Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

A 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 epidermal growth factor receptor (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 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 mitogen-activated protein-kinase kinase (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 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before 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 before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.     

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.     

Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion

Prior studies have identified recurrent oncogenic mutations in colorectal adenocarcinoma and have surveyed exons of protein-coding genes for mutations in 11 affected individuals. Here we report whole-genome sequencing from nine individuals with colorectal cancer, including primary colorectal tumors and matched adjacent non-tumor tissues, at an average of 30.7× and 31.9× coverage, respectively. We identify an average of 75 somatic rearrangements per tumor, including complex networks of translocations between pairs of chromosomes. Eleven rearrangements encode predicted in-frame fusion proteins, including a fusion of VTI1A and TCF7L2 found in 3 out of 97 colorectal cancers. Although TCF7L2 encodes TCF4, which cooperates with β-catenin in colorectal carcinogenesis, the fusion lacks the TCF4 β-catenin-binding domain. We found a colorectal carcinoma cell line harboring the fusion gene to be dependent on VTI1A-TCF7L2 for anchorage-independent growth using RNA interference-mediated knockdown. This study shows previously unidentified levels of genomic rearrangements in colorectal carcinoma that can lead to essential gene fusions and other oncogenic events.     

RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E)

Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.     

BRAF mutation predicts sensitivity to MEK inhibition

The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members. Using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumour growth in BRAF mutant xenografts, whereas RAS mutant tumours were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumours, and offer a rational therapeutic strategy for this genetically defined tumour subtype.