MTAP-deleted Cells Research Articles

AMG 193, a Clinical Stage MTA-Cooperative PRMT5 Inhibitor, Drives Antitumor Activity Preclinically and in Patients With MTAP-Deleted Cancers.

One of the most robust synthetic lethal interactions observed in multiple functional genomic screens has been dependency on PRMT5 in cancer cells with MTAP deletion. We report the discovery of the clinical stage MTA-cooperative PRMT5 inhibitor AMG 193, which preferentially binds PRMT5 in the presence of MTA and has potent biochemical and cellular activity in MTAP-deleted cells across multiple cancer lineages. In vitro, PRMT5 inhibition induces DNA damage, cell cycle arrest, and aberrant alternative mRNA splicing in MTAP-deleted cells. In human cell line and patient-derived xenograft models, AMG 193 induces robust antitumor activity and is well tolerated with no impact on normal hematopoietic cell lineages. AMG 193 synergizes with chemotherapies or the KRAS G12C inhibitor sotorasib in vitro, and combination treatment in vivo significantly inhibits tumor growth. AMG 193 is demonstrating promising clinical activity, including confirmed partial responses in patients with MTAP-deleted solid tumors from an ongoing phase 1/2 study.

Abstract 3227: Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM

Abstract Background: Homozygous deletion of MTAP is one of the most frequent genetic alterations in various solid tumors including glioblastoma (GBM), mesothelioma, pancreatic cancer and lung cancer. While MTAP-deleted cancers are associated with a poor prognosis, there are no approved drugs for the treatment of patients with MTAP deficiency. MAT2A has emerged as a potential target for selective anticancer effects in MTAP-deleted cancers. However, development of brain-penetrating MAT2A inhibitors are yet to be reported in preclinical or clinical stages despite of high MTAP deletion fraction in GBM and brain metastatic cancers. Here, we present highly potent and selective MAT2A inhibitors capable of brain penetration for MTAP deleted cancer therapy. Methods: A MAT2A biochemical enzyme assay was performed with phosphonate colorimetric kit. To evaluate in vitro and in vivo activities of compounds, S-adenosylmethionine (SAM) and symmetrical dimethyl arginine (SDMA) levels were measured using LC-MS/MS and high-content screening systems. Cell growth inhibition assay was evaluated to confirm the selectivity in isogenic pairs of HCT116 WT and MTAP-deleted cells. In vivo efficacy was conducted using the MTAP-deleted HCT116 xenograft. Results: To develop novel brain penetrating scaffolds, we focused on interaction of hydrogen bond acceptor in the scaffolds with arginine residue Arg313 in MAT2A protein and the improvement of physicochemical properties, including logP and logBB. A series of MAT2A inhibitors significantly inhibited MAT2A enzymatic activity, resulting in the reduction of intracellular SAM level, the direct product of MAT2A. These compounds showed highly potent inhibition of SDMA and cell proliferation in MTAP-deleted cells, while displaying minimal effect on MTAP-WT cells, showing >100-fold selectivity towards MTAP-deletion over MTAP-WT. Furthermore, anti-proliferative activity of MAT2A inhibitors were observed in MTAP-deleted cancer cells across various cancer types, including GBM, breast cancer, and non-small cell lung cancer. Remarkably, mouse pharmacokinetic study revealed high level of plasma and brain exposure with outstanding brain-to-plasma ratio (> 0.75). Robust tumor growth inhibition as well as reduction in tumor SAM levels were observed as monotherapy in an MTAP-deleted HCT116 xenograft model. Conclusion: We have identified novel brain-penetrating MAT2A inhibitors displaying encouraging in vitro and in vivo pharmacological profiles, indicating the potential use of these compounds as therapeutic agents for MTAP deleted brain metastatic cancer and GBM. Citation Format: Soyoung Ki, Jinhee Kim, Jungtae Na, Ilkyoo Koh, Hyunho Cho, Ho Yeon Lee, Hoiyun Jung, Gyeonghi Cho, Mijin Moon, Yongje Shin, Sook-Kyung Park. Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM [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 3227.

Abstract B098: Unbiased in vitro and in vivo drug anchor screens identify mechanisms of resistance and sensitization for MTA-cooperative PRMT5 inhibitors in MTAP-deleted cancer models

Abstract Homozygous deletion of the MTAP gene is one of the most common genetic alterations in cancer, affecting 10-15% of all human cancer. Clinical MTA-cooperative PRMT5 inhibitors, including TNG908 and TNG462, were developed to leverage the well-characterized synthetic lethal interaction between PRMT5 inhibition and MTAP deletion. Indeed, the first disclosed clinical data for an MTA-cooperative PRMT5 inhibitor demonstrated the ability of TNG908 to selectively inhibit PRMT5 in MTAP-deleted tumors while sparing adjacent normal, MTAP-proficient cells in patients. To further explore the mechanism of action of MTA-cooperative PRMT5 inhibitors in MTAP-deleted cells, we employed multiple CRISPR-based editing platforms (CRISPRn, CRISPRi and CRISPRa) to conduct unbiased MTA-cooperative PRMT5 inhibitor anchor screens in vitro and in vivo. A panel of cancer models representing multiple histologies revealed potential PRMT5 inhibitor sensitization and resistance pathways that also include mechanisms that are specific to MTA-cooperative inhibitors. Specifically, the results of these screens identify 1) the effect of MTA/SAM metabolism on MTA-cooperative PRMT5 inhibitors, and 2) identification of potential sub-stratification strategies for patients with MTAP-deleted cancer, and 3) the activity of PRMT5 in anti-apoptotic pathways and synergy between PRMT5 and BCLxL inhibition. Citation Format: Steven Lombardo, Matthew R Tonini, Lauren Grove, Silvia Fenoglio, James Tepper, Binzhang Shen, Hannah Stowe, Shangtao Liu, Samuel R Meier, Ashley H Choi, Tenzing Khendu, Yi Yu, Kevin M Cottrell, John P Maxwell, Jannik N Andersen, Alan Huang, Kimberly J Briggs, Teng Teng. Unbiased in vitro and in vivo drug anchor screens identify mechanisms of resistance and sensitization for MTA-cooperative PRMT5 inhibitors in MTAP-deleted cancer models [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 B098.

Abstract B018: MTA-cooperative PRMT5 inhibitors selectively modulate RNA splicing in MTAP-deleted cancer cells across histologies

Abstract PRMT5 is a type II arginine methyltransferase that forms an active complex with methylosome protein 50 (MEP50) to catalyze the symmetric dimethylation (SDMA) of arginine residues in proteins that regulate biological roles including modulation of apoptosis, DNA damage response and RNA processing. Some of the best characterized PRMT5 substrates are SNRPB, SNRPD1 and SNRPD3, which are necessary for the formation of the spliceosome and therefore RNA splicing fidelity. The development of MTA-cooperative PRMT5 inhibitors, including the clinical stage inhibitors TNG908 and TNG462, have been shown in preclinical studies to selectively inhibit PRMT5 in MTAP-deleted cancer cells while sparing normal, MTAP-intact cells. Consistent with this selective, on-target mechanism, treatment with TNG908 in preclinical studies results in increased aberrant RNA splicing in MTAP-deleted cells relative to MTAP-intact cells. As MTAP loss occurs in 10-15% of all human cancer, the identification of a signature of alternative splicing events may report pharmacodynamic activity of PRMT5 inhibitors and potentially predict patient response. Similar splicing alterations caused by PRMT5 inhibition were identified in preclinical MTAP-deleted cancer models representing glioblastoma, non-small cell lung cancer, among others, suggesting the events are histology-agnostic. Additionally, titration of exogenous MTA, an endogenous inhibitor of PRMT5, recapitulated the observed splicing events in preclinical MTAP-proficient cells. Similar findings were observed utilizing a pharmacological inhibitor of MTAP suggesting that the identified alternative splicing events are dependent on accumulation of MTA. Collectively, these data suggest that a PRMT5-dependent RNA splicing signature can be developed to identify solid tumors that may best respond to MTA-cooperative PRMT5 inhibitors as well as monitor their pharmacodynamic activity. Citation Format: Matthew R Tonini, Samuel R Meier, Shangtao Liu, Kevin M Cottrell, John P Maxwell, Jannik N Andersen, Alan Huang, Luisa Cimmino, Kimberly J Briggs. MTA-cooperative PRMT5 inhibitors selectively modulate RNA splicing in MTAP-deleted cancer cells across histologies [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 B018.

EXTH-63. MTA-COOPERATIVE PRMT5 INHIBITORS ARE EFFICACIOUS IN MTAP-DELETED MALIGNANT PERIPHERAL NERVE SHEATH TUMOR MODELS

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with limited treatment options and poor survival rates, necessitating the development of novel therapeutics. Approximately 25-50% of MPNST harbor loss of the enzyme methylthioadenosine phosphorylase (MTAP) due to a passenger deletion driven by loss of the proximal tumor suppressor gene, CDKN2A. PRMT5 was identified as a selective dependence in MTAP-deleted cells due to the accumulation of the substrate methylthioadenosine (MTA), which is itself an endogenous PRMT5 inhibitor. TNG908 and TNG462 are clinical stage MTA-cooperative PRMT5 inhibitors that demonstrate selectivity for MTAP-deleted cells over MTAP-intact cells of 15X and 45X, respectively. Previous reports show both molecules drive durable tumor regressions in xenograft models of various MTAP-deleted cancer histologies. Here, our objectives are to examine the activity of TNG908 and TNG462 in preclinical MPNST models. METHODS The proliferation effects of MTA-cooperative PRMT5 inhibitors, TNG908 or TNG462, and a SAM-cooperative PRMT5 inhibitor, GSK3326595, on MTAP-deleted and MTAP-intact MPNST cell lines were determined using CellTiter-Glo (CTG) assays. TNG908 and TNG462 were further profiled in two MTAP-deleted MPNST patient-derived xenograft (PDX) models, WU-356 and WU-386. RESULTS Incubation with the MTA-cooperative PRMT5 inhibitors, TNG908 and TNG462, selectively decreased the proliferation of MTAP-deleted MPNST cell lines relative to MTAP-intact MPNST cell lines. TNG908 and TNG462 drove dose-dependent antitumor activity including tumor regressions in the MTAP-deleted MPNST PDX models, WU-356 and WU-386, at well-tolerated doses. CONCLUSIONS The clinical stage MTA-cooperative PRMT5 inhibitors TNG908 (NCT05275478) and TNG462 (NCT05732831) are efficacious in MPNST models in vitro and in vivo and are therefore promising therapeutic agents for patients with MTAP-deleted MPNST.

DDDR-33. TNG908, A BRAIN-PENETRANT MTA-COOPERATIVE PRMT5 INHIBITOR, IS EFFICACIOUS IN PRECLINICAL MTAP-DELETED MODELS INCLUDING GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. The median overall survival (OS) of GBM patients is poor (1-2 years) with standard of care therapies, demonstrating the significant need for the development of novel therapeutics. TNG908 is a clinical stage MTA-cooperative PRMT5 inhibitor that is selectively active in MTAP-deleted cells by leveraging a synthetic lethal interaction. Approximately 40% of GBM tumors have MTAP loss due to a co-deletion event with the proximal tumor suppressor gene, CDKN2A. In preclinical in vitro studies, TNG908 was 15-fold more potent in MTAP-deleted cancer cell lines than in MTAP WT cells. TNG908 has high passive permeability and is neither a substrate for P-glycoprotein nor Breast Cancer Resistant Protein (BCRP) efflux transporters. Consistent with these favorable attributes, TNG908 demonstrated in vivo brain penetration in multiple preclinical models, including non-human primates and mice. TNG908 on-target pharmacodynamic activity was determined by dose-dependent decreases in SDMA-modified protein levels in a GBM subcutaneous xenograft model. TNG908 demonstrated dose-dependent antitumor activity in multiple hyper- and hypomethylated GBM subcutaneous models, including cell lines and patient-derived xenograft models. Despite 6-fold reduced Kpuu in rodents (Kpuu ~0.15) relative to non-human primates (Kpuu 0.9), oral administration of TNG908 drove near tumor stasis and increased median survival by 3-fold in a highly aggressive murine GBM orthotopic model. In summary, TNG908 is a potent, brain-penetrant, MTA-cooperative PRMT5 inhibitor that drives strong antitumor activity in preclinical models of MTAP-deleted GBM. TNG908 is currently enrolling patients with MTAP-deleted tumors including glioblastoma in a Phase I/II clinical trial (NCT05275478).

Abstract 1073: PRMT5 inhibition synergizes with a natural small molecule compound to kill MTAP-deleted cells and suppress tumor growth

Abstract Homozygous deletion of MTAP occurs in about 15% of all human cancers, such as glioblastoma, pancreatic cancer, mesothelioma, urothelial bladder carcinoma, and lung squamous cell carcinoma. PRMT5 inhibitors show activity against MTAP-deleted cancer cells in culture and xenografts with a mechanism that relies on the significant elevation of the MTAP substrate, methylthioadenosine (MTA). Previously, we have shown that unlike cells in culture, MTA levels in MTAP-deleted primary human GBM tumors are not significantly higher than in MTAP-intact tumors. Therefore, combining the PRMT5 inhibitor with another drug may be required to increase the therapeutic window and clinical efficacy of a PRMT5 inhibitor in MTAP-deleted patients. Here, we identified a natural small molecular chemical compound with a good safety profile that synergizes with a PRMT5-MTA complex inhibitor, which boosts the efficacy of MTAP-deleted selective cell killing in the presence of MTA sequestering cells. This combination therapy significantly increases the potency of PRMT5 inhibitor treatment in MTAP-deleted cells across various tumor cell lines and lowers the IC50 of PRMT5 inhibitor treatment. In vivo, PRMT5 inhibitor combination treatment leads to smaller tumor volumes in MTAP-deleted CDX tumors (U87, glioma cell line) compared to PRMT5 inhibitor monotherapy. In summary, our proposed combination therapy of PRMT5 inhibition with a natural compound may increase the therapeutic window and clinical efficacy of PRMT5 inhibitors leading to better treatment options for patients harboring MTAP-deleted cancer. Citation Format: Yasaman Barekatain, Kyle LaBella, Hikaru Sugimoto, Kristen Harris, Sunada Khadka, Florian Muller, Raghu Kalluri. PRMT5 inhibition synergizes with a natural small molecule compound to kill MTAP-deleted cells and suppress tumor growth [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 1073.

Extracellular 5′-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5′-deoxy-5′-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.

Abstract 1807: The discovery and preclinical characterization of the MTA cooperative PRMT5 inhibitor AM-9747

Abstract Homozygous deletion of the tumor suppressor gene CDKN2A and the neighboring MTAP gene located at chr9p21 occurs in 10-15% of human cancers. Deletion of MTAP, an enzyme in methionine and adenine salvage pathways, results in accumulation of its substrate MTA, which is structurally similar to SAM, the substrate methyl donor for the type II methyltransferase PRMT5. In MTAP deleted cells, MTA competes with SAM for binding to PRMT5, placing PRMT5 in a partially inhibited or hypormorphic state. Multiple studies using shRNAi knockdown have shown that tumor cell lines harboring MTAP deletions are vulnerable to PRMT5 inhibition. PRMT5 inhibitors that have advanced to clinical studies do not selectively target the MTA-bound form of PRMT5, and the preclinical activity of these molecules is not enriched in MTAP-deleted tumor cells lines. Moreover, the therapeutic window of these molecules is narrow, presumably due to the inhibition of PRMT5 in normal cells. We set out to identify PRMT5 inhibitors that bind cooperatively with MTA, with the goal of selectively targeting PRMT5 in MTAP-deleted tumors. A DNA encoded library screen was conducted to identify small molecules that preferentially bind to PRMT5 in the presence of MTA. The subsequent optimization of screening hits to improve potency, MTA-cooperativity, and pharmacokinetic properties led to the identification of AM-9747. The nature of the MTA cooperativity of AM-9747 was interrogated by multiple biophysical methods and structural biology experiments. Following treatment with AM-9747, the levels of SDMA marks were lower in HCT116 MTAP-deleted cells (IC50 = 0.0002 μM) compared to HCT116 MTAP-WT cells (IC50 = 0.050 μM). AM-9747 selectively inhibited the proliferation of HCT116 MTAP-deleted cells (IC50 = 0.027 μM) compared to HCT116 MTAP-WT cells (IC50 = 0.63 μM). The profiling of AM-9747 in an expanded panel of tumor cell lines demonstrated that AM-9747 inhibited the proliferation of most MTAP-deleted cells, with minimal effects on MTAP-WT cells. In vitro mechanism of action studies demonstrated that treatment with AM-9747 induces DNA damage, as illustrated by increased phosphorylation of H2AX, and an arrest in the G2/M phase of the cell cycle in MTAP-deleted cells. In vivo, oral administration of AM-9747 selectively inhibits SDMA and tumor growth in HCT116 MTAP-deleted tumor xenografts, compared to HCT116 MTAP-WT xenografts. Furthermore, treatment with AM-9747 inhibits the growth of multiple MTAP-deleted tumor xenograft models, including BXPC3 (PDAC) and DOHH2 (DLBCL). AM-9747 was profiled against a panel of over twenty PDX models, with greater than 50% tumor growth inhibition observed in the majority of PDX models harboring deletion of the MTAP gene. Our data with AM-9747 indicates that PRMT5 inhibitors that selectively target PRMT5 in cooperation with MTA may represent a novel and compelling therapeutic strategy for the treatment of MTAP-deleted cancers. Citation Format: Brian Belmontes, Antonia Policheni, Siyuan Liu, Katherine Slemmons, Jodi Moriguchi, Hayley Ma, Daniel Aiello, Yajing Yang, Mikkel Vestergaard, Sanne Cowland, Jan Anderson, Ian Sarvary, Nuria Tamayo, Liping Pettus, Susmith Mukund, Leszek Pope, Jennifer R. Allen, Sanne Glad, Matthew Bourbeau, Paul E. Hughes. The discovery and preclinical characterization of the MTA cooperative PRMT5 inhibitor AM-9747 [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 1807.

Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers.

The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP-deleted cancers. Here, we report the discovery of development candidate MRTX1719. MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP-deleted cells compared to MTAP-wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP-deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)phthalazin-1(2H)-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719.

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP’s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

Abstract 2714: Targeting MAT2A in CDKN2A/MTAP-deleted cancers

Abstract While deletions of the p16/CDKN2A tumor suppressor were first discovered more than 30 years ago, therapeutics that selectively target such tumors have proven elusive. Recent work utilizing functional genomics has identified a synthetic lethal vulnerability that arises due to co-deletion of the adjacent metabolic gene, methylthioadenosine phosphorylase (MTAP). Loss of MTAP in these tumors leads to an accumulation of MTAP substrate 5'-methylthioadenosine (MTA), which partially inhibits the arginine methyltransferase PRMT5 and sensitizes tumors to shRNA-mediated depletion of PRMT5 and the upstream metabolic enzyme, methionine adenosyltransferase 2 alpha (MAT2A). To investigate the therapeutic potential of this finding, we utilized a biophysical binding screen followed by iterative structure-guided design to make the first highly potent, selective, and orally bioavailable inhibitors of MAT2A. MAT2A inhibitor treatment leads to potent inhibition of the growth of HCT116 MTAP-/- cells while sparing isogenic HCT116 MTAP+/+ cells. Tumor xenograft studies similarly demonstrated MTAP-selective growth inhibition in HCT116 MTAP-/- tumors compared to isogenic HCT116 MTAP+/+ tumors. Further, MTAP-deletion correlated with MAT2A inhibitor efficacy across a panel of >300 cell lines in vitro, and MAT2A inhibitor treatment was efficacious in a variety of MTAP-deleted patient-derived xenografts in vivo. Having demonstrated that potent MAT2A inhibitors selectively block the proliferation of MTAP-deleted cells and tumors, we sought to investigate the mechanism by which these effects arise. Using methylation proteomics we noted that MAT2A inhibitor treatment leads to selective inhibition of PRMT5 methylation activity in MTAP-deleted cancers in vitro and in vivo. RNA-seq analyses revealed that MAT2A inhibition leads to substantial defects in RNA splicing in MTAP-deleted cancers, consistent with published findings that PRMT5-mediated methylation of splicing complex proteins is critical for their function. MAT2A inhibitor treatment led to a substantial increase in detained introns, which were enriched in genes involved in cell cycle regulation and DNA damage response, thus implicating dysregulated splicing in the antiproliferative effects of MAT2A inhibition in MTAP-deleted cancer cells. Furthermore, we demonstrated substantial drug-drug synergy between MAT2A inhibitors and select agents inhibiting cell cycle progression or DNA repair. Importantly we validated key combination findings in vivo, including demonstration of synergy with the MAT2A inhibitor AG-270 and anti-mitotic taxanes. AG-270 is the first MAT2A inhibitor to enter clinical development and is under investigation in a Phase I trial that is currently enrolling patients with MTAP-deleted solid tumors (NCT03435250). Our findings suggest clinically-applicable combination strategies which may further enhance the efficacy of AG-270 in malignancies with this genetic lesion. Citation Format: Katya Marjon, Peter Kalev, Marc Hyer, Mark Fletcher, Peili Zhang, Elia Aguado-Fraile, Everton Mandley, Zenon Konteatis, Jeremy Travins, Kevin Marks. Targeting MAT2A in CDKN2A/MTAP-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2714.

PRMT1 loss sensitizes cells to PRMT5 inhibition.

PRMT5 is an arginine methyltransferase that accounts for the vast majority of the symmetric methylation in cells. PRMT5 exerts its function when complexed with MEP50/WDR77. This activity is often elevated in cancer cells and correlates with poor prognosis, making PRMT5 a therapeutic target. To investigate the PRMT5 signaling pathway and to identify genes whose loss-of-function sensitizes cancer cells to PRMT5 inhibition, we performed a CRISPR/Cas9 genetic screen in the presence of a PRMT5 inhibitor. We identified known components of the PRMT5 writer/reader pathway including PRMT5 itself, MEP50/WDR77, PPP4C, SMNDC1 and SRSF3. Interestingly, loss of PRMT1, the major asymmetric arginine methyltransferase, also sensitizes cells to PRMT5 inhibition. We investigated the interplay between PRMT5 and PRMT1, and found that combinatorial inhibitor treatment of small cell lung cancer and pancreatic cancer cell models have a synergistic effect. Furthermore, MTAP-deleted cells, which harbor an attenuated PRMT5–MEP50 signaling pathway, are generally more sensitive to PRMT1 inhibition. Together, these findings demonstrate that there is a degree of redundancy between the PRMT5 and PRMT1 pathways, even though these two enzymes deposit different types of arginine methylation marks. Targeting this redundancy provides a vulnerability for tumors carrying a co-deletion of MTAP and the adjacent CDKN2A tumor suppressor gene.

Targeting the insulin-like growth factor-1 receptor in MTAP-deficient renal cell carcinoma

Renal cell carcinoma (RCC) has emerged as a metabolic disease characterized by dysregulated expression of metabolic enzymes. Patients with metastatic RCC have an unusually poor prognosis and near-universal resistance to all current therapies. To improve RCC treatment and the survival rate of patients with RCC, there is an urgent need to reveal the mechanisms by which metabolic reprogramming regulates aberrant signaling and oncogenic progression. Through an integrated analysis of RCC metabolic pathways, we showed that methylthioadenosine phosphorylase (MTAP) and its substrate methylthioadenosine (MTA) are dysregulated in aggressive RCC. A decrease in MTAP expression was observed in RCC tissues and correlated with higher tumor grade and shorter overall survival. Genetic manipulation of MTAP demonstrated that MTAP expression inhibits the epithelial-mesenchymal transition, invasion and migration of RCC cells. Interestingly, we found a decrease in the protein methylation level with a concomitant increase in tyrosine phosphorylation after MTAP knockout. A phospho-kinase array screen identified the type 1 insulin-like growth factor-1 receptor (IGF1R) as the candidate with the highest upregulation in tyrosine phosphorylation in response to MTAP loss. We further demonstrated that IGF1R phosphorylation acts upstream of Src and STAT3 signaling in MTAP-knockout RCC cells. IGF1R suppression by a selective inhibitor of IGF1R, linsitinib, impaired the cell migration and invasion capability of MTAP-deleted cells. Surprisingly, an increase in linsitinib-mediated cytotoxicity occurred in RCC cells with MTAP deficiency. Our data suggest that IGF1R signaling is a driver pathway that contributes to the aggressive nature of MTAP-deleted RCC.

Abstract 3507: Targeting the insulin-like growth factor-1 receptor in MTAP-deleted renal cell carcinomas

Abstract Renal cell carcinoma (RCC) has emerged as a metabolic disease characterized by dysregulated expression of metabolic enzymes. Patients with metastatic RCC have an unusually poor prognosis and near-universal resistance to all current therapies. To ultimately improve cancer treatment and survival rate, there is an urgent need to reveal the mechanisms by which metabolic enzymes and aberrant pathways regulate oncogenic signaling. Through an integrated two-step analysis of RCC metabolic pathways, we previously identified dysregulated expression of methylthioadenosine phosphorylase (MTAP) in aggressive RCC. An accumulation of cellular methylthioadenosine has been observed in MTAP-deleted cancer cells. Interestingly, we found a decreased of protein-methylation level with concomitant with an increase in tyrosine phosphorylation after MTAP knockout. Next, we performed a phospho-kinase antibody array screen and identified the type 1 insulin-like growth factor-1 receptor (IGF1R) as the top-one candidate with upregulated tyrosine phosphorylation in response to MTAP loss. IGF1R phosphorylation acted upstream of Src and STAT3 signaling in MTAP-knockout RCC cells. Reduction of phospho-IGF1R by a selective inhibitor of IGF-1R, linsitinib, inhibited the cell migration and invasion capability of MTAP-deleted cells. Data from cell viability assays showed that MTAP loss enhanced linsitinib-mediated cytotoxicity in RCC cells. Our data suggest that IGF1R signaling is a driver pathway to confer aggrieves nature of MTAP-deleted renal cell carcinomas. Citation Format: Jihao Xu, Wen-Hsin Chang, Carissa Huang, David Yang, Ching-Hsien Chen. Targeting the insulin-like growth factor-1 receptor in MTAP-deleted renal cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3507.

RIOK1 kinase activity is required for cell survival irrespective of MTAP status.

Genotype specific vulnerabilities of cancer cells constitute a promising strategy for the development of new therapeutics. Deletions of non-essential genes in tumors can generate unique vulnerabilities which could be exploited therapeutically. The MTAP gene is recurrently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. Recent studies have uncovered an increased dependency of MTAP-deleted cancer cells on the function of a PRMT5 containing complex, including WDR77, PRMT5 and the kinase RIOK1. As RIOK1 kinase activity constitutes a potential therapeutic target, we wanted to test if MTAP deletion confers increased sensitivity to RIOK1 inhibition. Using CRISPR/Cas9-mediated genome engineering we generated analog sensitive alleles of RIOK1 in isogenic cell lines differing only by MTAP status. While we were able to independently confirm an increased dependency of MTAP-deleted cells on PRMT5, we did not detect a differential requirement for RIOK1 kinase activity between MTAP-proficient and deficient cells. Our results reveal that the kinase activity of RIOK1 is required for the survival of cancer cell lines irrespective of their MTAP status and cast doubt on the therapeutic exploitability of RIOK1 in the context of MTAP-deleted cancers.

Abstract IA24: PRMT5 as a therapeutic target in MTAP-deleted cancers

Abstract An understanding of driver genetic events in tumorigenesis has led to the development of targeted therapies with clinical benefit for subsets of patients with melanoma, non-small cell lung cancer, and other malignancies. The identification of new cancer vulnerabilities may provide additional therapeutic opportunities for malignancies refractory to standard treatments and may expand the use of targeted therapies to a broader patient population. To identify new cancer vulnerabilities and associated genetic features, we have integrated insights about recurrent genetic alterations (from the Cancer Cell Line Encyclopedia) with cancer cell line dependencies identified from genome-scale RNA interference (from Project Achilles). Using this approach, we find that cancer cell lines harboring a highly-recurrent alteration (homozygous deletion of the gene encoding methylthioadenosine phosphorylase or MTAP) show selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77, core components of the methylosome. Homozygous loss of MTAP occurs as a passenger event in cancer due to its proximity to the commonly-deleted tumor suppressor gene CDKN2A. MTAP is lost in >40% of glioblastomas and in 25-30% of melanomas, urothelial carcinomas, and pancreatic adenocarcinomas (in addition to other cancers). MTAP cleaves methylthioadenosine (MTA) to generate precursor substrates for methionine and adenine salvage pathways, which are compromised with MTAP loss. While MTAP deletion in cancer has long been recognized as a potential vulnerability (as tumors with MTAP loss are dependent on de novo purine synthesis), therapeutic strategies to exploit MTAP loss have not been successfully implemented. Our findings credential PRMT5 as a previously-unrecognized potential therapeutic target in MTAP-deleted cancers. We have performed mechanistic studies to confirm a role for MTAP in modulating sensitivity to genetic depletion of PRMT5 or WDR77. Furthermore, we propose a mechanism by which homozygous MTAP deletion confers a selective dependency on PRMT5 and WDR77. We show that loss of MTAP leads to increased intracellular levels of MTA, which in turn specifically inhibits the catalytic activity of PRMT5. Since PRMT5 activity is essential for cell viability, MTAP-deleted cells may have some degree of basal inhibition of PRMT5 activity that confers heightened sensitivity to further reduction in PRMT5 activity with gene depletion. Given the frequency of homozygous MTAP loss in cancer, our findings suggest a potential role for PRMT5 inhibition in multiple malignancies, either as a single agent or possibly in combination with other therapies. As MTAP loss is not mutually exclusive of oncogenic drivers in cancer (such as activating BRAF mutations in melanoma or EGFR mutations in lung cancer), it may theoretically be possible to combine PRMT5 inhibition with targeting of oncogenic drivers in an effort to augment anti-tumor activity and possibly delay or prevent the emergence of drug resistance. Citation Format: Frederick H. Wilson. PRMT5 as a therapeutic target in MTAP-deleted cancers [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA24.

Abstract LB-307: MTAP deletions in cancer create vulnerability to a MAT2A/PRMT5/RIOK1 axis

Abstract Homozygous deletions of the CDKN2A tumor suppressor locus occur in approximately 15% of all cancer and represent a substantial therapeutic need(1). Targeted therapies selective for loss of CDKN2A have proven elusive, so we sought to evaluate whether the frequent co-deletion of the adjacent metabolic gene methylthioadenosine phosphorylase (MTAP) leads to collateral vulnerabilities. We conducted an shRNA screen in isogenic cells that vary only in their MTAP-deletion status, and identified an axis of targets that become vulnerable upon MTAP loss. Central in this axis is the arginine methyltransferase, PRMT5. Targeting PRMT5 with doxycycline-inducible shRNA impaired the growth of MTAP-deleted HCT116 colon carcinoma cells without impact on the growth of MTAP-wt HCT116 cells. Having identified this synthetic lethality between PRMT5 and MTAP, we sought next to elucidate the mechanistic basis for this relationship. Untargeted metabolomics revealed that MTA, the substrate of the MTAP enzyme reaction, accumulates dramatically in MTAP-deleted cancers. Biochemical profiling across the methyltransferase enzyme family revealed that MTA is a potent and selective inhibitor of PRMT5. Consistent with this in vitro finding, MTA accumulation in MTAP-deleted cancers led to reduced basal PRMT5 methylation. This selective vulnerability extends beyond PRMT5 to methionine adenosyltransferase 2, a (MAT2A), the metabolic enzyme upstream of PRMT5. MAT2A was the top hit in the shRNA screen, and targeting of MAT2A with inducible shRNA led to selective inhibition of growth in MTAP-deleted HCT116 cells in vitro and in vivo. Targeting of MAT2A led to substantial reduction of PRMT5 methylation activity in MTAP-deleted cells, indicating that PRMT5 activity is controlled by levels of substrate SAM and inhibitory metabolite MTA. To assess the therapeutic relevance of this axis in settings with endogenous deletion of the CDKN2A/MTAP locus, we depleted MAT2A in a panel of cancer cell lines and observed selective growth inhibition in the lines with endogenous deletion of CDKN2A/MTAP. Lastly, we noted that RIO Kinase 1 (RIOK1), a PRMT5-interacting protein(2), also scored in the shRNA screen. Doxycycline-inducible shRNA studies validated RIOK1 as an MTAP-selective target, and additional siRNA studies indicated that multiple PRMT5 co-complex members are selectively essential in MTAP-deleted cells. Thus, PRMT5 is the central node in an axis of targets that could be exploited therapeutically in cancers with deletion of CDKN2A/MTAP.

MTAP Deletions in Cancer Create Vulnerability to Targeting of the MAT2A/PRMT5/RIOK1 Axis.

Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.

544 DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy in breast cancer

Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.