Published OnlineFirst October 21, 2020; DOI: 10.1158/1535-7163.MCT-20-0259 MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS Reprogramming of Nucleotide Metabolism Mediates Synergy between Epigenetic Therapy and MAP Kinase Inhibition A C Tatiana Shorstova1, Jie Su1, Tiejun Zhao1, Michael Dahabieh1, Matthew Leibovitch1, Mariana De Sa Tavares Russo2, Daina Avizonis2, Shivshankari Rajkumar3, Ian R. Watson3, Sonia V. del Rincon 1, Wilson H. Miller Jr1, William D. Foulkes1,4, and Michael Witcher1 ABSTRACT ◥ Small cell carcinoma of the ovary, hypercalcemic type synergy is also observed in some SMARCA4-expressing ovarian (SCCOHT) is a rare but often lethal cancer that is diagnosed at a adenocarcinoma models intrinsically resistant to BETi. Mass spec- median age of 24 years. Optimal management of patients is not well trometry, coupled with knockdown of newly found targets such as defined, and current treatment remains challenging, necessitating thymidylate synthase, revealed that the repression of a panel of the discovery of novel therapeutic approaches. The identification of proteins involved in nucleotide synthesis underlies this synergy SMARCA4-inactivating mutations invariably characterizing this both in vitro and in vivo, resulting in reduced pools of nucleotide type of cancer provided insights facilitating diagnostic and thera- metabolites and subsequent cell-cycle arrest. Overall, our data peutic measures against this disease. We show here that the BET indicate that dual treatment with BETi and MEKi represents a inhibitor OTX015 acts in synergy with the MEK inhibitor cobime- rational combination therapy against SCCOHT and potentially tinib to repress the proliferation of SCCOHT in vivo. Notably, this additional ovarian cancer subtypes. Introduction others (3). The identification of a central role for SMARCA4 mutations in the pathogenesis of this tumor (4, 5), and subsequent use of Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is SMARCA4 (BRG1) IHC, with or without using antibodies raised an aggressive malignant tumor with a dismal prognosis (1). The mean against SMARCA2 (BRM) has greatly facilitated the diagnosis (6). age at diagnosis is approximately 24 years, and most patients die within Most SMARCA4 mutations in SCCOHT are deleterious resulting in 2 years of diagnosis. For SCCOHT, treatment generally involves a complete loss of protein expression, being confirmed by IHC in surgery and adjuvant chemotherapy, most commonly platinum- almost 100% of cases (4, 6). SMARCA4 and its paralog SMARCA2 are based compounds. Despite combination chemotherapy approaches, essential ATPase components of the multisubunit SWI/SNF (SWItch/ however, the prognosis still remains poor with overall 5-year survival Sucrose Non-Fermentable) chromatin remodeling complex and mod- rates of only 16% (2). Moving forward, personalized therapies for ify histone–DNA interactions by shifting or evicting nucleosomes to SCCOHT will require proper diagnosis and the identification of change the landscape of accessible regions on chromatin, thereby oncogenic drivers of these carcinomas. Differentiating SCCOHT from affecting transcriptional activation (7). SMARCA2 is concomitantly morphologically similar tumors is challenging. The “small” and “large” lost with SMARCA4 in almost all SCCOHT cases, and this profile now variants are fairly analogous (3), and SCCOHT also needs to be constitutes a molecular signature of the disease (6). distinguished from other primary and metastatic tumors that may Previously, it was shown that SMARCA4 and another bromodo- be found within the same tissue including nonepithelial ovarian main protein BRD4 independently associate with distal enhancer neoplasms and metastases from small cell lung carcinoma among elements of c-MYC in order to activate oncogene transcription, suggesting some redundancy between these two proteins in gene 1Departments of Oncology and Experimental Medicine, McGill University, Lady regulation and tumorigenesis (8). This led us to hypothesize that in Davis Institute and Segal Cancer Centre, Jewish General Hospital, Montreal, the absence of SMARCA4 and SMARCA2, BRD family members Quebec, Canada. 2Goodman Cancer Research Centre’s (GCRC) Metabolomics might represent essential proteins for driving transcriptional networks Facility, McGill University, Montreal, Quebec, Canada. 3Department of Biochem- involved in proliferation and survival (9). Thus, targeting SMARCA4- istry, Goodman Research Centre, McGill University, Montreal, Quebec, Canada. deficient cancers with bromodomain inhibitors (BETi), that target 4 Departments of Oncology and Human Genetics, McGill University, Lady Davis multiple BRD proteins, might effectively shut down this BRD-driven Institute and Segal Cancer Centre, Jewish General Hospital, Montreal, Quebec, fi Canada. oncogenic network. We demonstrated that SMARCA4/A2-de cient SCCOHT and non–small cell lung cancer (NSCLC) models were Note: Supplementary data for this article are available at Molecular Cancer acutely sensitive to BET inhibitors in vitro and in mouse models at Therapeutics Online (http://mct.aacrjournals.org/). very low doses (9). Notably, this work revealed that BETi efficacy Corresponding Author: Michael Witcher, Lady Davis Institute and Segal Cancer correlated with repression of PI3K and MAPK pathways. This is Centre, Jewish General Hospital, McGill University, 3755 Chemin de la Cote- Sainte-Catherine, Montreal, Quebec H3T1E2, Canada. Phone: 514-340-8222, ext. consistent with other studies suggesting that intrinsic resistance to 23363; Fax: 514-340-7502; E-mail: [email protected] BETi is conferred by constitutive signaling through receptor tyrosine kinase pathways including PI3K and MAPK (10–12). Recently, it was Mol Cancer Ther 2021;20:64–75 shown that NRAS-mutant melanoma models displayed resistance to doi: 10.1158/1535-7163.MCT-20-0259 BETi. In turn, combining BETi with MEKi led to decreased cell Ó2020 American Association for Cancer Research. proliferation in vitro and was also effective against melanomas AACRJournals.org | 64 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2021 American Association for Cancer Research. Published OnlineFirst October 21, 2020; DOI: 10.1158/1535-7163.MCT-20-0259 Combination BETi/MEKi Treatments in Ovarian Cancer carrying NRAS mutations in vivo (13). The precise mechanism Francois-Michel¸ Boisvert (Department of Anatomy and Cell Biology, through which this combination works, and whether this approach Universite de Sherbrooke). Next, the cells were exposed to 0.01% may be applicable to additional tumor types, remains unclear. DMSO, OTX015 (200 nmol/L), and cobimetinib (200 nmol/L) alone Here, we screened a range of inhibitors targeting PI3K and MAPK or in combination. After 24 hours of treatment, the cell pellets were pathways for potential synergy with BETi against a panel of frozen in liquid nitrogen and sent for the stable isotope labeling by SMARCA4-deficient and SMARCA4-expressing ovarian cancer cell amino acids in cell culture (SILAC) analysis to the Department of lines. Among all the tested compounds, we found a strong synergy Anatomy and Cell Biology, Universite de Sherbrooke. The analysis was between BETi (OTX015) and MEK inhibitors (cobimetinib and performed precisely as previously described (15). Perseus program was trametinib) at suboptimal doses in both ovarian cancer models. used for statistical analysis. The data were obtained from 3 replicates This combination also proved highly effective against orthotopic with log2 fold change > 0.5 and P value ≤ 0.05. Volcano plots were xenograft models of ovarian cancer. Using mass spectrometry to generated with Instant Clue. The data are available at the PRIDE assess changes in protein content after exposure to combination database (accession #PXD017581). therapy revealed that concurrent treatment with BETi/MEKi represses key enzymes involved in nucleotide metabolism. A con- Co-immunoprecipitation comitant decrease in nucleotide pools was validated using meta- Co-immunoprecipitation (co-IP) experiments were performed as bolomics profiling. This culminates in a reduced pool of nucleotide described previously (16). The HEK293T cells were transfected, using precursors and cell-cycle arrest. Overall, the combination of BETi/ polyethylenimine, with cDNAs encoding TYMS-Flag, Ubiquitin-HA, MEKi highlights a potential new therapeutic approach to treat and subsequently treated for 20 hours with MG132 (7 mmol/L), multiple subtypes of ovarian tumors. OTX015 (200 nmol/L), and/or Cobimetinib (200 nmol/L). Subse- quently, cells were collected, lysed, and immunoprecipitation carried out using antibodies against either FLAG or HA tags. Materials and Methods Cell culture In vivo experiments The OVK18 cell line was received from the RIKEN cell bank. Animal experiments were performed following guidelines of The SCCOHT1 cell line was a gift from Dr. Ralf Hass (Hannover the Canadian Council of Animal Care and approved by the Medical School, Hannover, Germany). OVCAR4, OVCAR3, SKOV3, Animal Resources Centre at McGill University. Five-week-old female IGROV1, and HEK293T were purchased from the ATCC. The cell NOD/SCID mice were injected with 5 Â 106 of OVK18 cells or 1 Â 107 lines were grown in RPMI-1640 medium supplemented with 10% FBS. of OVCAR4 cells in 1xPBS into left ovary via laparotomy. Treatments The culture medium for HEK293T cells was DMEM with 10% FBS. with vehicle, 20 mg/kg/day of OTX015 and 5 mg/kg/day of The cell lines were cultured for a maximum of 3 weeks. The