Published OnlineFirst February 9, 2018; DOI: 10.1158/1078-0432.CCR-17-1928 Cancer Therapy: Preclinical Clinical Cancer Research Ponatinib Shows Potent Antitumor Activity in Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) through Multikinase Inhibition Jessica D. Lang1,William P.D. Hendricks1, Krystal A. Orlando2, Hongwei Yin1, Jeffrey Kiefer1, Pilar Ramos1, Ritin Sharma3, Patrick Pirrotte3, Elizabeth A. Raupach1,3, Chris Sereduk1, Nanyun Tang1, Winnie S. Liang1, Megan Washington1, Salvatore J. Facista1, Victoria L. Zismann1, Emily M. Cousins4, Michael B. Major4, Yemin Wang5, Anthony N. Karnezis5, Aleksandar Sekulic1,6, Ralf Hass7, Barbara C. Vanderhyden8, Praveen Nair9, Bernard E. Weissman2, David G. Huntsman5,10, and Jeffrey M. Trent1 Abstract Purpose: Small cell carcinoma of the ovary, hypercalcemic type three SWI/SNF wild-type ovarian cancer cell lines. We further (SCCOHT) is a rare, aggressive ovarian cancer in young women identified ponatinib as the most effective clinically approved that is universally driven by loss of the SWI/SNF ATPase subunits RTK inhibitor. Reexpression of SMARCA4 was shown to confer SMARCA4 and SMARCA2. A great need exists for effective targeted a 1.7-fold increase in resistance to ponatinib. Subsequent therapies for SCCOHT. proteomic assessment of ponatinib target modulation in Experimental Design: To identify underlying therapeutic vul- SCCOHT cell models confirmed inhibition of nine known nerabilities in SCCOHT, we conducted high-throughput siRNA ponatinib target kinases alongside 77 noncanonical ponatinib and drug screens. Complementary proteomics approaches pro- targets in SCCOHT. Finally, ponatinib delayed tumor dou- filed kinases inhibited by ponatinib. Ponatinib was tested for bling time 4-fold in SCCOHT-1 xenografts while reducing efficacy in two patient-derived xenograft (PDX) models and one final tumor volumes in SCCOHT PDX models by 58.6% and cell-line xenograft model of SCCOHT. 42.5%. Results: The receptor tyrosine kinase (RTK) family was Conclusions: Ponatinib is an effective agent for SMARCA4- enriched in siRNA screen hits, with FGFRs and PDGFRs being mutant SCCOHT in both in vitro and in vivo preclinical models overlapping hits between drug and siRNA screens. Of multiple through its inhibition of multiple kinases. Clinical investigation potent drug classes in SCCOHT cell lines, RTK inhibitors were of this FDA-approved oncology drug in SCCOHT is warranted. only one of two classes with selectivity in SCCOHT relative to Clin Cancer Res; 24(8); 1932–43. Ó2018 AACR. 1Division of Integrated Cancer Genomics, Translational Genomics Research Introduction Institute (TGen), Phoenix, Arizona. 2Department of Pathology and Laboratory Medicine, Lineberger Cancer Center, University of North Carolina, Chapel Hill, Small cell carcinoma of the ovary, hypercalcemic type North Carolina. 3Collaborative Center for Translational Mass Spectrometry, (SCCOHT), a rare and aggressive form of ovarian cancer, is Translational Genomics Research Institute (TGen), Phoenix, Arizona. 4Depart- diagnosed in women at a median age of 24 years (range, 14 ment of Cell Biology and Physiology, Lineberger Cancer Center, University of months–47 years; ref. 1). Meta-analysis of 257 clinically anno- North Carolina, Chapel Hill, North Carolina. 5Department of Pathology and tated SCCOHT cases has shown a dismal 2-year survival rate Laboratory Medicine, University of British Columbia and Department of Molec- ular Oncology, British Columbia Cancer Research Centre, Vancouver, British less than 35%. The most effective treatment regimen based on this Columbia, Canada. 6Department of Dermatology, Mayo Clinic, Scottsdale, assessment is surgery, followed by aggressive high-dose chemo- Arizona. 7Department of Obstetrics and Gynecology, Hannover Medical School, therapy, radiation, and stem cell rescue (1, 2). The poor response Hannover, Germany. 8Department of Cellular and Molecular Medicine, University rates and extreme toxicity of this regimen necessitate identifica- of Ottawa, and Cancer Therapeutics Program, Ottawa Hospital Research Insti- tion of effective, targeted treatments for these young patients. tute, Ottawa, Ontario, Canada. 9Molecular Response, San Diego, California. SCCOHTs are characterized by inactivating germline and somatic 10Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, British Columbia, Canada. mutations in the tumor suppressor SMARCA4 (also known as BRG1), resulting in concomitant protein loss in nearly Note: Supplementary data for this article are available at Clinical Cancer – Research Online (http://clincancerres.aacrjournals.org/). all cases (3 8). These SMARCA4 alterations occur amidst other- wise diploid SCCOHT genomes and very rare secondary muta- J.D. Lang and W.P.D. Hendricks contributed equally to this article. tions in other cancer genes (4, 8). SMARCA4 is one of two Corresponding Author: Jeffrey M. Trent, Translational Genomics Research mutually exclusive ATPase subunits of the SWI/SNF chromatin Institute (TGen), 445 North 5th Street, Phoenix, AZ 85004. Phone: 602-343- remodeling complex that plays a central role in regulation of 8419; Fax: 602-343-8448; E-mail: [email protected] transcriptional programs associated with differentiation. The doi: 10.1158/1078-0432.CCR-17-1928 alternative SWI/SNF ATPase, SMARCA2 (also known as BRM), Ó2018 American Association for Cancer Research. is also absent in SCCOHT due to epigenetic silencing (3, 7). Thus, 1932 Clin Cancer Res; 24(8) April 15, 2018 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst February 9, 2018; DOI: 10.1158/1078-0432.CCR-17-1928 Ponatinib Is Potent in Small Cell Carcinoma of the Ovary by class, only RTK inhibitors (PD-161570) and metal chelators Translational Relevance (ammonium pyrrolidine dithiocarbamate) were selective hits Pathogenic mutations in SWI/SNF chromatin-remodeling in SMARCA4-mutant SCCOHT cells relative to three SWI/SNF complex members occur in approximately 20% of cancers, but wild-type ovarian cancer cell lines. Subsequent evaluation of a no targeted therapies that exploit a tumor's dependence on panel of FGFR/PDGFR–selective RTK inhibitors highlighted SWI/SNF dysfunction have yet shown clinical effect. Small cell ponatinib as the most potent tested agent in SCCOHT cell lines, carcinoma of the ovary, hypercalcemic type (SCCOHT) is a and an inducible SMARCA4 SCCOHT cell line demonstrates that rare and aggressive form of ovarian cancer affecting young restoration of SMARCA4 expression reduces the sensitivity of women. It is characterized by mutational inactivation of SCCOHT cells to ponatinib. These observations align with prior SMARCA4 and epigenetic silencing of SMARCA2, the mutually data in SWI/SNF–mutant rhabdoid tumor models in which exclusive ATPases of the SWI/SNF complex. Here, we demon- expression of the ponatinib target FGFR was shown to be asso- strate potency of the FDA-approved oncology drug ponatinib, ciated with SMARCB1 and SMARCA4 loss, conveying sensitivity a receptor tyrosine kinase (RTK) inhibitor whose targets to RTK inhibitors including ponatinib (26, 27). Furthermore, include PDGFRs, FGFRs, and EphAs, in SMARCA4-mutant activation of downstream AKT was shown to be elevated in SCCOHT cell and animal models. This work suggests that SMARCB1-deficient tumor models (28, 29) and dual inhibition ponatinib exploits SCCOHT's dependence on RTK signaling in of PDGFRa and FGFR1 in MRT cell lines was more effective than the context of SWI/SNF dysregulation and that ponatinib may specific inhibitors (30). Here, we additionally show via proteomic be effective in SCCOHT treatment. Preclinical identification of assessment of target engagement and downstream signaling that an effective, approved oncology drug holds promise for rap- ponatinib's effects in SCCOHT are also mediated by dependence idly improving outcomes for these young patients and war- on signaling of multiple RTKs. Finally, we show the ability of rants clinical investigation. ponatinib to significantly delay tumor progression in a cell line xenograft model of SCCOHT and markedly reduce tumor growth of patient-derived xenograft (PDX) models of SCCOHT, thereby prioritizing ponatinib for a clinical trial in SCCOHT patients. these tumors are driven by a unique genotype that fuels broad transcriptional dysregulation through SWI/SNF dysfunction. Several other tumor types are also universally characterized by Materials and Methods inactivation of SWI/SNF complex members, including thoracic Cell lines sarcomas bearing SMARCA4 mutation and SMARCA2 loss (9), BIN67 and SCCOHT-1 (SCCOHT), G401 and G402 (MRT), malignant rhabdoid tumors (MRT) that are universally charac- H522 (lung adenocarcinoma), and OVCAR-3, OVCAR-5, and terized by SMARCB1 (also known as SNF5) mutations alongside OVCAR-8 (SWI/SNF-wildtype high-grade serous ovarian cancer) SMARCA2 silencing and expression loss in 70% of cases (10, 11), cells were maintained in RPMI1640 (Thermo Fisher Scientific) and renal medullary cancers also characterized by SMARCB1 loss supplemented with 10% FBS (Thermo Fisher Scientific) and 1% (12, 13). Other cancers with a significant proportion of SWI/SNF penicillin/streptomycin (Thermo Fisher Scientific). A427 (lung mutations include ovarian clear cell carcinomas and endome- adenocarcinoma) and HepG2 (hepatocellular carcinoma) cells trioid carcinomas (50% and 30% with ARID1A loss, respec- were maintained in EMEM (Thermo Fisher Scientific) supple- tively;