Summary A main limitation of therapies that selectively target kinase signaling 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 cancers1. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug2. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood3-8. 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 but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK. 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. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

The EGFR-targeted antibodies cetuximab and panitumumab are used to treat metastatic colorectal cancers. Mutations in KRAS , NRAS, and BRAF and amplification of ERBB2 and MET drive primary ( de novo ) resistance to anti-EGFR treatment. Recently, the emergence of alterations in the same genes was detected in patients who responded to EGFR blockade and then relapsed. These results illuminate a striking overlap between genes that, when mutated, drive primary and secondary resistance to anti-EGFR antibodies. Remarkably, although the mechanisms of resistance are genetically heterogeneous, they biochemically converge on key signaling pathways. This knowledge is being translated in the rational design of additional lines of therapy.Signifi cance: Anti-EGFR-targeted therapies are used for the treatment of metastatic colorectal cancer. Molecular heterogeneity impairs their effi cacy by fuelling de novo and acquired resistance. In this review, we highlight how genetically distinct resistance mechanisms biochemically converge on a limited number of signaling pathways that can be therapeutically intercepted.

Highlights d Cancer-associated fibroblasts contribute to pancreatic cancer heterogeneity d Cancer cells can have a double-positive phenotype: proliferation and invasion d High CAF abundance linked with DP cells enriched for MAPK and STAT3 co-signaling d Intra-tumoral gland types provide tissue heterogeneity linked with clinical outcome

Entrectinib is a fi rst-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profi ling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1 , p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confi rmed that either mutation renders the TRKA kinase insensitive to entrectinib. These fi ndings can be immediately exploited to design next-generation TRKA inhibitors. SIGNIFICANCE:We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identifi cation of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the fi rst time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements. Cancer Discov; 6(1);[36][37][38][39][40][41][42][43][44]

KRAS and NRAS were also detected in plasma samples from patients who developed resistance to anti-EGFR antibodies. A mouse xeno-transplant from a CRC patient who responded and subsequently relapsed upon EGFR therapy showed exquisite sensitivity to combinatorial treatment with MEK and EGFR inhibitors. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which reactivate ERK signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in CRC patients who become refractory to anti-EGFR therapies.

Purpose: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models.Experimental Design: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance.Results: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab.Conclusions: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.

Purpose: KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy. Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C-activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. Results: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K-AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT-mTOR pathway inhibitors. The G12CiþPI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. Conclusions: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

Most KRAS G12C mutant non-small cell lung cancer (NSCLC) patients experience clinical benefit from selective KRAS G12C inhibition, while patients with colorectal cancer (CRC) bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRAS G12C inhibitors in CRC, we examined the effects of AMG510 in KRAS G12C CRC cell lines. Unlike NSCLC cell lines, KRAS G12C CRC models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In CRC lines, KRAS G12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRAS G12C blockade, we identify EGFR signaling as the dominant mechanism of CRC resistance to KRAS G12C inhibitors. The combinatorial targeting of EGFR and KRAS G12C is highly effective in CRC cells, patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat KRAS G12C CRC patients.