Abstract B171: BX-795 enhances the efficacy of crizotinib in colorectal cancer cells by altering the activity of aurora kinases

Abstract

Abstract Background: Metastatic Colorectal cancer (mCRC) is driven by various genetic mutations that, in turn, present potentially actionable targets. Recent improvements in combining therapies have shown modest benefits in patients with BRAF mutated mCRC. Several efforts focusing on various KRAS inhibitors are ongoing in mCRC with outcomes pending. With no significant advances to improve outcomes in most patients with mCRC, it is important to identify novel therapies that have the potential to significantly improve the outcomes of these patients. Aims: To perform high-throughput screening (HTS) using clinically relevant drugs to identify novel and effective therapeutic combinations for patients with KRAS/BRAF mutated mCRC. Methods: We performed unbiased HTS with ~250 drugs that were either currently in clinical trials or approved by the FDA. 57 drugs that decreased cell proliferation in both 2D and 3D cultures and were effective in both HCT116 (KRAS mutated) and HT29 (BRAF mutated) cell lines, were termed as “pan-active” drugs. These drugs were divided into 9 classes based on targets they inhibit. Using the BLISS model, synergistic drug combinations were identified by HTS where the top three effective drugs from each class of “pan-active” drugs were paired with the top three drugs of all other classes. Effects of one such combination (BX-795 with crizotinib) were further validated in vitro by cell growth assays and measuring changes in apoptotic mediators by western blotting (WB) and flow cytometry (FACS). Mechanisms of synergy were examined by Reverse Phase Protein Array (RPPA) followed by WB and immunostaining of CRC cells to assess inhibition of downstream targets. Results: Pairwise HTS studies identified the PDK1/TBK1 inhibitor BX-795 to be synergistic with the MET/ALK inhibitor crizotinib in multiple CRC cell lines. This combination was further validated in vitro by MTT and colony formation assays in various KRAS and BRAF mutated CRC cell lines. Analyses of markers for apoptosis by WB and FACS using FITC-Annexin V and PI confirmed that the drug combination increased cell death in multiple CRC cell lines as compared to single agents. Unbiased RPPA indicated that the combination led to very strong and unexpected inhibition of the Aurora kinases along with decreases in the expected AKT/mTOR pathway in multiple CRC cell lines when compared to monotherapies. Immunostaining revealed significant mitotic defects in CRC cells treated with the drug combination but not with single agents suggesting that mitotic defects was a possible mechanism of cell death. Further, combining an Aurora kinase A inhibitor with crizotinib enhanced the efficacy of crizotinib validating the proposed mechanism. Conclusions: Our unbiased HTS and validation studies demonstrated that combining BX-795 with crizotinib is a potential novel effective therapeutic combination for patients with mCRC. Our in vitro studies serve as the basis for future preclinical and, if warranted, clinical studies to determine the efficacy of this drug combination in patients with mCRC. Citation Format: Susmita Ghosh, Fan Fan, Reid Powell, Yong Sung Park, Clifford Stephan, Jason Roszik, Lee M Ellis, Rajat Bhattacharya. BX-795 enhances the efficacy of crizotinib in colorectal cancer cells by altering the activity of aurora kinases [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B171.