PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a major cause of cancer deaths which is curable if detected early. Unfortunately, many CRC tumors are diagnosed at more advanced stages where cure rates remain low with 20% five-year survival. The epidermal growth factor receptor (EGFR) is a major FDA-approved therapeutic target in metastatic CRC that may be underutilized. To date, it is still difficult to identify patients who may benefit from EGFRi therapy. In fact, the only identified biomarkers for EGFRi therapy are mutations in downstream genes that activate the RAS pathway (KRAS (40%), NRAS (5%), and possibly BRAF (10%)). Unfortunately, these biomarkers are negative predictors that are only ~50% accurate in predicting non-responders. Thus, accurate prediction of EGFRi sensitivity is still problematic. We have recently employed a novel hybrid approach using a predictive cetuximab sensitivity (CTX-S) gene expression signature score in 468 CRC tumors with coincident global gene expression and exome sequencing data to identify mutations beyond those in KRAS/BRAF/NRAS that might predict EGFRi responders. We found that tumors harboring the combination of APC + TP53 mutations are predicted to be the most sensitive to cetuximab (CTX). Whereas most CRC tumors (70%) have truncating mutations in APC and mutations in TP53 are present in 50% of CRC tumors, co-mutation of both genes is present in ~25% of CRC and heretofore has never been rigorously measured in clinical trials. Thus, we hypothesize co- mutated APC + TP53 as a new composite biomarker to identify CTX sensitive tumors that would allow greater response rates in WT RAS/RAF patients and potentially extend therapy to some mutant KRAS patients. To understand and validate the full potential of this novel biomarker, here we seek to determine the cellular impact of the APC and TP53 mutations that increase the tumor sensitivity to EGFR inhibition. Further, we seek to identify additional early/intermediate biomarkers of response correlating with the presence of these mutations. Finally, we will determine if these mutations alter the gene expression–based consensus molecular subtype (CMS). Tumors with CMS2 are predicted to have greater sensitivity to EGFR inhibition and appear to strongly correlate with APC + TP53 mutations in our human datasets. We seek to determine if mutations in APC + TP53 can convert molecular subtypes CMS1,3,4 to CMS2, coincident with enhancing EGFRi sensitivity. This application will provide the necessary pre-clinical validation of novel molecular biomarkers to best predict which CRC tumors will be responsive to EGFRi, and to support subsequent future clinical validation that may lead to expanded application of an under-used, FDA-approved targeted therapy.