capacity, expression of iTreg suppression markers, and enzymatic activity of PRMT5 using histone symmetric arginine di-methylation (H3R2me2s) as a read out. We used ChIP sequencing on Th1 cells, iTregs, and Th1-like iTregs to identify H3R2me2s-bound genes unique to Th1-like iTregs, then validated targets using CHiP-qPCR. We knocked down PRMT5 to validate its contribution to Th1-like iTreg lineage commitment. Finally we tested the therapeutic potential of Th1-like iTregs using a Th1-mediated mouse model of AA. Exposing iTregs to the Th1 cytokine, interleukin-12 (IL-12), during early events of differentiation conveyed increased suppressive function. We observed increased PRMT5 enzymatic activity, as measured by H3R2me2s, in Th1-like iTregs, which was downregulated in iTregs. Using ChIP-sequencing we discovered that H3R2me2s is abundantly bound to the Sirt1 promoter region in Th1-like iTregs to negatively regulate its expression. Furthermore, administering Th1-like iTregs to AA mice provided a survival benefit. Knocking down PRMT5 in Th1-like iTregs concomitantly reduced their suppressive capacity, supporting the notion that PRMT5 is important for the superior suppressive capacity and stability of Th1-like iTregs. Conclusively, therapeutic administration of Th1-like iTregs in a mouse model of AA significantly extended their survival and they may have therapeutic potential.

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.

The potential and challenges of targeting MTAP-negative cancers beyond synthetic lethality.

Approximately 15% of cancers exhibit loss of the chromosomal locus 9p21.3 - the genomic location of the tumour suppressor gene CDKN2A and the methionine salvage gene methylthioadenosine phosphorylase (MTAP). A loss of MTAP increases the pool of its substrate methylthioadenosine (MTA), which binds to and inhibits activity of protein arginine methyltransferase 5 (PRMT5). PRMT5 utilises the universal methyl donor S-adenosylmethionine (SAM) to methylate arginine residues of protein substrates and regulate their activity, notably histones to regulate transcription. Recently, targeting PRMT5, or MAT2A that impacts PRMT5 activity by producing SAM, has shown promise as a therapeutic strategy in oncology, generating synthetic lethality in MTAP-negative cancers. However, clinical development of PRMT5 and MAT2A inhibitors has been challenging and highlights the need for further understanding of the downstream mediators of drug effects. Here, we discuss the rationale and methods for targeting the MAT2A/PRMT5 axis for cancer therapy. We evaluate the current limitations in our understanding of the mechanism of MAT2A/PRMT5 inhibitors and identify the challenges that must be addressed to maximise the potential of these drugs. In addition, we review the current literature defining downstream effectors of PRMT5 activity that could determine sensitivity to MAT2A/PRMT5 inhibition and therefore present a rationale for novel combination therapies that may not rely on synthetic lethality with MTAP loss.

PRMT5 facilitates angiogenesis and EMT via HIF-1α/VEGFR/Akt signaling axis in lung cancer.

Abnormal angiogenesis is a critical factor in tumor growth and metastasis, and protein arginine methyltransferase 5 (PRMT5), a prominent type II enzyme, is implicated in various human cancers. However, the precise role of PRMT5 in regulating angiogenesis to promote lung cancer cell metastasis and the underlying molecular mechanisms are not fully understood. Here, we show that PRMT5 is overexpressed in lung cancer cells and tissues, and its expression is triggered by hypoxia. Moreover, inhibiting or silencing PRMT5 disrupts the phosphorylation of the VEGFR/Akt/eNOS angiogenic signaling pathway, NOS activity, and NO production. Additionally, inhibiting PRMT5 activity reduces HIF-1α expression and stability, resulting in the down-regulation of the VEGF/VEGFR signaling pathway. Our findings indicate that PRMT5 promotes lung cancer epithelial-mesenchymal transition (EMT), which might be possibly through controlling the HIF-1α/VEGFR/Akt/eNOS signaling axis. Our study provides compelling evidence of the close association between PRMT5 and angiogenesis/EMT and highlights the potential of targeting PRMT5 activity as a promising therapeutic approach for treating lung cancer with abnormal angiogenesis.

Abstract 2675: Vactosertib combined treatment with T1-44, a PRMT5 activity inhibitor, improves the survival and inhibits tumor invasion of murine pancreatic cancer model

Abstract Pancreatic cancer is the most lethal type of cancer and the third leading cause of cancer death with the lowest 5-year survival rate. Heterogeneity, difficulty in diagnosis, and rapid metastatic progression are the causes of high mortality in pancreatic cancer. Recent studies have shown that Protein arginine methyltransferase 5 (PRMT5) is overexpressed in pancreatic cancers, and these patients have a worse prognosis. Therefore, PRMT5 as an anti-cancer target has gained considerable interest. In this study, we investigated whether inhibition of PRMT5 activity was synergistic with blockade of TGF-β1 signaling, which plays an important role in the construction of the desmoplastic matrix and invasiveness in pancreatic cancer and induces therapeutic vulnerability. Compared with T1-44, a selective inhibitor of PRMT5 activity, the combination of T1-44 with the TGF-β1 signaling inhibitor vactosertib significantly reduced tumor size and surrounding tissue invasion and significantly improved long-term survival. RNA sequencing analysis of mouse tumors revealed that the combination of T1-44 and vactosertib significantly altered the expression of genes involved in cancer progression, such as cell migration, extracellular matrix, and apoptotic processes. These data demonstrate that the combination therapy of T1-44 with vactosertib is synergistic for pancreatic cancer, suggesting that this novel combination therapy has value in the treatment strategy of patients with pancreatic cancer. Citation Format: Eunji Hong, Sujin Park, Seok Hee Park, Nick B. La Thangue, Seong-Jin Kim. Vactosertib combined treatment with T1-44, a PRMT5 activity inhibitor, improves the survival and inhibits tumor invasion of murine pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2675.

Combination treatment of T1-44, a PRMT5 inhibitor with Vactosertib, an inhibitor of TGF-β signaling, inhibits invasion and prolongs survival in a mouse model of pancreatic tumors

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal type of cancer and the third leading cause of cancer death with the lowest 5-year survival rate. Heterogeneity, difficulty in diagnosis, and rapid metastatic progression are the causes of high mortality in pancreatic cancer. Recent studies have shown that Protein arginine methyltransferase 5 (PRMT5) is overexpressed in pancreatic cancers, and these patients have a worse prognosis. Recently, PRMT5 as an anti-cancer target has gained considerable interest. In this study, we investigated whether inhibition of PRMT5 activity was synergistic with blockade of TGF-β1 signaling, which plays an important role in the construction of the desmoplastic matrix in pancreatic cancer and induces therapeutic vulnerability. Compared with T1-44, a selective inhibitor of PRMT5 activity, the combination of T1-44 with the TGF-β1 signaling inhibitor Vactosertib significantly reduced tumor size and surrounding tissue invasion and significantly improved long-term survival. RNA sequencing analysis of mouse tumors revealed that the combination of T1-44 and Vactosertib significantly altered the expression of genes involved in cancer progression, such as cell migration, extracellular matrix, and apoptotic processes. In particular, the expression of Btg2, known as a tumor suppressor factor in various cancers, was markedly induced by combination treatment. Ectopic overexpression of Btg2 inhibited the EMT response, blocking cell migration, and promoted cancer cell death. These data demonstrate that the combination therapy of T1-44 with Vactosertib is synergistic for pancreatic cancer, suggesting that this novel combination therapy has value in the treatment strategy of patients with pancreatic cancer.

PRMT5 Inhibition Reduces Autoinflammation in a Murine Model of Secondary Hemophagocytic Lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare but frequently fatal autoinflammatory syndrome characterized by dysregulated immune activation, excessive production of proinflammatory cytokines and hyperactivation of T cells and macrophages leading to tissue damage. Primary HLH occurs due to predisposing genetic mutations in the perforin/granzyme pathway, while secondary HLH can be triggered by infections, rheumatologic disorders, or malignancies. HLH has a poor overall prognosis despite appropriate therapy, and new avenues targeting pathologic disease mechanisms are urgently needed. The protein arginine methyltransferase 5 (PRMT5) enzyme has been identified as an important mediator in inflammatory T cell subsets in autoimmune disease and graft versus host disease. We hypothesized that PRMT5 activity is relevant to inflammation loops that drive autoinflammation observed in HLH. Methods: We explored inhibition of type II protein arginine methyltransferase enzyme, PRMT5, in a murine model of secondary HLH. HLH was triggered in C57BL/6-J mice (Jackson Laboratories) by repeated injections of CpG 1826 (Integrated DNA Technologies) and an IL-10 receptor-blocking antibody (clone 1B1.3A; Bio X Cell) on days 0, 2, 4, and 7. Injected mice were left untreated or were treated with ruxolitinib 90 mg/kg (validated treatment of HLH) orally twice daily, starting on Day 4, or increasing doses of oral inhibitor of PRMT5, PRT382 (0.25, 1 or 2 mg/kg daily), starting on Day 0. Mice were weighed daily. On day 9, mice were euthanized and analyzed for spleen and liver weight, serum IFNγ levels and immune profiling of lymphoid and myeloid splenocyte subsets. Splenocytes were left unstimulated, or stimulated with lipopolysaccharide (LPS, myeloid cell stimulation) or CD3/CD28-directed antibodies (T cell stimulation) and analyzed by flow cytometry. Data analysis was performed by unsupervised ViSNE clustering in Cytobank software. Statistical analysis was performed by unpaired t test. Results: We observed that 1 mg/kg PRT382 lessened the hallmarks of secondary HLH, compared to the untreated HLH cohort, including weight loss (21% reduction, p-value 0.2746), the development of splenomegaly (71% reduction, p-value <0.001) and hepatomegaly (61% reduction, p-value <0.05), similarly to ruxolitinib (p-values <0.01, <0.001, and 0.0795, respectively). Furthermore, mice with untreated HLH showed a 17X increase in serum IFNγ levels compared to controls (p-value <0.01), while mice treated with ruxolitinib showed a 10X increase (p-value <0.05 compared to controls, 0.2577 compared to untreated HLH), and mice treated with 1 mg/kg PRT382 showed undetectable serum IFNγ (p-value 0.2104 compared to controls, <0.01 compared to untreated HLH). Flow cytometric analysis of mouse splenocytes showed that HLH stimulated the expansion of neutrophils (CD11b+/Ly6Clo/Ly6G+) and inflammatory monocytes (CD11b+/Ly6C+/Ly6G-). Ruxolitinib partially counteracted this expansion. PRT382 inhibited expansion of these myeloid cell subsets in a dose-dependent manner and also limited production of IFNγ and TNFα by myeloid cells in response to stimulation with LPS in presence of Golgi blockade with Brefeldin A. Furthermore, PRT382 limited the upregulation of CD25 on the surface of T cells in response to T cell stimulation, although no change in cytokine production by T cells was observed. Conclusions: PRMT5 inhibition counteracts pro-inflammatory features of pathologic hyper-inflammation driven by myeloid immune cell subsets in a model of secondary HLH. This may represent a novel avenue for urgently needed immune modulatory targeted treatment of HLH. Studies of PRMT5 expression and targeting in models of primary HLH are underway.

Constitutive PRMT5 Expression Drives Spontaneous Lymphoblastic Lymphoma In Vivo

Protein arginine methyltransferase 5 (PRMT5) is a highly conserved enzyme that catalyzes monomethylation and symmetric dimethylation of arginine residues on many target proteins, including histones and non-histone proteins (P53, P65/NFκB, E2F1, AKT and spliceosome proteins). (Shailesh, 2018) PRMT5 is overexpressed in numerous solid and hematologic malignancies. (Yang, 2013) It is associated with several oncogenic mechanisms, from driving the malignant phenotype to promoting tumor cell survival and invasiveness. While preliminary data has shown PRMT5 to be upregulated in T-ALL/LBL, the mechanism by which PRMT5 promotes T-ALL/LBL has yet to be established. (Mei, 2019) Preclinical transgenic models of leukemia and lymphoma driven by a variety of oncogenes (MYC, NOTCH) have identified PRMT5 activity as a critical component for the driver phenotype. (Li, 2015) To test the hypothesis that overexpression of this enzyme is capable of malignant driver activity, we developed a transgenic (Tg) mouse model designed to over-express PRMT5 in the lymphoid compartment. (Figure 1) Tg mice were created by injecting a linearized pBH-Eμ-PRMT5 vector into FVB/N pronuclei and implanted into pseudo-pregnant FVB/N mice. We obtained five founder lines crossed with wild-type FVB/N mice to obtain the F1 generation. RT-PCR for PRMT5 showed preferential overexpression in Tg B lymphocytes compared to control FVB/N mice (p<0.01) but also overexpression in T and NK cell compartments due to leaky Eμ phenotype. We observed a high percentage of F1 progeny from all five founder lines to develop spontaneous lymphoproliferative disease compared to control mice (p<0.001). Immunophenotypic and pathologic evaluation was performed by flow cytometry, immunohistochemistry, and clonality by IgH and TCR Vβ PCR analysis. Pathologic features showed a range of lymphoid neoplasms, with pre-T lymphoblastic immunophenotype being the most common. Tumor-bearing animals demonstrated diffuse involvement of all organs, peripheral blood, and bone marrow. Pre-T lymphoblastic lymphomas (LBL) showed expression of cytoplasmic CD3, surface CD4 (dim), TdT, and variable Ki67 with constitutive overexpression of PRMT5, MYC, CYCLIN D1, Ki67, and CD99. We developed a T-LBL cell line (Tg813) derived from a thymic tumor that displayed clonal TCR Vβ.17 gene rearrangement, intracellular TdT and CD3 expression with dim surface CD4 expression, immunophenotypic characteristics consistent with T-ALL/LBL. This cell line was maintained in culture and successfully passaged in immune-competent FVB mice, which led to disseminated disease, suggesting that PRMT5 drives an immune escape program in vivo. Preliminary data demonstrate that tumor-bearing animals display a prominent population of normal and neoplastic T-cell subsets with strong PD1 expression. PD1 expression in T-ALL/LBL has been linked with oncogenic function (Yang, 2019) and, in normal T-cell subsets, is a marker for exhaustion. (Lee, 2015) In addition, treatment of our Tg813 in vitro model with PRMT5 knockdown (CRISPR CAS9) and selective inhibition with a SAM-competitive inhibitor (C220 and PRT382, Prelude Therapeutics) showed a reduction of global symmetric dimethyl arginine, loss of the PRMT5 epigenetic mark H4R3(Sme2) and decreased proliferation with cell cycle arrest by day 6, findings consistent with PRMT5-dependent driver activity. Engraftment of Tg813 into SCID and immunocompetent FVB/N mice led to disseminated lymphomas (mean: 21 days). Tg813 engrafted animals treated with C220 (10mg/kg) showed improved survival compared to vehicle-treated mice (p<0.001). This preclinical model supports the hypothesis that PRMT5 over-expression provides cancer driver activity and a unique opportunity to study the role of this oncogene in an immune-competent, in vivo model system. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-76. PRMT5 INHIBITION SENSITIZES GLIOBLASTOMA NEUROSPHERES TO TEMOZOLOMIDE

Abstract INTRODUCTION The median survival of Glioblastoma (GBM) patients is less than two years with the standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) chemotherapy. Protein Arginine Methyltransferase 5 (PRMT5), which regulates cellular functions by symmetrically di-methylating arginine residues, is overexpressed in GBM. Inhibiting PRMT5 induces apoptosis in differentiated and senescence in stem-like GBM tumor cells. We inhibited PRMT5 in GBM neurospheres to determine if PRMT5 inhibition would enhance TMZ’s antitumor effect. METHODS We depleted PRMT5 activity, in vitro, using target-specific siRNA or LLY-283 and combined these with TMZ treatment. We evaluated the antitumor effect of this combination using cell viability assay, cell cycle analysis, apoptosis assay, and western blot. RESULTS TMZ reduced the viability of GBMNS with PRMT5 knockdown significantly more than the viability of PRMT5 intact GBMNS. The combination of TMZ and PRMT5 knockdown elevated the expression of cleaved caspase 3 and caspase3/7 indicating that PRMT5 knockdown enhanced the apoptotic effects of TMZ. Cell cycle analysis showed that depleting PRMT5 abrogated TMZ-induced G2/M cell cycle arrest. Further, treatment of PRMT5-depleted GBMNS with TMZ increased ɣ-H2AX expression compared PRMT5 intact GBMNS treated with TMZ, suggesting that PRMT5 depletion enhanced TMZ-induced DNA damage. PRMT5 knockdown also inhibited the symmetric di-methylation of RUVBL1 that is required for homologous recombination repair of TMZ treatment-related DNA damage. CONCLUSION Overall, PRMT5 inhibition sensitized GBMNS to TMZ and enhanced TMZ-related DNA damage and cytotoxicity. These findings support further development of this potential therapeutic combination.

Downregulation of PRMT5 by AMI-1 enhances therapeutic efficacy of compound kushen injection in lung carcinoma in vitro and in vivo.

Protein arginine methyltransferase 5 (PRMT5) is overexpressed in lung carcinoma, which promotes tumor cell proliferation, survival, migration and invasion. Compound Kushen injection (CKI) is a mixture of natural compounds extracted from Kushen and Baituling, which are mainly used to stop in cancer pain and bleeding. Here we found that cell viability and colony formation were inhibited after the incubation of AMI-1. Meanwhile, AMI-1 suppressed cell migration, enhanced apoptosis, induced cell cycle arrest, inhibited PRMT5 expression and histone H3R8 and H4R3 symmetric di-methylation (H3R8me2s and H4R3me2s) accumulation, down-regulated the expression of eukaryotic translation initiation factor 4E (eIF4E) in lung carcinoma cells. Moreover, AMI-1 suppressed tumor growth, decreased H3R8me2s and H4R3me2s accumulation, down-regulated eIF4E expression and increased p53 expression in lung carcinoma xenografts of BALB/c nude mice. Of note, combined and CKI markedly enhanced the anticancer efficacy CKI in lung carcinoma. The above findings demonstrated that AMI-1 has established antineoplastic activity and this role may be associated with affecting the function of eIF4E via inhibiting PRMT5 activity or protein levels in lung carcinoma. This study highlights evidence of novel selective anticancer activity of AMI-1 in combination with CKI in lung carcinoma.

Abstract 1031: PRMT5 inhibition alters mitochondrial dynamics in mantle cell lymphoma, creating vulnerability to BH3 mimetic compounds

Abstract Mantle cell lymphoma (MCL) is an aggressive and incurable blood cancer comprising 5% of all non-Hodgkin lymphomas diagnosed annually. The median age of diagnosis is 68yo, and while many patients initially respond to frontline treatment, relapse is common. There is an unmet need to develop novel therapeutic strategies for the treatment of MCL. Our group has identified protein arginine methyltransferase 5 (PRMT5) as a key driver of MCL pathogenesis. PRMT5 symmetrically dimethylates arginine residues on a number of proteins (P53, E2F1, P65) and histones (H4R3, H3R8, H2AR3), which support tumorigenesis. Selectively inhibiting PRMT5 has shown significant anti-tumor activity in preclinical MCL models, and a Phase 1 clinical trial with PRT543 (Prelude), a novel PRMT5 inhibitor, is underway. While exploring pathways that converge with PRMT5 activity as potential avenues for combination treatment, we identified the intrinsic apoptotic pathway as an attractive target in MCL. BCL2 family proteins either promote or inhibit intrinsic apoptosis at the outer mitochondrial membrane through a dynamic set of binding interactions. Prior work has shown PRMT5 inhibition to drive the expression of multiple pro-death BCL2 family gene products (BAX, BAK, and BBC3/PUMA) in MCL. We hypothesized that combining PRMT5 inhibition with BH3 mimetics, compounds that target pro-survival BCL2 proteins, would induce synergistic cell death in MCL. Selective PRMT5 inhibition with PRT382 inhibits the viability of MCL cell lines with an IC50 below 1uM in eight of nine lines (IC50 44.8nM - 1905.5nM). BH3 mimetics navitoclax (BCL2, BCLXL, and BCLw inhibitor), A852 (BCLXL inhibitor), and AMG176 (MCL1 inhibitor) were found to have IC50s below 1uM in five, two, and four of the nine cell lines respectively. We chose six cell lines to test combination treatment ranging in sensitivity to BH3 mimetics and expression of BCL2 family proteins (CCMCL1, Z-138, UPN1, Granta 519, Mino, and Maver1). Synergistic decreases in viability were tested via MTS assay and analyzed with the Loewe model of synergy. Mino, which showed sensitivity to all three mimetics, exhibited a synergistic reduction in viability with combination PRT382 treatment. Granta 519 and Z-138 exhibited a similar effect with the combination of PRT382 and navitoclax or A852. These observations were confirmed through BH3 profiling, supporting MCL cell line dependence on BCL2, BCLXL, BCLw and MCL1, and increased sensitivity to BCL2 family protein targeting with PRMT5 inhibition. Two patient derived xenograft models were tested ex vivo after treatment with 10mg/kg of the PRMT5 inhibitor PRT382 or vehicle. iBH3 flow-based analysis revealed increased sensitivity of ex vivo PDX cells to pan BCL2, BCLXL, and MCL1 inhibition. These results provide rationale for combining BH3 mimetics and PRMT5 inhibition in clinical trials as a novel treatment strategy for MCL. Citation Format: Claire Hinterschied, Fiona Brown, Janani Ravikrishnan, JoBeth Helmig-Mason, Kris Vaddi, Peggy Scherle, Jennifer Woyach, Selina Chen-Kiang, Oliver Elemento, Jihye Paik, Robert Baiocchi. PRMT5 inhibition alters mitochondrial dynamics in mantle cell lymphoma, creating vulnerability to BH3 mimetic compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1031.

Synthesis and Activity of Triazole-Adenosine Analogs as Protein Arginine Methyltransferase 5 Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is an attractive molecular target in anticancer drug discovery due to its extensive involvement in transcriptional control, RNA processing, and other cellular pathways that are causally related to tumor initiation and progression. In recent years, various compounds have been screened or designed to target either the substrate- or cofactor-binding site of PRMT5. To expand the diversity of chemotypes for inhibitory binding to PRMT5 and other AdoMet-dependent methyltransferases, in this work, we designed a series of triazole-containing adenosine analogs aimed at targeting the cofactor-binding site of PRMT5. Triazole rings have commonly been utilized in drug discovery due to their ease of synthesis and functionalization as bioisosteres of amide bonds. Herein, we utilized the electronic properties of the triazole ring as a novel way to specifically target the cofactor-binding site of PRMT5. A total of about 30 compounds were synthesized using the modular alkyne-azide cycloaddition reaction. Biochemical tests showed that these compounds exhibited inhibitory activity of PRMT5 at varying degrees and several showed single micromolar potency, with clear selectivity for PRMT5 over PRMT1. Docking-based structural analysis showed that the triazole ring plays a key role in binding to the characteristic residue Phe327 in the active pocket of PRMT5, explaining the compounds’ selectivity for this type-II enzyme. Overall, this work provides new structure–activity relationship information on the design of AdoMet analogs for selective inhibition of PRMT5. Further structural optimization work will further improve the potency of the top leads.

PRMT5 confers lipid metabolism reprogramming, tumour growth and metastasis depending on the SIRT7-mediated desuccinylation of PRMT5 K387 in tumours.

The protein arginine methyltransferase 5 (PRMT5), which is highly expressed in tumour tissues, plays a crucial role in cancer development. However, the mechanism by which PRMT5 promotes cancer growth is poorly understood. Here, we report that PRMT5 contributes to lipid metabolism reprogramming, tumour growth and metastasis depending on the SIRT7-mediated desuccinylation of PRMT5 K387 in tumours. Mass spectrometric analysis identified PRMT5 lysine 387 as its succinylation site. Moreover, the desuccinylation of PRMT5 K387 enhances the methyltransferase activity of PRMT5. SIRT7 catalyses the desuccinylation of PRMT5 in cells. The SIRT7-mediated dessuccinylation of PRMT5 lysine 387 fails to bind to STUB1, decreasing PRMT5 ubiquitination and increasing the interaction between PRMT5 and Mep50, which promotes the formation of the PRMT5-Mep50 octamer. The PRMT5-Mep50 octamer increases PRMT5 methyltransferase activity, leading to arginine methylation of SREBP1a. The symmetric dimethylation of SREBP1a increases the levels of cholesterol, fatty acid, and triglyceride biogenesis in the cells, escaping degradation through the ubiquitin-proteasome pathway. Functionally, the desuccinylation of PRMT5 K387 promotes lipid metabolism reprogramming, tumour growth and metastasis in vitro and in vivo in tumours.

Ribavirin inhibits the growth and ascites formation of hepatocellular carcinoma through downregulation of type I CARM1 and type II PRMT5

Type I co-activator-associated arginine methyltransferase 1 (CARM1) and type II protein arginine methyltransferase 5 (PRMT5) are highly expressed in multiple cancers including liver cancer and their overexpression contributes to poor prognosis, thus making them promising therapeutic targets. Here, we evaluated anti-tumor activity of ribavirin in hepatocellular carcinoma (HCC). We found that ribavirin significantly inhibited the proliferation of HCC cells in a time- and dose-dependent manner. Furthermore, ribavirin suppressed the growth of subcutaneous and orthotopic xenograft of HCC in mice, decreased vascular endothelial growth factor (VEGF) and peritoneal permeability to reduce ascites production, and prolonged the survival of mice in HCC ascites tumor models. Mechanistically, ribavirin potently down-regulated global protein expression of CARM1 and PRMT5, and concurrently decreased accumulation of H3R17me2a and H3R8me2s/H4R3me2s. However, ribavirin did not affect the activity and mRNA levels of both CARM1 and PRMT5 in vivo and in vitro HCC cells. In addition, our ChIP results shown that ribavirin inhibited CARM1 which in turn decreased the H3R17me2a, binds to the eukaryotic translation initiation factor 4E (eIF4E) and VEGF promoter region, and reduced the relative mRNA expression level of eIF4E and VEGF in HCC cells. Our findings suggested a potential therapeutic strategy for patients with HCC through inhibition of the abnormal activation/expression of both CARM1 and PRMT5.

Abstract P039: A phase 1 dose escalation study of protein arginine methyltransferase 5 (PRMT5) inhibitor PRT543 in patients with advanced solid tumors and lymphoma

Abstract Background: PRMT5 catalyzes symmetric arginine dimethylation of protein substrates with important roles in cancer cell growth/survival. PRT543 is a potent, selective, oral PRMT5 inhibitor with robust preclinical efficacy (Bhagwat AACR 2020). An open-label Phase I study of PRT543 in unselected patients (pts) with advanced solid tumors and hematologic malignancies (NCT03886831) is ongoing. Dose escalation results from pts with solid tumors and lymphoma are presented herein. Materials and Methods: This study assesses safety, pharmacokinetics, pharmacodynamics, and preliminary tumor response (REClST v1.1) of PRT543 administered at varying schedules beginning with twice weekly (BIW) and increasing to once daily (QD) with doses ranging from 5-50 mg in 28-day cycles. Dose escalation followed a 3+3 design. Serum symmetric dimethylarginine (sDMA) and intron retention, a marker of PRMT5-mediated mRNA splicing fidelity, were assessed as measures of PRMT5 target engagement and function, respectively. Results: As of 18 June 2021, 49 unselected pts with measurable disease refractory to established therapies had enrolled (17 pts BIW, 11 pts 5x/week [wk], 21 pts QD). Median number of prior lines of systemic therapies was 3. Six of 21 pts dosed at 25-50 mg QD experienced dose limiting toxicity (DLT) of thrombocytopenia, and 1 pt of 6 dosed at 45 mg 5x/wk had DLT of fatigue. Most frequent treatment-related adverse events (TRAEs), any grade, in all regimens were fatigue (n=20, 41%), nausea (n=14, 29%), thrombocytopenia (n=13, 27%), and anemia (n=12, 24%). Grade ≥3 TRAEs in all regimens occurring in ≥5 pts included thrombocytopenia (n=10, 20%) and anemia (n=6, 12%). Cytopenias were reversible and managed with dose modifications. 44 pts discontinued treatment, mainly due to disease progression, with 2 due to AEs (Grade 3 thrombocytopenia and Grade 4 cholangitis). The 45 mg 5x/wk regimen was selected as the expansion dose. PRT543 demonstrated dose-dependent increases of Cmax (nM) and AUC (nM.hr). At the expansion dose, T½ was 15 hrs, and plasma drug exposures (Cmax, 2142 nM; AUC, 26,293 nM.hr) exceeded those for preclinical efficacy models. Serum sDMA decreased in a dose-dependent manner reaching 77% reduction with 50 mg QD and 69% reduction with the expansion dose. Increased intron retention was seen in peripheral blood mononuclear cells. A complete response (CR) has been maintained for &amp;gt;1 yr in a pt with homologous recombination deficiency-positive (HRD+) ovarian cancer who remains on study therapy. An additional 5 pts exhibited stable disease ≥6 mo. Conclusions: PRT543 was well tolerated with a favorable safety profile. Dose-dependent inhibition of target engagement and functional activity of PRMT5 were observed. PRT543 demonstrated encouraging clinical activity with a CR in HRD+ ovarian cancer and prolonged stable disease in multiple pts. An expansion phase in biomarker-selected solid tumor cohorts is ongoing. Citation Format: Meredith McKean, Manish R. Patel, Robert Wesolowski, Renata Ferrarotto, Eytan M. Stein, Alexander N. Shoushtari, David Mauro, John Viscusi, Peggy Scherle, Neha Bhagwat, William Sun, Rachel Chiaverelli, Eric Mintah, Shekeab Jauhari, Laura Finn, Neil D. Palmisiano, Robert A. Baiocchi. A phase 1 dose escalation study of protein arginine methyltransferase 5 (PRMT5) inhibitor PRT543 in patients with advanced solid tumors and lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P039.

PRMT5 Regulates Cell Pyroptosis By Silencing CASP1 in Multiple Myeloma

Protein arginine methyltransferase 5 (PRMT5), a histone methyltransferase responsible for the symmetric dimethylation of histone H4 on Arg 3 (H4R3me2s), is an enzyme that participates in tumor cell progression in a variety of hematological malignancies. However, the biological functions of PRMT5 in multiple myeloma (MM) and the underlying molecular mechanisms remain unclear. In this study, we conducted a bioinformatics analysis and found that PRMT5 expression was significantly upregulated in MM. In vitro and in vivo phenotypic experiments revealed that knockdown of PRMT5 expression enhanced cell pyroptosis in MM. Moreover, we found that CASP1 expression was negatively correlated with PRMT5 expression, and repressing PRMT5 expression rescued both the phenotype and expression markers (N-GSDMD, IL-1b and IL-18). Inhibition of PRMT5 activity increased CASP1 expression and promoted MM cell pyroptosis. Finally, high expression of PRMT5 or low expression of CASP1 was correlated with poor overall survival in MM. Collectively, our results provide a mechanism by which PRMT5 regulates cell pyroptosis by silencing CASP1 in MM. DisclosuresNo relevant conflicts of interest to declare.

A Phase 1 Dose Escalation Study of Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor PRT543 in Patients with Myeloid Malignancies

A Phase 1 Dose Escalation Study of Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor PRT543 in Patients with Myeloid Malignancies

Discovery and Pharmacological Characterization of JNJ-64619178, a Novel Small-Molecule Inhibitor of PRMT5 with Potent Antitumor Activity.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.