Methylthioadenosine Phosphorylase Gene Research Articles

6483 Diaphyseal Medullary Stenosis With Malignant Fibrous Histiocytoma: A Rare Skeletal Dysplasia/Sarcoma Syndrome

Abstract Disclosure: A. Grover: None. C.R. Ferreira: None. R.I. Gafni: None. S. Jumani: None. M.T. Collins: None. K.L. Roszko: None. I.R. Hartley: None. Introduction: Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is a rare autosomal dominant syndrome associated with pathologic fractures, myopathy, premature hair graying, and aggressive osteosarcomas. DMS-MFH has been linked to genetic variants in the methylthioadenosine phosphorylase (MTAP) gene, which encodes an enzyme in the methionine salvage and polyamine synthesis pathway. This pathway is implicated in other skeletal fragility disorders, such as Snyder-Robinson syndrome, suggesting a still undefined role of this pathway in bone biology. We describe a kindred with DMS-MFH and identify diagnostic challenges. Case: A 53-year-old perimenopausal woman presented with four fragility fractures over one year. Relevant history included premature hair graying, esophageal strictures, and nephrolithiasis. The patient’s bone formation markers were slightly elevated; bone resorption markers were within reference range. Her only osteoporosis risk factor was perimenopausal status. Bone series showed a diffuse heterogeneous appearance of the long bones with a mixed lytic/sclerotic appearance. DEXA scan revealed osteopenia. Family history was significant for 2 sons with aggressive osteosarcomas diagnosed in their late teens; one died at age 20. The other son is currently 39-years-old with recurrent osteosarcomas in three different limbs. The family also has a multigenerational history of severe bone fragility, muscle weakness, and premature hair graying. The patient, her living son, and his father underwent genome sequencing that identified a variant in MTAP (c.885A>G; p.(R295=)) in the patient and son. This variant was previously reported in three other families with DMS-MFH and has been shown to alter splicing. Notably, commercial genetic testing did not report this as a relevant variant, and diagnosis required independent analysis of the raw sequence data. Based on these findings, the patient and her son were diagnosed with DMS-MFH. Given the bone fragility with elevated bone turnover markers we treated the patient with yearly zoledronic acid infusions with no subsequent fractures. Surveillance for osteosarcoma is also being performed with annual whole-body MRI. Conclusion: DMS-MFH has been reported in only five families to date. The sixth family described here has clinical characteristics similar to the other kindreds. Due to high risk of aggressive osteosarcoma, timely diagnosis is imperative to decrease morbidity and mortality. Commercial genome sequencing may miss this diagnosis, so targeted genetic testing should be pursued. As more cases are identified, characterization of affected families may enhance our understanding of the natural history of the disease and guide surveillance and treatment strategies. Presentation: 6/2/2024

Endogenous adenine is a potential driver of the cardiovascular-kidney-metabolic syndrome.

Mechanisms underlying the cardiovascular-kidney-metabolic (CKM) syndrome are unknown, although key small molecule metabolites may be involved. Bulk and spatial metabolomics identified adenine to be upregulated and specifically enriched in coronary blood vessels in hearts from patients with diabetes and left ventricular hypertrophy. Single nucleus gene expression studies revealed that endothelial methylthioadenosine phosphorylase (MTAP) was increased in human hearts with hypertrophic cardiomyopathy. The urine adenine/creatinine ratio in patients was predictive of incident heart failure with preserved ejection fraction. Heart adenine and MTAP gene expression was increased in a 2-hit mouse model of hypertrophic heart disease and in a model of diastolic dysfunction with diabetes. Inhibition of MTAP blocked adenine accumulation in the heart, restored heart dysfunction in mice with type 2 diabetes and prevented ischemic heart damage in a rat model of myocardial infarction. Mechanistically, adenine-induced impaired mitophagy was reversed by reduction of mTOR. These studies indicate that endogenous adenine is in a causal pathway for heart failure and ischemic heart disease in the context of CKM syndrome.

Structure-Based Design and Optimization of Methionine Adenosyltransferase 2A (MAT2A) Inhibitors with High Selectivity, Brain Penetration, and In Vivo Efficacy.

Synthetic lethality has recently emerged as a new approach for the treatment of mutated genes that were previously considered undruggable. Targeting methionine adenosyltransferase 2A (MAT2A) in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene leads to synthetic lethality and thus has attracted significant interest in the field of precise anticancer drug development. Herein, we report the discovery of a series of novel MAT2A inhibitors featuring a pyrazolo[3,4-c]quinolin-4-one skeleton based on structure-based drug design. Further optimization led to compound 39, which has a high potency for inhibiting MAT2A and a remarkable selectivity for MTAP-deleted cancer cell lines. Compound 39 has a favorable pharmacokinetic profile with high plasma exposure and oral bioavailability, and it exhibits significant efficacy in xenograft MTAP-depleted models. Moreover, 39 demonstrates excellent brain exposure with a Kpuu of 0.64 in rats.

PRIMROSE: A modular phase 1/2a study of AZD3470, an MTA-cooperative PRMT5 inhibitor, in patients with MTAP deficient advanced solid tumors.

TPS3179 Background: Protein arginine methyltransferase 5 (PRMT5) is an epigenetic enzyme that catalyzes symmetric dimethylation of arginine substrates, regulating multiple cell processes. Deletion of the methylthioadenosine phosphorylase gene MTAP in tumor cells results in accumulation of the metabolite methylthioadenosine (MTA), a partial inhibitor of PRMT5, potentially rendering tumor cells susceptible to further PRMT5 targeting. Clinical activity of first-generation, non-selective PRMT5 inhibitors (PRMT5i) is limited by bone marrow toxicity due to lack of selectivity between normal cells and MTAP-deficient tumor cells. Second-generation PRMT5i, including AZD3470, should selectively target MTAP-deficient tumor cells whilst sparing normal cells. AZD3470 preferentially binds PRMT5 in the presence of MTA in MTAP-deficient tumor cells, inhibiting PRMT5 methylation activity, and is less effective in MTAP-proficient cells. PRIMROSE (NCT06130553) is a first-in-human, Phase 1/2a, open-label, multicenter study of AZD3470 as monotherapy and in combination with anti-cancer agents in patients with MTAP-deficient advanced / metastatic solid tumors. Methods: Eligible patients are aged ≥18 years with MTAP-deficient advanced or refractory solid tumors, ≥1 prior line of treatment in the recurrent / metastatic setting, ≥1 measurable lesion per RECIST v1.1, ECOG performance status 0/1, adequate organ and bone marrow function, and no prior treatment with PRMT5i. Module 1 will evaluate AZD3470 monotherapy. Part A (dose escalation) will enroll up to ~54 patients and follow a modified toxicity probability interval-2 (mTPI-2) design with once-daily dosing in 21-day cycles. Treatment will be given until progression or discontinuation criteria are met. Part B (dose optimization and expansion) will expand specific patient populations and / or tumor types, with up to ~30 patients per cohort; dosing will be based on safety and tolerability data from Part A. Further modules for combination treatments may be added based on emerging supportive data. The primary objectives are to assess safety and tolerability, including dose-limiting toxicities, and to determine the recommended phase 2 dose of AZD3470. The secondary objective is evaluation of efficacy, including objective response rate, duration of response, disease control rate, change in tumor size, progression-free survival, and overall survival. An additional secondary objective in Module 1 is to assess pharmacokinetics. The study is planned to take place in ~20 centers across eight countries. The study was opened to enrollment in December 2023. Clinical trial information: NCT06130553 .

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

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

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

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

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

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

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

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

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

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

HGG-02. METABOLIC TARGETING OF HIGH-GRADE GLIOMAS WITH MTAP LOSS

Abstract Malignant transformation of pediatric glioma is often associated with homozygous deletion of CDKN2A. With CDKN2A loss, MTAP which is in the same chromosomal location, 9p21, is homozygously deleted in a majority of HGGs and some LGGs. The MTAP gene codes for methylthioadenosine phosphorylase (MTAP) protein which plays an important role in methionine salvage pathway. Absence of MTAP results in accumulation of 5’-methylthioadenosine (MTA), de novo purine synthesis, and tumor cell dependency on protein arginine methyltransferase 5 (PRMT5) and its substrate provider, methionine adenosyltransferase II alpha (MAT2A). We hypothesize that drugs targeting PRMT5, MAT2A, and de novo purine synthesis, in combination with drugs targeting hyper-activated RAS/MAPK pathway, will be effective in inhibiting MTAP-null glioma growth. Patient-derived glioma cells were validated for MTAP loss using genome sequencing, RNA sequencing and western blot. MTA accumulation caused by MTAP loss was validated through high-performance liquid chromatography. Cells were subjected to monotherapy and combination drug therapy with PRMT5, MAT2A and de novo purine synthesis inhibitors, in addition to drugs targeting RAS/MAPK signaling pathway. Additionally, MTAP-null (MTAPnull) in vivo models are being developed.We show accumulation of MTA in MTAPnull cells compared to MTAP-wild type (MTAPWT) cells, caused by loss of MTAP that cleaves MTA to adenine and 5-methylthioribose-1-phosphate (MTR). Accumulation of MTA makes MTAPnull glioma cells more sensitive to MAT2A inhibitor AG-270 in comparison to MTAPWT cells. Additionally, dual-drug therapies involving MTA and AG-270 result in additive effect on MTAPnull cells and antagonistic effect on MTAPWT cells. De novo purine synthesis inhibitor and drugs targeting the RAS/MAPK pathways also exhibited cell growth inhibition of MTAPnull cells. Overall, metabolic targeting of HGGs bearing CDKN2A/MTAP loss is indispensable for targeted therapy and for minimizing toxicity towards surrounding normal cells lacking similar mutations.

Methylthioadenosine phosphorylase deficiency in tumors: A compelling therapeutic target.

The methionine salvage pathway is responsible for recycling sulfur-containing metabolites to methionine. This salvage pathway has been found to be implicated in cell apoptosis, proliferation, differentiation and inflammatory response. Methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-methylthioadenosine, a by-product produced from polyamine biosynthesis. The MTAP gene is located adjacent to the cyclin-dependent kinase inhibitor 2A gene and co-deletes with CDKN2A in nearly 15% of tumors. Moreover, MTAP-deleted tumor cells exhibit greater sensitivity to methionine depletion and to the inhibitors of purine synthesis. In this review, we first summarized the molecular structure and expression of MTAP in tumors. Furthermore, we discussed PRMT5 and MAT2A as a potential vulnerability for MTAP-deleted tumors. The complex and dynamic role of MTAP in diverse malignancies has also been discussed. Finally, we demonstrated the implications for the treatment of MTAP-deleted tumors.

Abstract 1145: PH020-803: an MTA-cooperative and brain-penetrable PRMT5 inhibitor that selectively targets MTAP-deleted tumors

Abstract Background: Methylthioadenosine phosphorylase (MTAP) gene deletion occurs in ~10% of human cancers and is enriched in non-small cell lung cancer, pancreatic cancer and brain cancer. Since MTAP is the only enzyme responsible for methylthioadenosine (MTA) degradation, homozygous deletion of MTAP gene leads to intracellular accumulation of MTA. MTA competes with S-adenosylmethionine (SAM, the methyl donor) and results in partial inhibition of protein arginine methyltransferase 5 (PRMT5) activity. Recent studies reported that tumor cells with MTAP loss are vulnerable to PRMT5 inhibition. Based on the synthetic lethality interaction, an MTA cooperative PRMT5 inhibitor has the potential to selectively target MTAP-deleted tumors and avoid hematologic toxicity of substrate-competitive (such as GSK3326595) or SAM-competitive (such as JNJ64619178) PRMT5 inhibitors. Methods: A pair of HCT116 isogenic cell lines (MTAP-/- and MTAP+/+) were used to determine the effects of PH020-803 on cell proliferation and intracellular symmetric dimethylarginine (SDMA) content. Human CD34+ hematopoietic stem cells (HSCs) were collected to evaluate hematological toxicity. Intravenous injection of PH020-803 in rats were performed to assess the brain-penetration capability. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with tumor harboring MTAP gene deletion. The in vitro and in vivo pharmacokinetic and safety properties were assessed with corresponding assay methods. Results: PH020-803 dramatically decreased SDMA content and inhibited proliferation in HCT116 MTAP-/- cells (SDMA, IC50=1.04 nM; proliferation, IC50=19 nM), but had very weak effect on HCT116 MTAP+/+ cells (SDMA, IC50=535.5 nM; proliferation, IC50=1620 nM). In contrast, both GSK3326595 and JNJ64619178 had no obvious selectivity (proliferation: 595, IC50=8 nM and 35 nM in MTAP-/- cells and MTAP+/+ cells, respectively; 178, IC50=1 nM and 3 nM, respectively). PH020-803 mildly inhibited CD34+ HSC (IC50=900.2 nM), compared to potent cytotoxicity of GSK3326595 (IC50=15.8 nM). PH020-803 showed excellent brain penetration with a Kp value of 0.16. In two CDX models (HCT116 MTAP-/- and LU99), PH020-803 at a dose of 100 mg/kg QD potently inhibited tumor growth without hematologic toxicity observed. A 10-day repeated dosing study in mice (300 mg/kg QD) suggested that red blood cell and reticulocyte counts significantly decreased in GSK3326595 group, but not in PH020-803 group. Caco-2 and MDCK-MDR1 assays demonstrated excellent membrane permeability of PH020-803 without obvious efflux. Accordingly, PH020-803 possessed good pharmacokinetic profile in mice and rats with absolute bioavailability at 74% and 58%, respectively. Conclusion: The present data suggest that PH020-803 is an MTA-cooperative and brain-penetrable PRMT5 inhibitor that selectively targets MTAP-deleted tumors. Citation Format: Feng Gao, Bin Liu, Liandong Jing, Yongyong Wu, Pengzhi Zhang, Shuai Yuan, Hui Zhao, Yu Gao, Zhizhong Li, Xiaofan Wang, Yongqi Guo. PH020-803: an MTA-cooperative and brain-penetrable PRMT5 inhibitor that selectively targets MTAP-deleted tumors [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 1145.

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 503: ISM3412, a novel and selective MAT2A inhibitor for the treatment of cancer

Abstract The enzyme methionine adenosyltransferase 2α (MAT2A) has an important role in metabolism and epigenetics as the major producer of the universal methyl donor S-adenosyl methionine (SAM). Despite broad cellular functions, MAT2A was recently implicated as a synthetic lethal target in cancers that have a deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is found in approximately 15% of all cancers. Here, we report ISM3412 is a novel potent MAT2A inhibitor with high selectivity. In enzymatic assays, ISM3412 demonstrated a strong inhibition of MAT2A with an IC50 of 19.4 nM. In both HCT116 MTAP-/- cells and HCT116 parental cells, the compound inhibited SAM production, with IC50 values of 5.97 nM and 3.66 nM, respectively. Additionally, it inhibited SDMA production in HCT116 MTAP-/- cells (IC50 of 6.37 nM), but not in HCT116 parental cells (IC50 > 2000 nM). Consistent with the large SDMA inhibitory potency difference between the HCT116 isogenic cell pair, ISM3412 also exhibited very good selectivity in cellular proliferation assays. While it inhibited cellular growth in HCT116 MTAP-/-cells with an IC50 of 199nM, its inhibitory potency on HCT116 parental cells was very weak (IC50 of >21117nM), revealing more than a 100-fold selectivity in inhibiting growth in this isogenic pair cell line. Furthermore, while ISM3412 strongly inhibited proliferation in multiple cancer cell lines with natural MTAP deficiency (including pancreatic cancer, bladder cancer, and NSCLC), the same was not observed in multiple MTAP-intact cell lines (IC50 > 30 uM). Consistent with the in vitro data, ISM3412 demonstrated robust mono-therapeutic efficacy in an MTAP-/- HCT116 CDX model with tumor growth inhibition of 66% at 3 mg/kg QD. Similarly, ISM3412 also demonstrated robust single-agent efficacy in CDX models of pancreatic cancer, bladder cancer and NSCLC, as well as in a PDX model of esophageal cancer. Notably, ISM3412 showed improved efficacy when combined with docetaxel, compared to the individual monotherapy. ISM3412, at 10 uM, showed no obvious agonistic or antagonistic effect on 44 selected targets in a mini safety panel, demonstrating its selectivity as a MAT2A inhibitor. In addition to its strong biological potency and selectivity, ISM3412 exhibited favorable drug-likeness properties, including low molecular weight, good solubility and permeability, and low plasma protein binding. In vivo PK data revealed low clearance and high oral-bioavailability. Further, it was well tolerated with no significant hepatobiliary toxicity. In conclusion, ISM3412 is a novel and highly selective MAT2A inhibitor for MTAP-deficient cancers, and merits clinical evaluation. Citation Format: Qingyuan Meng, Xiao Ding, Xiaosong Liu, Hailong Wang, Ling Wang, Junwen Qiao, Hua Cao, Yushu Yin, Congcong Zhu, Xing Liang, Xin Cai, Yuanyuan Xu, Chenxi Xu, Shan Chen, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM3412, a novel and selective MAT2A inhibitor for the treatment of cancer [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 503.

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.

Copy Number Alterations in CDKN2A/2B and MTAP Genes Are Associated With Low MEF2C Expression in T-cell Acute Lymphoblastic Leukemia.

The molecular heterogeneity of T-cell acute lymphoblastic leukemia (T-ALL) makes this disease complex. Early T-cell precursor ALL (ETP-ALL) is a recognized subtype of T-ALL associated with a high probability of induction failure with conventional therapy. Higher expression of myocyte enhancer factor 2C (MEF2C) and the absence of a biallelic deletion (ABD) are the designated markers for the ETP-ALL. Co-deletion of the contiguous genes cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) and the methylthioadenosine phosphorylase (MTAP) cluster, located at 9p21.3, is another common alteration in T-ALL and confers poor response to treatment. We used real-time polymerase chain reaction (PCR) analysis to assess MEF2C mRNA expression and ABD status. Copy number alterations (CNAs) in key genes previously reported to be altered in T-ALL were assessed using multiple ligation probe amplification (MLPA). We observed that CNAs in this co-deletion cluster of CDKN2A/B and MTAP genes exhibited low MEF2C expression while ABD was associated with CNA in the Abelson murine leukemia 1 (ABL1) gene. Assessment of MEF2C expression based on immunophenotype revealed that its association with CDKN2A/2B alteration is present in non-immature immunophenotype. Additionally, ABD was associated with copy number alterations of T-cell acute lymphocytic leukemia protein 1 (TAL1), myeloblastosis (MYB), and LIM domain only 2 (LMO2) genes in immature immunophenotypes. Further, STIL::TAL1fusion was associated with low expression ofMEF2C. These associations may help explain the difficulties in assessing disease heterogeneity and the prognostic importance of 9p21.3 alterations in T-ALL.

Abstract 1397: A phenotypic-based drug discovery approach against tumors with MTAP loss

Abstract The Methylthioadenosine phosphorylase (MTAP) gene is in chromosome 9q21 location, which includes p16/CDKNA and is homozygously deleted in about 15% of all cancers, including a large proportion of gliomas, mesotheliomas and T cell lymphomas. MTAP cleaves methylthioadenosine (MTA), a byproduct of polyamine synthesis, to regenerate methionine and adenine. Several functional genomics screens have identified the downstream genes MAT2A and PRMT5 as synthetically lethal with MTAP loss. These two targets have been the subject of several drug discovery efforts. Our approach was based on a forward chemical genetic approach, in which we employed two isogenic cell lines, PANC1, with MTAP loss and PAM cells where MTAP is re-introduced. A library of over 200,000 compounds was screened for hits that affected the viability of PANC1 cell, whereas spared PAM cells. One hit series was found to reproducibly inhibit the proliferation of PANC1 cells with EC50s in the low single digit uM range and one order of selectivity over PAM cells. Phenotype-based optimization resulted in improved analogs, with prototype MTB-22252 exhibiting an EC50 of 47nM in PANC1 cells and 15-fold selectivity. An affinity probe was designed based on an analog of MTB-22252 bearing a biotin moiety via a linker and an azide group for photoactivatable cross-linking with potential interacting proteins. This probe maintained cellular activity and selectivity and it was used in an MS-proteomics experiment in PANC1 cells. The top protein identified by this experiment showed high enrichment over DMSO control and was effectively competed out by MTB-22252. MTB2252 and analogs thermo-stabilized this protein by up to 50C in PANC1 cells. PANC1 cells expressed high levels of this protein, whereas insensitive PAM cell did not express it at all. In additional cell line pairs, sensitivity to MTB-2252 correlated well with the expression levels of this protein. Reintroduction of MTAP in cells lacking MTAP and this new protein, resulted in loss of expression of the new protein, suggesting a regulatory connection. In a wider cell panel of over 130 cell lines, there was correlation of sensitivity with MTAP loss and even better correlation with the expression levels of this protein. Some of the most sensitive cell lines included several colorectal, glioma and hematologic cancer cell lines. In vivo evaluation of MTB-9655 against some of the most sensitive tumor cell lines is on-going. Citation Format: Andreas Goutopoulos, Iris Amanati, Avital Hay-Koren, Ali Fattaey, Philippe Nakache, Dimitri Kovalerchik, Dikla Paz, Simone Botti, Hanna Oppenheimer, Omri Erez, Irit Fainer. A phenotypic-based drug discovery approach against tumors with MTAP loss [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 1397.

Clinical significance of 9P21 gene combined with BAP1 and MTAP protein expression in diagnosis and prognosis of mesothelioma serous effusion.

The diagnostic value of the 9P21 gene determined using fluorescence in situ hybridization (FISH) combined with BRCA1-associated protein 1 (BAP1) and methylthioadenosine phosphorylase (MTAP) expression detection by immunohistochemistry, was investigated in serous effusion samples of malignant mesothelioma. A total of 70 serous disease samples with serous effusion were collected from June 2017 to June 2020. Following biopsy specimen pathological diagnosis, samples were divided into malignant mesothelioma and benign mesothelioma. Differential expression of BAP1 and MTAP genes were identified in mesothelioma and mesenchymal hyperplasia. The 9P21 gene fragment was lost in mesothelioma. The positive rates of FISH, BAP1 and MTAP in biopsy specimens were 98.00, 94.00 and 90.00%. The specificity of the three were 96.00, 85.71 and 77.27%, the sensitivity were 90.00, 95.92 and 93.75%, and the positive rate of the combined detection of the three was 93.33%. The positive rate of serous fluid samples detected by the three methods (9P21 FISH probe combined with BAP1 and MTAP expression detected immunohistochemically) was 96.00, 92.00 and 88.00%, the specificity were 90.00, 77.27 and 71.43%, the sensitivity was 96.00, 93.75 and 89.80%, and the positive rate of the three combined detections was 91.33%. It was demonstrated that there was a high consistency between serous fluid samples and biopsy samples. According to clinicopathological analysis, sex, age, lesion site, Ki67 had little association with the occurrence and development of malignant mesothelioma, while asbestos exposure history was closely associated to the occurrence of mesothelioma. A high level of BAP1 gene was positively associated with the prognosis of mesothelioma, while a high level of MTAP gene was negatively associated with the prognosis of mesothelioma (P<0.05). Therefore, 9P21 FISH probe combined with BAP1 and MTAP can be used as a new method for the detection of malignant mesothelioma, and provide an important basis for the early diagnosis of mesothelioma.

Leveraging Structure-Based Drug Design to Identify Next-Generation MAT2A Inhibitors, Including Brain-Penetrant and Peripherally Efficacious Leads.

Inhibition of the S-adenosyl methionine (SAM)-producing metabolic enzyme, methionine adenosyltransferase 2A (MAT2A), has received significant interest in the field of medicinal chemistry due to its implication as a synthetic lethal target in cancers with the deletion of the methylthioadenosine phosphorylase (MTAP) gene. Here, we report the identification of novel MAT2A inhibitors with distinct in vivo properties that may enhance their utility in treating patients. Following a high-throughput screening, we successfully applied the structure-based design lessons from our first-in-class MAT2A inhibitor, AG-270, to rapidly redesign and optimize our initial hit into two new lead compounds: a brain-penetrant compound, AGI-41998, and a potent, but limited brain-penetrant compound, AGI-43192. We hope that the identification and first disclosure of brain-penetrant MAT2A inhibitors will create new opportunities to explore the potential therapeutic effects of SAM modulation in the central nervous system (CNS).

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer.

5'-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.