Integrative Kinome Profiling Identifies Mtorc1/2 Inhibition As Treatment Strategy in Ovarian Clear Cell Carcinoma
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Published OnlineFirst April 23, 2018; DOI: 10.1158/1078-0432.CCR-17-3060 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Integrative Kinome Profiling Identifies mTORC1/2 Inhibition as Treatment Strategy in Ovarian Clear Cell Carcinoma Joseph J. Caumanns1, Katrien Berns2, G. Bea A. Wisman1, Rudolf S.N. Fehrmann3, Tushar Tomar1, Harry Klip1, Gert J. Meersma1, E. Marielle Hijmans2, Annemiek M.C. Gennissen2, Evelien W. Duiker4, Desiree Weening5, Hiroaki Itamochi6, Roelof J.C. Kluin2, Anna K.L. Reyners3, Michael J. Birrer7, Helga B. Salvesen8, Ignace Vergote9, Els van Nieuwenhuysen9, James Brenton10, E. Ioana Braicu11, Jolanta Kupryjanczyk12, Beata Spiewankiewicz13, Lorenza Mittempergher2, Rene Bernards2, Ate G.J. van der Zee1, and Steven de Jong3 Abstract Purpose: Advanced-stage ovarian clear cell carcinoma Results: We identified several putative driver mutations in (OCCC) is unresponsive to conventional platinum-based kinases at low frequency that were not previously annotated in chemotherapy. Frequent alterations in OCCC include del- OCCC. Combining mutations and copy-number alterations, eterious mutations in the tumor suppressor ARID1A and 91% of all tumors are affected in the PI3K/AKT/mTOR path- activating mutations in the PI3K subunit PIK3CA.Inthis way, the MAPK pathway, or the ERBB family of receptor study, we aimed to identify currently unknown mutated tyrosine kinases, and 82% in the DNA repair pathway. Strong kinases in patients with OCCC and test druggability of p-S6 staining in patients with OCCC suggests high mTORC1/2 downstream affected pathways in OCCC models. activity. We consistently found that the majority of OCCC cell Experimental Design: In a large set of patients with OCCC lines are especially sensitive to mTORC1/2 inhibition by (n ¼ 124), the human kinome (518 kinases) and additional AZD8055 and not toward drugs targeting ERBB family of cancer-related genes were sequenced, and copy-number altera- receptor tyrosine kinases or DNA repair signaling. We subse- tions were determined. Genetically characterized OCCC cell quently demonstrated the efficacy of mTORC1/2 inhibition in lines (n ¼ 17) and OCCC patient–derived xenografts (n ¼ 3) all our unique OCCC patient–derived xenograft models. were used for drug testing of ERBB tyrosine kinase inhibitors Conclusions: These results propose mTORC1/2 inhibi- erlotinib and lapatinib, the PARP inhibitor olaparib, and the tion as an effective treatment strategy in OCCC. Clin Cancer mTORC1/2 inhibitor AZD8055. Res; 24(16); 3928–40. Ó2018 AACR. Introduction ian cancer. The majority of patients with OCCC are diagnosed at In the United States, ovarian cancer is the fifth-leading cause of an early stage (57%–81% at stage I/II) and have better overall cancer deaths in women (1). Ovarian clear cell carcinoma survival compared with stage-matched high-grade serous (HGS) (OCCC) is the second most common subtype of epithelial ovar- ovarian cancer, the most common subtype of ovarian cancer. In 1Department of Gynecologic Oncology, Cancer Research Center Groningen, Medical University, Berlin, Germany. 12Department of Pathology and Laboratory University Medical Center Groningen, University of Groningen, Groningen, the Diagnostics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Netherlands. 2Division of Molecular Carcinogenesis, The Netherlands Cancer Oncology, Warsaw, Poland. 13Department of Gynecologic Oncology, Maria Institute, Amsterdam, the Netherlands. 3Department of Medical Oncology, Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Cancer Research Center Groningen, University Medical Center Groningen, Poland. University of Groningen, Groningen, the Netherlands. 4Department of Pathology and Medical Biology, Cancer Research Center Groningen, University Medical Note: Supplementary data for this article are available at Clinical Cancer Center Groningen, University of Groningen, Groningen, the Netherlands. Research Online (http://clincancerres.aacrjournals.org/). 5Department of Genetics, Cancer Research Center Groningen, University Med- ical Center Groningen, University of Groningen, Groningen, the Netherlands. In memory of H. Klip and H.B. Salvesen. 6Department of Obstetrics and Gynecology, Iwate Medical University School of Corresponding Author: Steven de Jong, Cancer Research Center Groningen, Medicine, Morioka, Iwate, Japan. 7Center for Cancer Research, The Gillette University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 Center for Gynecologic Oncology, Massachusetts General Hospital, Harvard GZ Groningen, the Netherlands. Phone: 31-50-3612964; Fax: 31-50-3614862; Medical School, Boston, Massachusetts. 8Department of Obstetrics and Gyne- E-mail: [email protected] cology, Haukeland University Hospital, Bergen, Norway. 9Department of Gynae- cology and Obstetrics, Leuven Cancer Institute, University Hospitals Leuven, doi: 10.1158/1078-0432.CCR-17-3060 Leuven, Belgium. 10Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom. 11Department of Gynecology, Charite Ó2018 American Association for Cancer Research. 3928 Clin Cancer Res; 24(16) August 15, 2018 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst April 23, 2018; DOI: 10.1158/1078-0432.CCR-17-3060 Kinome Profiling Identifies mTORC1/2 Sensitivity in OCCC research in OCCC cell lines demonstrated sensitivity to PI3K/ Translational Relevance mTOR dual inhibitors and AKT inhibitors, although PIK3CA Advanced-stage ovarian clear cell carcinoma (OCCC) is less mutations did not predict sensitivity to these inhibitors (16, 17). responsive to platinum-based chemotherapy compared with In the present study, we aimed to identify novel targetable high-grade serous ovarian carcinoma. Our in-depth analyses mutations by means of high-coverage sequencing of all protein of a large set of patients with OCCC reveal numerous genomic kinase genes, referred to as the kinome, and of a subgroup of alterations related to activation of mTORC1/2. High sensitiv- cancer-related genes in a large set of OCCC. In addition, we ity, especially to inhibitors targeting both mTORC1 and determined copy-number gains and losses in kinases and other mTORC2, is observed in a large OCCC cell line panel. Similar genes of OCCC tumors using high-coverage single-nucleotide results are obtained in OCCC patient–derived xenografts, polymorphism (SNP) arrays. To detect kinase mutations and signifying the clinical implications of these genomic altera- CNA at both high and low frequencies, we used a large cohort tions. Targeting of mTORC1 in combination with standard of 124 untreated primary OCCC tumors and most of the available chemotherapy did not improve overall survival in patients OCCC cell lines (n ¼ 17). Finally, we functionally validated with OCCC. Therefore, mTORC1/2 inhibitors, currently eval- several candidate targets in OCCC cell lines and unique OCCC uated in phase II clinical trials, are proposed for the treatment patient-derived xenograft (PDX) models. Our results indicate of OCCC. Based on the mutational landscape in OCCC, our in mTORC1/2 inhibition as an approach to guide future develop- vitro results, and the toxicity observed with dual inhibitors of ment of therapeutic strategies for OCCC. PI3K and mTORC1/2, future treatment combinations of mTORC1/2 inhibitors with either PI3K or MEK inhibitors Materials and Methods could be considered to improve clinical benefit for patients Sample collection with OCCC. Primary tumor samples from 124 patients with OCCC and 47 paired control blood samples were prospectively collected from Belgium, Germany, Norway, Poland, the Netherlands, the UK, and the United States. All patients gave written informed consent for samples to be collected, and the corresponding ethical review contrast, patients with OCCC diagnosed at late stage respond boards approved the study. Tumor samples had to contain 40% poorly to standard platinum-based chemotherapy compared with tumor cells, of which 70% was OCCC, as determined by patients with late-stage HGS ovarian carcinoma (2). In recent experienced gynecologic oncology pathologists. We obtained years, genetic studies in relatively small patient groups have 17 human OCCC cell lines: TOV21G (ATCC); RMG1, RMG2, revealed the mutational landscape in OCCC. The SWI-SNF chro- OVMANA, OVTOKO, and HAC2 (JCRB Cell Bank); JHOC5 matin remodeling complex DNA-binding AT-rich interactive (RIKEN Cell Bank); OVCA429 (Cell Biolabs); OVSAYO, TUOC1, domain 1A gene (ARID1A) has been shown to be deleteriously KK, OVAS, SMOV2, and KOC7C (Dr. Hiroaki Itamochi, mutated in 40% to 57% of patients with OCCC, the highest Tottori University School of Medicine, Tottori, Japan); ES2 percentage found in any cancer (3, 4). Loss of ARID1A protein, (Dr. Els Berns, Erasmus MC, Rotterdam, the Netherlands); TAYA being a key component of the complex, may affect the expression (Dr. Yasushi Saga, Jichi Medical University, Yakushiji, of many genes (5). Activation of the PI3K/AKT/mTOR pathway, Shimotsuke-shi, Tochigi, Japan); and OV207 (Dr. Vijayalakshmi implicated in survival, protein synthesis, and proliferation, is Shridhar, Mayo Clinic). All cell lines were maintained in RPMI another major player in OCCC. PIK3CA, encoding the catalytic supplemented with 10% FCS, 100 mg/mL Penicillin/Streptomy- domain of PI3K, contains activating mutations in 30% to 40% of cin and 2 mM L-glutamine. All cell lines were tested by short patients with OCCC,