Abstract B03: Targeting the KIF11/KIF15/TPX2 axis to develop new therapies for ovarian cancer

Abstract

Abstract Unlimited genetic and epigenetic diversity across the patient population, and within the tumor and among metastatic lesions of an individual patient, presents a challenge in developing targeted therapies to treat epithelial ovarian cancer (EOC). To find the Achilles heel of ovarian cancers, we designed an RNAi-based screen (>6,000 genes) to identify points of molecular vulnerability. These screens identified KIF11 as essential to maintain tumor cell viability. KIF11 encodes kinesin Eg5 (KIF11), a motor protein required for microtubule antiparallel sliding during mitosis that has been targeted clinically. Although KIF11 inhibitors are well tolerated by EOC patients, clinical response rates were disappointing in heavily treated patients with advanced disease. We hypothesize one mechanism through which KIF11 inhibitors’ efficacy is muted is via functional compensation of mitotic spindle assembly by KIF15, a second motor kinesin. Using a synthetic lethal approach, we have shown that RNAi-mediated silencing of KIF15 has no effect on cell viability, but significantly sensitized cells by >10- to 100-fold to a class of KIF11 inhibitors (KIF11is). TPX2-targeted siRNAs reduce cell viability by 5-fold alone, and sensitize cells by 100-fold in the presence of KIF11i. Based on these observations, we designed a high-throughput screening assay using Alpha Technology©. Informed by previous predictions of KIF15-TPX2 binding domains, we designed peptides corresponding to the appropriate domains of KIF15 (aa1149-1388, GST-tagged) and TPX2 (aa346-747, His-tagged), for use in the AlphaScreen© assay. Following several optimization steps (e.g., buffer selection, protein titration, TruHits© assay validation), we demonstrate that KIF15 binds TPX2, producing a signal >30-fold greater than background signal. Further, peptides designed to mimic either KIF15 or TPX2 binding domains, but not scrambled controls, exhibited concentration-dependent disruption of the KIF15-TPX2 interaction. Next, we screened a FDA library (3,474 compounds) and bioactive (1,902 compounds) library and identified 122 hits; fresh compound stocks were tested at 4 concentrations (20, 10, 5, and 2.5 μM), resulting in 53 confirmed hits (28 FDA, 25 bioactive). To identify false positives that interfere with assay chemistry, we repeated the screen at 3 concentrations, replacing the His-TPX2 and GST-KIF15 proteins with a His-GST fusion. As an orthogonal screen, we performed a Homogenous Time Resolved Fluorescence© (HTRF)-based screen to reconfirm hit compounds at the aforementioned concentrations. From these combined data, we have identified 4 hit compounds (KU-P001 to KU-P004) that disrupt KIF15/TPX2 protein-protein interactions. To further prioritize our hits, we developed in vitro (EOC cell lines were exposed to increasing concentrations, up to 1 µM, of KIF11i for 4 months) and in vivo (patient-derived ascites xenograft models of EOC were treated with 3 cycles of either 5 mg/kg or 10 mg/kg of a KIF11i and were serially passaged and treated for 3 additional generations) models of KIF11i-resistance to evaluate their activities. In resistant EOC cells we observed a 3-fold increase in KIF15 protein expression as compared with parental cells; TPX2 protein expression remained unchanged. In vivo, both KIF15 (68% increase) and TPX2 (2-fold) protein expression was increased in resistant versus untreated PDX tumor cells. Combination studies of KU-P004 and ispinesib (KIF11i) in vitro revealed a 2- to 64-fold increase in cell death for both responsive and resistant EOC cells compared with KIF11i treatment alone. In summary, we have developed a high-throughput screening assay platform to identify inhibitors of the KIF15-TPX2 interaction and are using iterative medicinal chemistry to guide optimization and functional evaluation of hits and lead compounds to develop new combination therapies for women with ovarian cancer. Citation Format: Rebecca J. Wates, Anuradha Roy, Frank Schoenen, John Karanicolas, Scott Weir, Andrew Godwin. Targeting the KIF11/KIF15/TPX2 axis to develop new therapies for ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B03.