Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7 inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA, or p53. These cells are defective in apoptosis due to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target.

The Bcl-2 family protein Mcl-1 is often degraded in cancer cells subjected to effective therapeutic treatment, and defective Mcl-1 degradation has been associated with intrinsic and acquired drug resistance. However, a causal relationship between Mcl-1 degradation and anticancer drug responses has not been directly established, especially in solid tumor cells where Mcl-1 inhibition alone is insufficient to trigger cell death. In this study, we present evidence that Mcl-1 participates directly in determining effective therapeutic responses in colon cancer cells. In this setting, Mcl-1 degradation was induced by a variety of multi-kinase inhibitor drugs, where it relied upon GSK3β phosphorylation and FBW7-dependent ubiquitination. Specific blockade by genetic knock-in (KI) abolished apoptotic responses and conferred resistance to kinase inhibitors. Mcl-1-KI also suppressed the anti-angiogenic and anti-hypoxic effects of kinase inhibitors in the tumor microenvironment. Interestingly, these same inhibitors also induced the BH3-only Bcl-2 family protein PUMA, which is required for apoptosis. Degradation-resistant Mcl-1 bound and sequestered PUMA from other pro-survival proteins to maintain cell survival, which was abolished by small-molecule Mcl-1 inhibitors. Our findings establish a pivotal role for Mcl-1 degradation in the response of colon cancer cells to targeted therapeutics, and they provide a useful rational platform to develop Mcl-1-targeting agents that can overcome drug resistance.

Molecular targeted drugs are clinically effective anti-cancer therapies. However, tumours treated with single agents usually develop resistance. Here we use colorectal cancer (CRC) as a model to study how the acquisition of resistance to EGFR-targeted therapies can be restrained. Pathway-oriented genetic screens reveal that CRC cells escape from EGFR blockade by downstream activation of RAS-MEK signalling. Following treatment of CRC cells with anti-EGFR, anti-MEK or the combination of the two drugs, we find that EGFR blockade alone triggers acquired resistance in weeks, while combinatorial treatment does not induce resistance. In patient-derived xenografts, EGFR-MEK combination prevents the development of resistance. We employ mathematical modelling to provide a quantitative understanding of the dynamics of response and resistance to these single and combination therapies. Mechanistically, we find that the EGFR-MEK Combo blockade triggers Bcl-2 and Mcl-1 downregulation and initiates apoptosis. These results provide the rationale for clinical trials aimed at preventing rather than intercepting resistance.