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High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological cancer with few effective, targeted therapies. HGSOC tumors exhibit genomic instability with frequent alterations in the protein kinome; however, only a small fraction of the kinome has been therapeutically targeted in HGSOC. Using multiplexed inhibitor beads and mass spectrometry (MIB-MS), we mapped the kinome landscape of HGSOC tumors from patients and patient-derived xenograft (PDX) models. The data revealed a prevalent signature consisting of established HGSOC-driver kinases, as well as several kinases previously unexplored in HGSOC. Loss-of-function analysis of these kinases in HGSOC cells indicated MRCKA (also known as CDC42BPA) as a putative therapeutic target. Characterization of the effects of MRCKA knockdown in established HGSOC cell lines demonstrated that MRCKA was integral to signaling that regulated the cell cycle checkpoint, focal adhesion and actin remodeling, as well as cell migration, proliferation, and survival. Moreover, inhibition of MRCKA using the small molecule BDP9066 decreased cell proliferation and spheroid formation and induced apoptosis in HGSOC cells, suggesting that MRCKA may be a promising therapeutic target for the treatment of HGSOC.

// Niklas K. Finnberg 1 , Prashanth Gokare 1 , Avital Lev 1 , Sergei I. Grivennikov 2 , Alexander W. MacFarlane IV 3 , Kerry S. Campbell 3 , Ryan M. Winters 4 , Karen Kaputa 4 , Jeffrey M. Farma 5 , Abbas El-Sayed Abbas 6 , Luigi Grasso 7 , Nicholas C. Nicolaides 7 and Wafik S. El-Deiry 1 1 Department of Hematology/Oncology and Molecular Therapeutics Program, Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Fox Chase Cancer Center, Philadelphia, PA, USA 2 Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, USA 3 Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA, USA 4 Biosample Repository Facility, Fox Chase Cancer Center, Philadelphia, PA, USA 5 Division of General Surgery, Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA 6 Division of Thoracic Surgery, Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA 7 Morphotek Inc., Philadelphia, PA, USA Correspondence to: Wafik S. El-Deiry, email: // Keywords : 3D, organoid, colorectal cancer, immune cells, biomarkers Received : June 24, 2017 Accepted : July 20, 2017 Published : August 05, 2017 Abstract We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient’s peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo . Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

Introduction: Tumors harboring BRCA1 mutations initially respond well to platinum and PARP inhibitor therapy; however, resistance invariably arises and is a major clinical problem. The BRCA1 185delAG allele is a common founder mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. Experimental procedures: In this study, we utilized the SUM1315MO2 breast cancer cell line that harbors a BRCA1 185delAG mutation to study mechanisms of PARP inhibitor and platinum resistance. SUM1315MO2 cells were cultured in the presence of increasing concentrations of the PARP inhibitor rucaparib or cisplatin until rucaparib resistant (RR) and cisplatin resistant (CR) clones emerged. Results: DNA sequencing revealed that no BRCA1 gene reversion mutations were present in resistant cells. We next measured BRCA1 protein levels by Western blotting with antibodies specific for the N- and C-terminal domains of BRCA1. As a control, the wild-type BRCA1 protein expressed in MDA-MB-231 cells was detectable with both N- and C-terminal antibodies. In contrast, BRCA1 protein was undetectable in both SUM1315MO2 parental and resistant clones using the N-terminal specific antibody. However, the C-terminal specific antibody identified a more quickly migrating band of low abundance in parental cells, but with elevated expression in both RR and CR clones. We inferred that this protein was devoid of the extreme N-terminal RING domain-containing region that mediates interaction with BARD1. To investigate the functionality of the N-terminal truncated BRCA1 protein (Nt-BRCA1), we measured BRCA1 and RAD51 irradiation-induced focus formation by immunofluorescence. RR and CR clones demonstrated 2.25 - 2.75-fold increase (P = 0.024) in the number of cells with BRCA1 foci compared to parental cells. Similarly, we detected a 2 - 2.85-fold increase (P = 0.0325) in RAD51 foci formation in resistant clones compared to parental cells. Additionally, BRCA1 siRNA treated cells were 12- and 9-fold more sensitive to rucaparib compared to scrambled siRNA-treated control cells (P = 0.0002). Similarly, CR-1 cells treated with BRCA1 siRNAs were 1.56- and 1.7-fold more sensitive to cisplatin (P = 0.0346) compared to scrambled siRNA treated cells. Furthermore, N-terminal truncated BRCA1 proteins were detectable in a primary tumor from a germline BRCA1185delAG mutation carrier. Conclusions: Taken together, these results provide evidence for a novel, mutation location-dependent mechanism of PARP inhibitor and platinum resistance. Citation Format: Yifan Wang, Andrea Bernhardy, Emmanuelle Nicolas, Ryan Winters, Kathy Cai, Kelly Duncan, James Duncan, Maria Harrell, Elizabeth Swisher, Neil Johnson. BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2015-5467