Methionine Adenosyltransferase 2A Research Articles

Hepatic prohibitin 1 and methionine adenosyltransferase α1 defend against primary and secondary liver cancer metastasis

The liver is a common site of cancer metastasis, most commonly from colorectal cancer, and primary liver cancers that have metastasized are associated with poor outcomes. The underlying mechanisms by which the liver defends against these processes are largely unknown. Prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) are highly expressed in the liver. They positively regulate each other and their deletion results in primary liver cancer. Here we investigated their roles in primary and secondary liver cancer metastasis. We identified common target genes of PHB1 and MAT1A using a metastasis array, and measured promoter activity and transcription factor binding using luciferase reporter assays and chromatin immunoprecipitation, respectively. We examined how PHB1 or MAT1A loss promotes liver cancer metastasis and whether their loss sensitizes to colorectal liver metastasis (CRLM). Matrix metalloproteinase-7 (MMP-7) is a common target of MAT1A and PHB1 and its induction is responsible for increased migration and invasion when MAT1A or PHB1 is silenced. Mechanistically, PHB1 and MAT1A negatively regulate MMP7 promoter activity via an AP-1 site by repressing the MAFG-FOSB complex. Loss of MAT1A or PHB1 also increased MMP-7 in extracellular vesicles, which were internalized by colon and pancreatic cancer cells to enhance their oncogenicity. Low hepatic MAT1A or PHB1 expression sensitized to CRLM, but not if endogenous hepatic MMP-7 was knocked down first, which lowered CD4+ T cells while increasing CD8+ T cells in the tumor microenvironment. Hepatocytes co-cultured with colorectal cancer cells express less MAT1A/PHB1 but more MMP-7. Consistently, CRLM raised distant hepatocytes' MMP-7 expression in mice and humans. We have identified a PHB1/MAT1A-MAFG/FOSB-MMP-7 axis that controls primary liver cancer metastasis and sensitization to CRLM. Primary and secondary liver cancer metastasis is associated with poor outcomes but whether the liver has underlying defense mechanism(s) against metastasis is unknown. Here we examined the hypothesis that hepatic prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) cooperate to defend the liver against metastasis. Our studies found PHB1 and MAT1A form a complex that suppresses matrix metalloproteinase-7 (MMP-7) at the transcriptional level and loss of either PHB1 or MAT1A sensitizes the liver to metastasis via MMP-7 induction. Strategies that target the PHB1/MAT1A-MMP-7 axis may be a promising approach for the treatment of primary and secondary liver cancer metastasis.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

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

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

Hematopoietic Stem/Progenitor Cell Intrinsic SAM Synthesis Is Required to Prevent p53 Pathway Activation By Maintaining DNA Stability

S-adenosylmethionine (SAM) is a principle methyl group donor to maintain epigenetic status. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a (Methionine adenosyltransferase 2a). It has been reported that SAM reduction by methionine restriction or Mat2a inhibition suppresses the proliferation of leukemia cells to attract attentions as a potential therapeutic target for leukemia. To develop the therapy targeting methionine metabolism in leukemia, it is essential to elucidate the significance of methionine metabolism in non-leukemic hematopoiesis. However, still little is known about the importance of SAM synthesis in hematopoiesis, including hematopoietic stem/progenitor cells (HSPCs), in vivo. By using Mx1-Cre mediated Mat2a gene deletion in mice ( Mat2a fl/fl;Mx1-Cre mice), we investigated the roles of Mat2a in hematopoiesis in vivo. Reduction of Mat2a activity in Mat2a knockout (KO) mice in bone marrow (BM) was confirmed by ultra-high performance liquid chromatography tandem mass spectrometry analysis. Mat2a KO mice displayed severe pancytopenia in peripheral blood (PB). Mature cell fractions (Myeloid cells, B cells and T cells) and HSPCs (HSCs, MPP2s, MPP3s and MPP4s) in BM were also significantly decreased in Mat2a KO mice, suggesting that Mat2a is essential for the maintenance of hematopoiesis from HSPCs in vivo. To exclude the possible contributions of the effects induced by Mat2a KO in non-hematopoietic cells, we performed BM transplantation analyses in which hematopoietic cell specific KO or non-hematopoietic cell specific KO were achieved. These analyses clearly confirmed that HSPC intrinsic SAM synthesis is inevitable for the maintenance of hematopoiesis. To reveal the mechanistic insights of the regulatory roles for Mat2a in HSPCs, we performed RNA sequencing analysis ( Mat2a WT vs KO in HSPCs). GSEA (Gene Set Enrichment Analysis) using the Hallmark gene sets showed that p53 pathway was most highly enriched in Mat2a KO. In depth, genes downstream of p53 ( Bax, Apaf1, Bbc3, Casp3, Casp8, Fas, Cdkn1a etc.) were up-regulated by Mat2a KO, causing cell cycle arrest and apoptosis in HSPCs. On the other hand, gene expressions of the upstream of p53 ( Atr, Atm, Chek1, Chek2 etc.) were unchanged, suggesting that p53 activity was not increased at mRNA expression levels. In line with this, mRNA expression levels of Trp53 was unchanged, but total protein amount of p53 was increased in Mat2a KO HSPCs. Since DNA stability is known as a critical regulator of p53 pathway, we analyzed gH2AX levels as DNA damage marker and found that gH2AX levels were increased in Mat2a KO HSPCs. To be noted, expressions of genes involved in Fanc pathway ( Fanca, Fancc, Fancd2, Fance, Fancg, Fancl, Fanci etc), which is responsible for the DNA maintenance, were broadly down-regulated in Mat2a KO HSPCs at transcriptomic levels. Therefore, the Fanc pathway repression by SAM synthesis inhibition could cause DNA damage and p53 pathway dependent loss of maintenance in Mat2a KO HSPCs. To further understand the importance of SAM synthesis under stress hematopoiesis. We analyzed Mat2a expression changes in HSPCs by irradiation (5.5 Gy/mouse). Intriguingly, Mat2a expression was up-regulated by irradiation. We also found that dietary methionine restriction (MR) in mice prevents its hematopoietic recovery from irradiation stress. To be noted, long term MR (24 weeks) did not show any particular compared to control diet. Therefore, methionine metabolism possesses significant roles at least in the situation where acute hematopoietic recovery is required. Collectively, in this study, we found that cell intrinsic SAM synthesis is required for the maintenance (proliferation and survival) of HSPCs in vivo. SAM might be required for the prevention of DNA damage and p53 pathway activation by supporting Fanc pathway gene expressions. Methionine and/or SAM availability might possess critical roles for the regeneration of non-leukemic hematopoiesis. Further investigation about the significance of SAM synthesis in non-leukemic hematopoiesis is required for the proper establishment of SAM synthesis targeting therapy in cancers.

Single Cell Multi-Omic Analysis of Neoplastic Plasma Cells across the Disease Spectrum Identifies Novel Pathobiologic Mediators and Potential Therapeutic Targets in Multiple Myeloma (MM)

Background: A challenge to developing a full understanding of MM pathobiology using bulk profiling techniques is the large degree of intra-tumoral heterogeneity from minor subclones that, despite their low representation, nonetheless contribute to therapy resistance and disease progression. Moreover, our current therapies tend to be targeted against pathways common to both neoplastic and normal plasma cells (PCs), indicating a need to define novel tumor biology modifiers that could potentially serve to improve risk stratification and as therapeutic targets. Methods: To overcome these limitations, we simultaneously evaluated individual PC single-cell transcriptome and B-cell receptor variable (VDJ) sequences from freshly enriched bone marrow biopsies. These included 33 patients with precursor conditions, 17 each with newly diagnosed symptomatic or relapsed/refractory disease (RRMM), and 3 controls using the 10x Genomics platform. A total of ~320,000 PCs were analyzed, yielding median per sample cell counts of 4,626 and ~300 million total reads. Identical immunoglobulin VDJ sequences defined monoclonal PCs, while diverse VDJ sequences at low frequencies defined polyclonal cells, allowing each patient to serve as their own control. Results: The percentage of monoclonal PCs in active MM was >84%, while precursor stages were more variable (40-100% in smoldering MM; 0.3-82% in monoclonal gammopathy of undetermined significance (MGUS)). Though we could not identify monoclonal PCs in two MGUS cases, we identified them by scRNA-seq in four MGUS patients whose bone marrow showed no clonal cells by standard clinical multiparametric flow cytometry. In differential gene expression analysis where samples were grouped based on the diagnosis, 501 dysregulated genes were identified in monoclonal PCs from symptomatic MM compared to polyclonal PCs. These included genes previously identified as disease modifiers, including Hepatocyte growth factor, Lactate dehydrogenase A, Dickkopf WNT signaling pathway inhibitor 1, CD27, and Lysosomal associated membrane protein family member 5, providing validation for our approach. Next, we identified 279 genes that were commonly dysregulated in monoclonal PCs from symptomatic vs. asymptomatic MM. We also performed unsupervised hierarchical clustering of the top 1000 most variable genes and defined three diagnostic categories. Significant ontology terms characterizing these categories broadly involved antigen processing and recognition in Group 1 (enriched in precursor stages); transcription and translation control in Group 2 (enriched in active MM); and cell division and DNA metabolism in Group 3 (enriched with RRMM) ( A). We focused on dysregulated targets that: showed differential expression in monoclonal vs. polyclonal PCs; expression in most patients within a diagnostic category; a correlation with outcomes in the CoMMpassstudy; and novelty in MM. Among these were Mitotic arrest deficient 2 like 1 (MAD2L1), a component of the mitotic spindle assembly checkpoint, and Methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of S-adenosylmethionine, a key methyl donor in multiple cellular processes, from methionine and ATP. Notably, MAD2L1 inhibition with M2I1, and MAT2A targeting with the allosteric inhibitor AG-270, both produced a dose- and time-dependent inhibition in cell proliferation and reduction in myeloma cell line viability. Importantly, the effects of AG-270 ( B) were associated with induction of markers of apoptosis, including Caspase cleavage, and a reduction of Histone H3 dimethylation. Moreover, they occurred at clinically relevant concentrations based on the known pharmacokinetics of this drug in Phase I testing. Conclusions: Our combined single-cell transcriptomics and VDJ sequencing allowed a greater understanding of heterogeneity among neoplastic PCs compared to each patient's own polyclonal PCs. These analyses can identify putative novel mediators of disease pathobiology that impact prognosis, and that can serve as potential new therapeutic targets using either novel agents, or drug repurposed from other therapeutic areas, setting the stage for their translation to the clinic.

THU486 Identification Of Metabolic And Molecular Mechanisms Driving Estrogen Receptor Positive Breast Cancer Disparities Using A Machine-Learning Pipeline

Abstract Disclosure: A. Santaliz Casiano: None. D. Mehta: None. H. Patel: None. G. Rauscher: None. J. Kim: None. J.M. Frasor: None. K. Hoskins: None. Background: African American (AA) women in the United States have a 40% higher breast cancer mortality rate compared with Non-Hispanic White (NHW) women. The survival disparity is particularly striking among ER+ breast cancer cases. The purpose of this study is to examine whether there are racial differences in metabolic pathways typically activated in patients with ER+ positive breast cancer. Methods: We collected pretreatment plasma from AA and NHW ER+ breast cancer cases and cancer-free controls to conduct an untargeted metabolomics analysis using gas chromatography mass spectrometry (GC-MS) to identify metabolites that may be altered in the different racial groups. Statistical methods combined with multiple feature selection and prediction models were employed to identify race-specific altered metabolic signatures. This was followed by the identification of altered metabolic pathways with a focus in AA patients with breast cancer. The clinical relevance of the identified pathways was further examined in PanCancer Atlas breast cancer data set from TCGA. Results: We identified differential metabolic signatures between NHW and AA patients. In AA patients, we observed changes in metabolites associated with amino acid metabolism, while fatty acid metabolism was significantly enriched in NHW patients. By mapping these metabolites to potential epigenetic regulatory mechanisms, this study identified significant association with regulators of metabolism such as methionine adenosyltransferase 1A (MAT1A), DNA Methyltransferases and Histone methyltransferases for AA individuals, and Fatty acid Synthase (FASN) and Monoacylglycerol lipase (MGL) for NHW individuals. Specific gene NELFE with histone methyltransferase activity was associated with poor survival exclusively for AA individuals. Conclusion: We employed a comprehensive and novel approach that integrates multiple machine learning methods and statistical methods, coupled with human functional pathway analyses. The metabolic profile of plasma samples identified may help elucidate underlying molecular drivers of disproportionately aggressive ER+ tumor biology in AA women, and may ultimately lead to identification of novel therapeutic targets. To our knowledge, this is a novel finding that describes a link between metabolic alterations and epigenetic regulation in AA breast cancer, and underscores the need for detailed investigations into the biological underpinnings of breast cancer health disparities. Presentation: Thursday, June 15, 2023

P08 - Methionine Adenosyltransferase 2a (MAT2A) is a driver and an actionable therapeutic target in androgen-indifferent prostate tumors

P08 - Methionine Adenosyltransferase 2a (MAT2A) is a driver and an actionable therapeutic target in androgen-indifferent prostate tumors

Discovery of novel methionine adenosyltransferase 2A (MAT2A) allosteric inhibitors by structure-based virtual screening

Methionine adenosyltransferase 2A (MAT2A) has been indicated as a drug target for oncology indications. Clinical trials with MAT2A inhibitors are currently on-going. Here, a structure-based virtual screening campaign was performed on the commercially available chemical space which yielded two novel MAT2A-inhibitor chemical series. The binding modes of the compounds were confirmed with X-ray crystallography. Both series have acceptable physicochemical properties and show nanomolar activity in the biochemical MAT2A inhibition assay and single-digit micromolar activity in the proliferation assay (MTAP -/- cell line). The identified compounds and the relating structural data could be helpful in related drug discovery projects.

Identification of metabolic pathways contributing to ER+ breast cancer disparities using a machine-learning pipeline

African American (AA) women in the United States have a 40% higher breast cancer mortality rate than Non-Hispanic White (NHW) women. The survival disparity is particularly striking among (estrogen receptor positive) ER+ breast cancer cases. The purpose of this study is to examine whether there are racial differences in metabolic pathways typically activated in patients with ER+ breast cancer. We collected pretreatment plasma from AA and NHW ER+ breast cancer cases (AA n = 48, NHW n = 54) and cancer-free controls (AA n = 100, NHW n = 48) to conduct an untargeted metabolomics analysis using gas chromatography mass spectrometry (GC–MS) to identify metabolites that may be altered in the different racial groups. Unpaired t-test combined with multiple feature selection and prediction models were employed to identify race-specific altered metabolic signatures. This was followed by the identification of altered metabolic pathways with a focus in AA patients with breast cancer. The clinical relevance of the identified pathways was further examined in PanCancer Atlas breast cancer data set from The Cancer Genome Atlas Program (TCGA). We identified differential metabolic signatures between NHW and AA patients. In AA patients, we observed decreased circulating levels of amino acids compared to healthy controls, while fatty acids were significantly higher in NHW patients. By mapping these metabolites to potential epigenetic regulatory mechanisms, this study identified significant associations with regulators of metabolism such as methionine adenosyltransferase 1A (MAT1A), DNA Methyltransferases and Histone methyltransferases for AA individuals, and Fatty acid Synthase (FASN) and Monoacylglycerol lipase (MGL) for NHW individuals. Specific gene Negative Elongation Factor Complex E (NELFE) with histone methyltransferase activity, was associated with poor survival exclusively for AA individuals. We employed a comprehensive and novel approach that integrates multiple machine learning and statistical methods, coupled with human functional pathway analyses. The metabolic profile of plasma samples identified may help elucidate underlying molecular drivers of disproportionately aggressive ER+ tumor biology in AA women. It may ultimately lead to the identification of novel therapeutic targets. To our knowledge, this is a novel finding that describes a link between metabolic alterations and epigenetic regulation in AA breast cancer and underscores the need for detailed investigations into the biological underpinnings of breast cancer health disparities.

DIPG-36. H3K27M HISTONE MUTANT GLIOMAS ARE SENSITIVE TO METHIONINE LOSS AND MAT2A INHIBITION THROUGH A NOVEL FEEDBACK MECHANISM BETWEEN AMD1 AND METTL16

Abstract Diffuse Midline gliomas (DMGs) are grade IV tumors by the World Health Organization. They are inoperable and resistant to chemo/radiotherapies resulting in a median survival of 8-11 months and a 5-year survival of <2%. DMG is an epigenetic disease characterized by mutations on histone H3.3 K27M resulting in global transcriptional reprogramming. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model using clinically relevant co-alterations in Olig2+ neural progenitor cells (NPCs). Using an unbiased systems biology approach, we identified a reliance of H3K27M but not isogenic controls to the amino acid methionine, and the enzymes methionine adenosyltransferase 2A (MAT2A), and adenosylmethionine decarboxylase 1 (AMD1). MAT2A is a master regulator of methionine metabolism that converts methionine into the universal methyl donor S-adenosylmethionine (SAM) which is later converted into decarboxylated SAM (dcSAM) by AMD1 for polyamine metabolism. We postulated that targeting methionine regulator MAT2A through genetic/pharmacological abrogation would selectively alter DMG viability by disrupting the methylome. We discovered a novel mechanism demonstrating H3K27M cells are sensitive to MAT2A loss independent of methylthioadenosine phosphorylase (MTAP) deletions but rather through AMD1 overexpression. The current paradigm shows that MAT2A protein expression is inversely correlated with cellular SAM concentrations as sensed by splicing complex and m6A reader methyltransferase-like protein 16 (METTL16). To investigate the molecular mechanism by which H3K27M represses MAT2A, we postulated that dcSAM, the resultant metabolite of AMD1, promote(s) high turnover of METTL16–MAT2A transcript interactions like SAM, thereby diminishing MAT2A transcript and protein expression. We found that exogenous dcSAM promoted MAT2A intron retention and lower mature transcript levels. Our findings demonstrate that H3K27M leads to increased AMD1 protein expression resulting in diminished MAT2A expression. Combinatorial treatments inhibiting MAT2A and AMD1 may presents exploitable therapeutic vulnerabilities in these gliomas.

MAT1A Suppression by the CTBP1/HDAC1/HDAC2 Transcriptional Complex Induces Immune Escape and Reduces Ferroptosis in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains a significant health burden globally due to its high prevalence and morbidity. C-terminal–binding protein 1 (CTBP1) is a transcriptional corepressor that modulates gene transcription by interacting with transcription factors or chromatin-modifying enzymes. High CTBP1 expression has been associated with the progression of various human cancers. In this study, bioinformatics analysis suggested the existence of a CTBP1/histone deacetylase 1 (HDAC1)/HDAC2 transcriptional complex that regulates the expression of methionine adenosyltransferase 1A (MAT1A), whose loss has been associated with ferroptosis suppression and HCC development. Thus, this study aims to investigate the interactions between the CTBP1/HDAC1/HDAC2 complex and MAT1A and their roles in HCC progression. First, high expression of CTBP1 was observed in HCC tissues and cells, where it promoted HCC cell proliferation and mobility while inhibiting cell apoptosis. CTBP1 interacted with HDAC1 and HDAC2 to suppress the MAT1A transcription, and silencing of either HDAC1 or HDAC2 or overexpression of MAT1A led to the inhibition of cancer cell malignancy. In addition, MAT1A overexpression resulted in increased S-adenosylmethionine levels, which promoted ferroptosis of HCC cells directly or indirectly by increasing CD8+ T-cell cytotoxicity and interferon-γ production. In vivo, MAT1A overexpression suppressed growth of CTBP1-induced xenograft tumors in mice while enhancing immune activity and inducing ferroptosis. However, treatment with ferrostatin-1, a ferroptosis inhibitor, blocked the tumor-suppressive effects of MAT1A. Collectively, this study reveals that the CTBP1/HDAC1/HDAC2 complex–induced MAT1A suppression is liked to immune escape and reduced ferroptosis of HCC cells.

Abstract 1747: Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

Abstract Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 412; RC, 12941 ± 1023; n=2; p=2.10e-8].Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p<.0001]. Both primary and recurrent GBM cells were subject to select MAT2A and AHCY inhibitor compounds and EC50s were determined. Recurrent cells demonstrated a vulnerability to TMZ-generated cell death both by genetically knocking down and pharmacologically inhibiting MAT2A and AHCY. MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to augment the practically negligible rate of remission for GBM and improve treatment outcomes for cancer patients. Citation Format: Emma Rowland, Jordan Walter, Anna Jermakowicz, Robert Suter, Rebecca Riggins, Nagi Ayad. Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy [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 1747.

Abstract 5804: Targeting methyl donor synthesis inhibits platinum-induced enrichment of ovarian cancer stem cells

Abstract Ovarian cancer (OC) is one of the deadliest gynecologic cancers in US. Platinum (Pt)-based chemotherapy can reprogram cancer cells to ovarian cancer stem cells (OCSCs) and contribute to incurable recurrence. We have previously shown that Pt-containing drugs, e.g., cisplatin, enrich for OCSCs and Pt-induced alterations in DNA methylation enhanced OCSC malignant properties. CSCs have been shown to be dependent on methionine (Met) in many cancers, and in the current study, we investigated the role of Met and the interplay between methylation and Met metabolism in OCSC enrichment. In high grade serous ovarian cancer cell lines (OVCAR5, OVCAR3), cisplatin treatment (IC50, 16hr) increased (p<0.01) the percentage of cells that were positive for aldehyde dehydrogenase (ALDH+ cells), a known OCSCs marker. Cisplatin treatment also increased (p<0.05) the whole cell level of 5-methylcytosine, indicating Pt-induced methylation changes. In addition, Met metabolism was altered by cisplatin, demonstrated by decreased (p<0.01) level of Met derivative, S-adenosyl-methionine (SAM), and increased (p<0.01) expression of Met utilization enzyme, methionine adenosyltransferase 2A (MAT2A). To investigate a possible role of Met metabolism in Pt-induced enrichment of OCSCs, OVCAR5 cells were cultured in complete or Met-depleted (Met-) media, treated with cisplatin or vehicle and examined for CSC properties, including ALDH+ cells and the ability to form spheroids under low-attachment conditions. Met depletion blocked (p<0.01) both the Pt-induced increase of ALDH+ cells and spheroid formation. Both Met- only and Met- in combination with cisplatin activated p38, determined by increased phosphorylated p38, which may have responded to Met depletion and promoted cell cycle arrest. In addition, MAT2A expression was higher (p<0.01) in ALDH+ compared to ALDH- OC cells. MAT2A inhibitor FIDAS-5 combined with cisplatin blocked the Pt-induced increase in ALDH+ cells (p<0.01), demonstrating a requirement for Met utilization in Pt-induced OCSC enrichment. As MAT2A is a therapeutic target in cancer, MAT2A inhibition could represent a therapeutic approach for inhibiting CSC in ovarian cancer. We are further investigating the mechanism of MAT2A-mediated inhibition on Pt-induced OCSCs enrichment by examining the OC methylome and cell cycle arrest. Citation Format: Shu Zhang, Tara X. Metcalfe, Christiane A. Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Targeting methyl donor synthesis inhibits platinum-induced enrichment of ovarian cancer stem cells. [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 5804.

Abstract 1095: Exploiting altered methionine metabolism to overcome treatment resistance in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive adult brain tumor and is uniformly fatal due to resistance to standard therapies such as radiation (RT) and chemotherapy. Our group and others have identified altered metabolism as a key mediator of GBM RT resistance. Methionine is an essential sulfur-containing amino acid that cells use to synthesize antioxidants, polyamines and S-adenosyl methionine (SAM), which drives intracellular methylation reactions. Methionine uptake is dramatically elevated in GBM compared to normal brain, but what GBMs use this methionine for, and whether it governs GBM treatment resistance, is unknown. Here, we find that RT acutely increases the levels of numerous methionine-related metabolites in multiple RT-resistant GBM models. To interrogate metabolic pathway activity, we used 13C5 methionine stable isotope tracing to show that GBMs respond to RT by activating the conversion of methionine to SAM, which is dependent on signaling through the DNA damage response. We developed in vivo methionine stable isotope tracing techniques to confirm these findings in orthotopic PDX models of GBM. Blocking the conversion of methionine to SAM, through pharmacologic inhibition of methionine adenosyltransferase 2A (MAT2A), slowed the repair of RT-induced DNA damage and increased cell death in GBM models following RT. These effects were especially pronounced in GBM models lacking the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP). Pharmacologic inhibition of MAT2A in flank and orthotopic in vivo GBM models depleted SAM levels and slowed tumor growth when combined with RT. Combining MAT2A inhibition with dietary methionine restriction and RT slowed GBM tumor growth even further. Together, our work demonstrates a new signaling link between DNA damage and methionine-driven SAM synthesis in GBM. Inhibiting SAM synthesis slows the repair of RT-induced DNA damage and augments RT efficacy. This therapeutic strategy may be especially effective in GBMs defective in methionine salvage and spare normal cortex in which methionine salvage is active. Citation Format: Navyateja Korimerla, Kari-Wilder Romans, Peter Kalev, Ayesha Kothari, Nathan Qi, Charles Evans, Maureen Kachman, Marc L Hyer, Katya Marjon, Taryn Sleger, Daniel R Wahl. Exploiting altered methionine metabolism to overcome treatment resistance in glioblastoma [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 1095.

Abstract 490: Discovery of novel, potent and orally available MAT2A inhibitors

Abstract The methylthioadenosine phosphorylase (MTAP) gene is co-deleted with CDKN2A in approximately 15% of all cancers, but targeting a loss of function mutant is not trivial due to the lack of a target protein. The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We describe here potent and orally bioavailable MAT2A inhibitors (“Euregen MAT2Ais”) with superior potency and selectivity compared to AG-270. Euregen MAT2Ais bind at the interface of two MAT2A subunits and exhibit low nanomolar IC50s in biochemical assay. These compounds demonstrated more than 20-fold selective for MTAP−/− cell lines over isogenic MTAP WT. Oral administration of Euregen MAT2Ais drived dose-dependent antitumor activity without meaningful body weight loss in HCT116 MTAP−/− xenograft model. Furthermore, Euregen MAT2Ais revealed excellent oral bioavailability and PK properties in pre-clinical species. We are optimizing multiple MAT2A inhibitors that exhibit activity across a wide range of cancers with MTAP-deletion. Citation Format: Xuxing Chen, Jing Li, Yanhong Chen. Discovery of novel, potent and orally available MAT2A inhibitors [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 490.

Abstract 6278: Combination therapeutic strategy with type I PRMT inhibition in cancer treatment

Abstract Type I protein arginine methyltransferases (PRMTs) are highly expressed and associated in various tumors. Targeting PRMTs as an epigenetic modulation could be valuable for treating cancer when combined with other therapies. We identified a series of orally bioavailable small molecule type I PRMT inhibitors with demonstrated single and combination anti-tumor efficacy in a variety of scenarios or models. Firstly, mutations of RNA splicing factors (SFs) commonly occur in hematologic malignancies and a variety of solid tumors, resulting in vulnerability to further disruption of the spliceosome. Inhibiting symmetric or asymmetric dimethylation of arginine could alter the RNA-binding specificity of the SFs and lead to the preferential killing of SF-mutant leukemias over wild-type counterparts (Fong et al., 2019). Therefore, targeting type I protein arginine methyltransferases (PRMTs) and PRMT5 concomitantly could minimize the compensatory effects and synergistically enhance antitumor activity. We validated the synergistic effects and mechanisms of combined treatment with a type I PRMT inhibitor and a PRMT5 inhibitor (JNJ-64619178, GSK3326595/EPZ015938 or an in-house PRMT5i) using cell-based assays and in vivo studies. Secondly, Methionine Adenosyltransferase 2A (MAT2A) inhibition decreases formation of S-adenosylmethionine (SAM), the cofactor of PRMTs, could in turn sensitize cancer cells to type I PRMT inhibition. In vitro and in vivo studies revealed that inhibitors of type I PRMT and MAT2A (AG-270) dosing combination indeed exhibited stronger anti-cancer activity than mono-treatment. Thirdly, the arginine methylation of receptor tyrosine kinases (RTKs) mediated by type I PRMT could increase the therapeutic sensitivity to RTK inhibitors like FLT3i. PRMT1 overexpression may contribute to AML stem/progenitor cells survival possibly through FLT3-ITD methylation, which may lead to enhanced activation of downstream signaling for cell survival and impact the regulation of self-renewal genes in leukemic initiating cells (LIC) (He et al, 2018). Hence, the combination treatment of FLT3 and type I PRMT inhibitors may provide a broader therapeutic benefit on FLT3 mutated and/or overexpressed Acute Myeloid Leukemia (AML). In vitro, strong combination synergy was observed with a remarkable induction of cell death. In vivo, the combination of type I PRMT inhibitor with different FLT3 inhibitors (Gilteritinib, Midostaurin, or CTS2016) led to deeper antitumor responses in a variety of FLT3-ITD AML models. Taken together, these findings support the co-administration of type I PRMT inhibitor with various therapies including epigenetic reprogramming, cancer metabolism modulation, or targeted therapies for receptor tyrosine kinases, which could benefit cancer patients as a promising therapeutic strategy. Citation Format: Hui Shi, Xingnian Fu, Yilin Liu, Qiugeng Ouyang, Meng Wang, Jiaxin Huang, Long Wang, Youzhen Wang, Yuan Mi, Haiping Wu. Combination therapeutic strategy with type I PRMT inhibition in cancer treatment. [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 6278.

Abstract 1637: MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations

Abstract Methylthioadenosine phosphorylase (MTAP) is co-deleted with the tumor suppressor CDKN2A in approximately 15% of solid tumors and can elicit dependency on methionine adenosyltransferase 2A (MAT2A) production of S-adenosyl methionine (SAM), the major methyl donor for cellular methyltransferase reactions. IDE397, a potent small molecule inhibitor of MAT2A, was developed to exploit this synthetic lethal relationship for therapeutic benefit in patients harboring MTAP-/- cancers and demonstrates selective anti-tumor activity in MTAP-/- models. Evaluation of IDE397 efficacy across a panel of 48 MTAP-/- PDX models revealed consistent tumor growth inhibition (TGI>60%) across diverse lineages, with distinct enrichment of tumor regressions in squamous lung cancers. To further enhance the efficacy of IDE397 within and among tumor lineages, we sought to identify rational combination partners. A three-pronged approach was employed that dovetailed molecular profiling of IDE397 drug effects in vivo, chemogenomic evaluation of selective drug sensitivities in MTAP-/- cell lines across the CCLE, and high-throughput in vitro drug combination screens. Unbiased pathway analysis of IDE397-dependent changes in MTAP-/- PDX transcriptomes revealed perturbations in pre-mRNA splicing, DNA damage repair, and mitotic spindle assembly as commonly enriched across diverse models. Evaluation of altered pre-mRNA splicing patterns suggested that many of these changes may be a consequence of selective impairment of PRMT5-dependent support of spliceosome activity. Notably, sensitivities to drugs that inhibit the fidelity of pre-mRNA splicing, genome stability, and microtubule stability were enriched in MTAP-/- cell lines, suggesting that the biological processes selectively altered by IDE397 in tumors are already partially impaired by MTAP deletion. Finally, an IDE397 in vitro drug combination viability screen of over 400 drugs returned taxanes, platins, topoisomerase inhibitors, splicing inhibitors, and anti-folates as selectively synergistic with IDE397 in MTAP-/- models. Synergy between IDE397 and pemetrexed was of particular interest given mechanistic convergence of the methionine salvage and folate cycle pathways on nucleotide synthesis in the context of MTAP-/-. Accordingly, this combination provided enhanced tumor growth inhibition in vivo in MTAP-/- NSCLC and bladder models and is currently under evaluation in phase 1. Collectively, these observations indicate that MAT2A inhibition can generate cell states in MTAP-/- tumor cells that are selectively vulnerable to approved chemotherapies and targeted therapies. These synergistic relationships may provide a predictive biomarker strategy for multiple IDE397 synthetic lethal combination therapies. Citation Format: Kimberline Y. Gerrick, Jenny Laraio, Kelsey Annen, Hoang Tran, Marcus M. Fischer, Yevgeniy Freyman, Geeta Sharma, Isaac Bishof, Stephen Federowicz, Divya Pankajakshan, Claire L. Neilan, Biju Mangatt, Anthony Mazurek, Mark R. Lackner, Michael White, Zineb Mounir. MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations [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 1637.

337 Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

OBJECTIVES/GOALS: Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. METHODS/STUDY POPULATION: Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. RESULTS/ANTICIPATED RESULTS: Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 0.012; RC, 12925 ± 0.023; n=2; p=2.10e-8]. Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p DISCUSSION/SIGNIFICANCE: MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to increase remission and reduce recurrence for GBM patients.

Design and Structural Optimization of Methionine Adenosyltransferase 2A (MAT2A) Inhibitors with High In Vivo Potency and Oral Bioavailability.

Inhibition of methionine adenosyltransferase 2A (MAT2A) in cancers with a deletion of methylthioadenosine phosphorylase (MTAP) gene leads to synthetic lethality, thus receiving significant interest in the field of precise cancer treatment. Herein, we report the discovery of a tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine fragment which occupies the MAT2A allosteric pocket. The lead compound 8 exhibited extremely high potency to inhibit MAT2A enzymatic activity (IC50 = 18 nM) and proliferation of MTAP-null cancer cells (IC50 = 52 nM). 8 had a favorable pharmacokinetic profile with a bioavailability of 116% in mice. More importantly, introducing an amide motif (28) to the core structure raised the plasma drug exposure from 11 718 to 41 192 ng·h·mL-1. 28 displayed a significantly better in vivo potency than AG-270, which is being evaluated in clinical trails, and induced -52% tumor regression in a xenograft MTAP-depleted colon tumor model.

Hyperphosphorylation of hepatic proteome characterizes nonalcoholic fatty liver disease in S-adenosylmethionine deficiency

Methionine adenosyltransferase 1a (MAT1A) is responsible for hepatic S-adenosyl-L-methionine (SAMe) biosynthesis. Mat1a -/- mice have hepatic SAMe depletion, develop nonalcoholic steatohepatitis (NASH) which is reversed with SAMe administration. We examined temporal alterations in the proteome/phosphoproteome in pre-disease and NASH Mat1a -/- mice, effects of SAMe administration, and compared to human nonalcoholic fatty liver disease (NAFLD). Mitochondrial and peroxisomal lipid metabolism proteins were altered in pre-disease mice and persisted in NASH Mat1a -/- mice, which exhibited more progressive alterations in cytoplasmic ribosomes, ER, and nuclear proteins. A common mechanism found in both pre-disease and NASH livers was a hyperphosphorylation signature consistent with casein kinase 2α (CK2α) and AKT1 activation, which was normalized by SAMe administration. This was mimicked in human NAFLD with a metabolomic signature (M-subtype) resembling Mat1a -/- mice. In conclusion, we have identified a common proteome/phosphoproteome signature between Mat1a -/- mice and human NAFLD M-subtype that may have pathophysiological and therapeutic implications.