Acquired Resistance to Anti EGFR Therapy in Colorectal Cancer and Paracrine Protection by KRAS Mutated Cells

The main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance. Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6/10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. KRAS mutated alleles were present in low prevalence (below 10% on average). Therefore in most of the resistant tumors KRAS mutations are present only in a subset of tumor cells. We hypothesized that small fraction of KRAS mutated cells are capable of protecting the rest of otherwise sensitive cells against cetuximab and that this could be achieved through paracrine protection. To test these hypotheses we first generated cetuximab-resistant variants of three colorectal cancer (CRC) cellular models (DiFi, Lim1215 and OXCO2) that are highly sensitive to EGFR inhibition. Resistant sublines and their clonal populations were then used to condition the medium in the presence of cetuximab. We found that the growth inhibitory effect of cetuximab on parental cells was significantly impaired if the medium was conditioned by their resistant counterparts. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancer and indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression. Relatively small number of KRAS mutant cells could render the entire tumor resistant due to active paracrine protection of surrounding sensitive cells. This ‘babysitting’ effect offers further therapeutic opportunity in cancers that develop secondary resistance to anti EGFR therapies.