Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists

Multivalent second-generation TRAIL-R2 agonists are currently in late pre-clinical development and early clinical trials. Herein, we use a representative 2(nd) generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models. Whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to supress MEDI3039 induced cell-death. High throughput drug-screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics anaysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell-lines and patient-derived xenografts, identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination-screening of TRAIL-R2, caspase-8, FADD and BID knockout models with 60 compounds with varying mechanisms-of-action identified 2 inhibitor of apoptosis proteins (IAPs) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8: MCL-1 as potential predictive biomarkers for second generation TRAIL-R2 agonists and loss of key effectors like FADD and caspase-8 as likely drivers of clinical resistance in solid tumors