Colorectal cancer (CRC) is a leading cause of cancer related death with PIK3CA mutations occurring in ~18% of all cases. Mutations in this gene lead to constitutive activation of the phosphoinositide-3 kinase (PI3K) oncogene. Previously we've shown that MTORC1/2 inhibition is sufficient to induce a therapeutic response both in vitro and in vivo with minimal induction of apoptosis. BCL-xL is a well-known negative regulator of apoptosis in solid tumors. We therefore investigated whether inhibition of the BCL-2 family, and more specifically BCL-xL, would enhance therapeutic response and induction of apoptosis. Murine-derived cancer organoids (MDCOs) were generated from invasive colon adenocarcinomas of Apc and Pik3ca transgenic mice (F1 (FVBxB6) Apcfl/+ Pik3caH1047R). MDCOs were allowed to mature for 24 hours, baseline brightfield imaging performed and therapeutic agents added at concentrations outlined below. Median relative change in organoid diameter after 48 hours of treatment was determined. In vivo response was measured in F1 (FVBxB6) Apcfl/+ Pik3caP110* mice as change in endoscopic tumor lumen occlusion over 14 days. Immunoblotting (IB) and immunofluorescence (IF) were utilized to evaluate for induction of apoptosis. Navitoclax (BCL-2/BCL-xL/BCL-w inhibitor, 250nM) was evaluated alone and in combination with a panel of MTORC1/2 inhibitors (BEZ-235 (BEZ), TAK-228 (TAK), copanlisib (Cop), 200nM). Navitoclax did not induce a treatment response as a single agent. Enhanced response was seen with the combination compared to the MTORC1/2 inhibitors alone (Bez 56% vs combo -100%, p<0.001; TAK -27% vs combo -100%, p<0.001; Cop -16% vs -100%; p<0.001). Results were confirmed in vivo with BEZ-235 (30mg/kg/day), navitoclax (80mg/kg/day), or the combination with the greatest reduction in lumen occlusion of colon tumors in the combination therapy (control +15%, navitoclax +1%, BEZ -15%, and combo -42%, p<0.003 BEZ vs combo). IB of cleaved PARP, a main cleavage target of cleaved caspase 3 (CC3) once apoptosis is induced, and IF of CC3 confirmed induction of apoptosis was highest in the combination therapy in both in vitro and in vivo studies. This induction was found as early as 6 hours post treatment in the MDCOs. To confirm inhibition of BCL-xL was the primary anti-apoptotic protein necessary for this induction of apoptosis, MDCOs were treated with copanlisib (200nM) alone or in combination with WEHI-539 (BCL-xL inhibitor, 250nM) or ABT-199 (BCL-2 inhibitor, 250nM). An enhanced sensitivity was observed when MTORC1/2 inhibition was combined with the inhibition of BCL-xL compared to BCL-2. These studies indicate that BCL-xL signaling reduces MTORC1/2 inhibitor response and targeting BCL-xL in combination with MTORC1/2 enhances both the treatment response and the induction of apoptosis in PIK3CA mutant CRC.
Citation Format: Rebecca A. DeStefanis, Alyssa DeZeeuw, Gioia Sha, Susan N. Payne, Christopher P. Babiarz, Devon Miller, Demetra K. Korkos, Cheri A. Pasch, Linda Clipson, Kristina Matkowskyj, Dustin A. Deming. BCL-xL inhibition enhances therapeutic response of MTORC1/2 inhibition and induction of apoptosis in PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 75.