ABSTRACT Purpose Novel covalent inhibitors of KRAS G12C have shown limited response rates in KRAS G12C mutant (MT) colorectal cancer (CRC) patients. Thus, novel KRAS G12C inhibitor combination strategies that can achieve deep and durable responses are needed. Experimental design Small molecule KRAS G12C inhibitors AZ’1569 and AZ’8037 were employed. To identify novel candidate combination strategies for AZ’1569, we performed RNA sequencing, siRNA and high-throughput drug screening. Top hits were validated in a panel of KRAS G12C MT CRC cells and in vivo . AZ’1569-resistant CRC cells were generated and characterised. Results Response to AZ’1569 was heterogeneous across the KRAS G12C MT models. AZ’1569 was ineffective at inducing apoptosis when used as single-agent or combined with chemotherapy or agents targeting the EGFR/KRAS/AKT axis. Using a systems biology approach, we identified the anti-apoptotic BH3-family member BCL2L1 /Bcl-xL as a top hit mediating resistance to AZ’1569. Further analyses identified acute increases in the pro-apoptotic protein BIM following AZ’1569 treatment. ABT-263 (Navitoclax), a pharmacological Bcl-2 family-inhibitor that blocks the ability of Bcl-xL to bind and inhibit BIM, led to dramatic and universal apoptosis when combined with AZ’1569. Furthermore, this combination also resulted in dramatically attenuated tumour growth in KRAS G12C MT xenografts. Finally, AZ’1569-resistant cells showed amplification of KRAS G12C , EphA2/c-MET activation, increased pro-inflammatory chemokine profile and cross-resistance to several targeted agents. Importantly, KRAS amplification and AZ’1569-resistance were reversible upon drug withdrawal, arguing strongly for the use of drug holidays in the case of KRAS amplification. Conclusions Combinatorial targeting of Bcl-xL and KRAS G12C is highly effective, suggesting a novel therapeutic strategy for KRAS G12C MT CRC patients.

Bcl-xL is a key mediator of apoptosis following KRASG12C inhibition in KRASG12C mutant colorectal cancer