Assessing the impact of genomic alterations on protein networks is fundamental in identifying the mechanisms that shape cancer heterogeneity. We have used isobaric labeling to characterize the proteomic landscapes of 50 colorectal cancer cell lines and to decipher the functional consequences of somatic genomic variants. The robust quantification of over 9,000 proteins and 11,000 phosphopeptides on average enabled the de novo construction of a functional protein correlation network, which ultimately exposed the collateral effects of mutations on protein complexes. CRISPR-cas9 deletion of key chromatin modifiers confirmed that the consequences of genomic alterations can propagate through protein interactions in a transcript-independent manner. Lastly, we leveraged the quantified proteome to perform unsupervised classification of the cell lines and to build predictive models of drug response in colorectal cancer. Overall, we provide a deep integrative view of the functional network and the molecular structure underlying the heterogeneity of colorectal cancer cells.@en

Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-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 TRAILR2 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 suppress MEDI3039-induced cell death. High-throughput drug screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics analysis 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 two inhibitor of apoptosis proteins (IAP) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BIDdeleted 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 such as FADD and caspase-8 as likely drivers of clinical resistance in solid tumors.@en