MAT2A Inhibition Blocks the Growth of MTAP-Deleted Cancer Cells by Reducing PRMT5-Dependent mRNA Splicing and Inducing DNA Damage

•Development of MAT2A inhibitors (MAT2Ai) AGI-24512 and AG-270 with improved potency•AGI-24152 reduce proliferation cancer cells tumors that lack MTAP•MAT2Ai PRMT5 activity affecting mRNA splicing inducing DNA damage•Antiproliferative effects are synergistic taxanes in vitro vivo The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor co-deleted CDKN2A approximately 15% all cancers. This co-deletion leads aggressive poor prognosis effective, molecularly targeted therapies. metabolic enzyme methionine adenosyltransferase 2? (MAT2A) was identified as a synthetic lethal target MTAP-deleted We report characterization potent substantially levels S-adenosylmethionine (SAM) demonstrate antiproliferative tumors. Using RNA sequencing proteomics, we inhibition mechanistically linked reduced protein arginine methyltransferase 5 (PRMT5) perturbations. further show damage mitotic defects ensue upon HCT116 MTAP?/? cells, providing rationale for combining clinical candidate antimitotic taxanes. has an important role metabolism epigenetics primary producer universal methyl donor (SAM). Despite this broad cellular role, recent work shown depletion selective effect cancers deletion separate gene, (Kryukov et al., 2016Kryukov G.V. Wilson F.H. Ruth J.R. Paulk J. Tsherniak A. Marlow S.E. Vazquez F. Weir B.A. Fitzgerald M.E. Tanaka M. al.MTAP confers enhanced dependency on cells.Science. 2016; 351: 1214-1218Crossref PubMed Scopus (207) Google Scholar; Marjon 2016Marjon K. Cameron M.J. Quang P. Clasquin M.F. Mandley E. Kunii McVay Choe S. Kernytsky Gross deletions create vulnerability targeting MAT2A/PRMT5/RIOK1 Axis.Cell Rep. 15: 574-587Abstract Full Text PDF (137) Mavrakis 2016Mavrakis K.J. McDonald 3rd, E.R. Schlabach M.R. Billy Hoffman G.R. deWeck Ruddy D.A. Venkatesan Yu McAllister G. al.Disordered MTAP/CDKN2A-deleted dependence PRMT5.Science. 1208-1213Crossref (202) Scholar). An explanation could be SAM-utilizing type II inhibited by metabolite, 5?-methylthioadenosine (MTA), which accumulates when MTAP deleted. Within high-MTA environment cancers, catalytic sensitive reduction SAM (Marjon While these results suggest may prove beneficial past efforts devise effective have been hampered presence large hydrophilic active site; large, open, highly hydrophobic allosteric binding pocket; adaptations potency (Quinlan 2017Quinlan C.L. Kaiser Bolanos B. Nowlin D. Grantner R. Karlicek-Bryant Feng J.L. Jenkinson Freeman-Cook Dann S.G. al.Targeting biosynthesis novel Mat2A.Nat. Chem. Biol. 2017; 13: 785-792Crossref (43) Methionine analogs such cycloleucine (Lombardini 1970Lombardini J.B. Coulter A.W. Talalay Analogues substrates reaction. Deductions concerning conformation methionine.Mol. Pharmacol. 1970; 6: 481-499PubMed Lombardini Sufrin, 1983Lombardini Sufrin Chemotherapeutic potential analogue tumor-derived adenosyltransferases.Biochem. 1983; 32: 489-495Crossref (29) Scholar) inhibit vitro, although their low structural simplicity raise concerns about specificity, pharmacokinetics limit use vivo. A moderately inhibitor, PF-9366, recently discovered However, PF-9366 treatment induces adaptation, including upregulation MAT2A, blunts its effects. developed de novo chemotype small-molecule potent, cell permeable, amenable testing. Our optimized potently blocking biosynthesis. pharmacologic validate demonstrating tumor growth Finally, describe biological consequences genotype-selective impact splicing, ultimately leading cell-cycle attenuated proliferation. Thus, represents successful application lethality therapeutic approach substantial subset patients loss (CDKN2A)/MTAP locus. Small-molecule were screening library >2,000 low-molecular-weight fragments ability bind using mass spectrometry-based ultrafiltration assay. Fragment hits confirmed orthogonal enzymatic surface plasmon resonance assays co-crystallized complex SAM. Further structure-guided design resulted discovery AGI-24512, improvement 3–6 orders magnitude compared previously reported (Figures 1A, 1B, S1A). To assess specificity monitored product after either or structurally related inactive compound 1A 1C). Treatment but not control, led dose-dependent decrease levels, suggesting on-target effect. Next, rate intracellular conversion stable isotope-labeled 13C5,15N-methionine 13C5,15N-SAM assessed directly measure production following short-term treatment. near complete at doses above IC50 1C 1D) 6.2 nM (Figure S1B). performed local pathway tracing U-13C5-methionine characterize pathways downstream S1C), revealing metabolite both wild-type (referred hereafter WT). uniquely reductions due biochemical properties Ki substrate MTA combined relatively affinity therefore tested whether would impair MTAP-null manner. Levels PRMT5-dependent symmetric dimethyl (SDMA) marks WT 1E 1F). Consistent prior results, basal SDMA lower cells. also observed significant 1G) (Singh 2004Singh V. Shi W. Evans G.B. Tyler P.C. Furneaux R.H. Almo S.C. Schramm V.L. Picomolar transition state human X-ray structure MT-immucillin-A.Biochemistry. 2004; 43: 9-18Crossref (62) Additionally, synergized dramatically marks. Dose-response studies quantitative assay revealed inhibits PRMT5-mediated 95 correlating 100 cell-growth In contrast, SAM-uncompetitive EPZ015666, irrespective status 1G 1H). induce date PRMT5. ATP-based readout time-lapse imaging confluence S2A). (IC50 96 h nM), no accumulation sensitized S2B). Scholar), Notably, feedback mechanism did prevent AGI-24512. hypothesized relative enabled maintain despite adaptation while initially decreased over time, self-limiting plateaued within 40 S2C). responsible sensitivity inhibition, (MTAPi) pretreatment necessary knockout (KO) HAP1 S2D S2E). selectively confirming our observations genetic tools function viable approach. explored panel 316 lines diverse origins 2B Table S1). most lines, limited lines. Across panel, predicted (GI50; p = 2.6 × 10?13) (GR) adjusted metric (GR, 2.2 ?M; 1.4 10?15) (this chosen arbitrarily; similar obtained additional GR metrics). Furthermore, unbiased bioinformatics analyses feature predictive only other features genes chromosome 9p21 2C S2F). significantly more than nonspecific cytotoxic agent staurosporine, indicative (Table data suggested AGI-24512-mediated likely target, generated drug-resistant establish increasing 10 weeks emergence population. Genome population indicated A276V point mutation S2G). Exogenous expression treatment-naïve mediated via S2H S2I). failed MAT2A-expressing showing occur S2J). pocket Scholar Agios file), it destabilize formation drug-enzyme complex. Initial assessment pharmacokinetic oral absorption short half-life Structure-guided optimization AG-270, orally bioavailable metabolically 3A). demonstrated SAM, MTAP?/?-selective activity, 50 35.3 nM, respectively 3A, S3A, S3B). Oral animals sustained high-level exposure xenograft S3C) corresponding S3D) well tolerated. test vivo, dosed once daily mice bearing subcutaneous xenografts S3E S3F). (tumor [TGI] 75%, < 0.01) tolerated, body weight <20% animals. tumor-bearing mice. comparable models S3G), supporting hypothesis reliance differences driving inhibition. negligible S3H). explore surveyed patient-derived (PDX) from variety histological types 3B S2). PDX grown immunocompromised animals, established treated 200 mg/kg maximum engagement isogenic pair work. Drug resulting histologically models, high drug intratumoral S3I S3J). Availability genotype non-small lung (NSCLC) cohort allowed us correlations between extent analysis highlighted that, similarly data, NSCLC greater 3C), equivalent 3D). Critically, measured immunohistochemistry provided proof-of-concept evidence regarding modulation extended setting. efficacy across distinguish any particular targeting, observe stasis regressions select 3E 3F). ?M Eurofins' 90 unrelated targets reveal exceeding 50% S3K). yielding models. Since combination regimens therapeutics often critical maximal patient benefit (Bayat Mokhtari 2017Bayat Homayouni T.S. Baluch N. Morgatskaya Kumar Das Yeger H. Combination therapy combating cancer.Oncotarget. 8: 38022-38043Crossref (669) set out develop rational approaches maximize responses treatment, incorporation thymidine analog 5-ethynyl-2?-deoxyuridine (EdU) immunofluorescence 4A 4B ). pronounced checkpoint activation detectable asynchronously proliferating alterations induced become obvious synchronized double-thymidine block synchronize pretreated 72 early S phase 4C). Upon release replicative block, progression AGI-24512-treated G1 into G2/M 4C 4D). correlated several regulators transition, cyclin B Aurora B, appearance marker, phosphorylated histone H3 S4A). CDKN1A/Cip1/p21 (inhibitor CDK4/6 E2F-driven transcription) consistent stabilization p21 upstream regulator p53 These changes accompanied chromosomal-level damage, micronuclei binucleated multinucleated 4E–4H). Both micronucleus confined absent Reduced rate, activation, chromosomal abnormalities triggering response (DDR). DDR induction, analyzed H2AX (?H2AX) foci marker double-stranded breaks 5A 5B found increase ?H2AX–positive sulfoxide control next sought identify trigger known modulate RNA/DNA hybrid structures R loops (Zhao 2016Zhao D.Y. Gish Braunschweig U. Li Y. Ni Z. Schmitges F.W. Zhong Liu Moffat al.SMN dimethylation polymerase C-terminal domain termination.Nature. 529: 48-53Crossref (113) (Paulsen 2009Paulsen R.D. Soni D.V. Wollman Hahn A.T. Yee M.C. Guan Hesley J.A. Miller Cromwell E.F. Solow-Cordero D.E. al.A genome-wide siRNA screen reveals processes mediate genome stability.Mol. Cell. 2009; 35: 228-239Abstract (375) used high-throughput R-loop-specific S9.6 antibody detect R-loop detected increases nuclear signal intensity 5C 5D). Additional dot blot S5A S5B). RNAse H major removal eukaryotes (Amon Koshland, 2016Amon J.D. Koshland RNase enables efficient repair damage.eLife. 5: e20533Crossref overexpression H1, catalytically mutant D145N, ?H2AX 5B), indicating essential Importantly, formation, direct EPZ015666 independent S5C–S5E). sufficient explain damage. findings, (K333R/Y334T) impaired S5F) dominant negative S5G) MAT2Ai S5H), MAT2Ai-induced arises Evidence suggests collision transcription replication machineries acting same template (Hamperl 2017Hamperl Bocek Saldivar J.C. Swigut T. Cimprich K.A. Transcription-replication conflict orientation modulates activates distinct responses.Cell. 170: 774-786.e19Abstract (224) Indeed, S-phase supported co-localization EdU collapsed fork pRPA32 S33 spliceosome SC35 S5I S5J). processivity efficiency, fiber analysis, showed increased number forks S5K S5L). Collectively, accumulate rates Given 3D), methylates multiple components complex, dysregulate (Bezzi 2013Bezzi Teo S.X. Muller Mok W.C. Sahu S.K. Vardy L.A. Bonday Z.Q. Guccione Regulation constitutive alternative Mdm4 pre-mRNA sensing spliceosomal machinery.Genes Dev. 2013; 27: 1903-1916Crossref (146) Braun 2017Braun C.J. Stanciu Boutz P.L. Patterson Calligaris Higuchi Neupane Fenoglio Cahill D.P. Wakimoto al.Coordinated regulatory detained introns oncogenic transcripts creates exploitable malignant glioma.Cancer 411-426.e11Abstract (87) MAT2A-inhibitor-treated changes. affected events 6A 6B ; S3). forms perturbed particularly containing intron (DI) 6B). reports DIs heavily regulated dysregulated influence expression, since DI-containing retained nucleus thus ineffectively translated (Boutz 2015Boutz Bhutkar Sharp P.A. Detained novel, widespread class post-transcriptionally spliced introns.Genes 2015; 29: 63-80Crossref majority upregulated 6B), involved repair. Proteomic downregulated Reactome Fanconi anemia (FA) affected, FANCL, FANCA, FANC