Synthetic Lethal Interaction with BCL-XL Blockade Deepens Response to Cetuximab in Patient-Derived Models of Metastatic Colorectal Cancer.

PURPOSE: Approximately 20% of patients with RAS wild-type metastatic colorectal cancer (mCRC) experience objective responses to the anti-EGFR antibody cetuximab, but disease eradication is seldom achieved. The extent of tumor shrinkage correlates with long-term outcome. We aimed to find rational combinations that potentiate cetuximab efficacy by disrupting adaptive dependencies on antiapoptotic molecules (BCL2, BCL-XL, MCL1). EXPERIMENTAL DESIGN: Experiments were conducted in patient-derived xenografts (PDX) and organoids (PDXO). Apoptotic priming was analyzed by BH3 profiling. Proapoptotic and antiapoptotic protein complexes were evaluated by co-immunoprecipitation and electroluminescence sandwich assays. The effect of combination therapies was assessed by caspase activation in PDXOs and by monitoring PDX growth. RESULTS: A population trial in 314 PDX cohorts, established from as many patients, identified 46 models (14.6%) with appreciable (>50% tumor shrinkage) but incomplete response to cetuximab. From these models, 14 PDXOs were derived. Cetuximab primed cells for apoptosis, but only concomitant blockade of BCL-XL precipitated cell death. Mechanistically, exposure to cetuximab induced upregulation of the proapoptotic protein BIM and its sequestration by BCL-XL. Inhibition of BCL-XL resulted in displacement of BIM, which was not buffered by MCL1 and thereby became competent to induce apoptosis. In five PDX models, combination of cetuximab and a selective BCL-XL inhibitor triggered apoptosis and led to more pronounced tumor regressions and longer time to relapse after treatment discontinuation than cetuximab alone. CONCLUSIONS: In mCRC tumors that respond to cetuximab, antibody treatment confers a synthetic-lethal dependency on BCL-XL. Targeting this dependency unleashes apoptosis and increases the depth of response to cetuximab.

Effective drug combinations in breast, colon and pancreatic cancer cells.

Combinations of anti-cancer drugs can overcome resistance and provide new treatments1,2. The number of possible drug combinations vastly exceeds what could be tested clinically. Efforts to systematically identify active combinations and the tissues and molecular contexts in which they are most effective could accelerate the development of combination treatments. Here we evaluate the potency and efficacy of 2,025 clinically relevant two-drug combinations, generating a dataset encompassing 125 molecularly characterized breast, colorectal and pancreatic cancer cell lines. We show that synergy between drugs is rare and highly context-dependent, and that combinations of targeted agents are most likely to be synergistic. We incorporate multi-omic molecular features to identify combination biomarkers and specify synergistic drug combinations and their active contexts, including in basal-like breast cancer, and microsatellite-stable or KRAS-mutant colon cancer. Our results show that irinotecan and CHEK1 inhibition have synergistic effects in microsatellite-stable or KRAS-TP53 double-mutant colon cancer cells, leading to apoptosis and suppression of tumour xenograft growth. This study identifies clinically relevant effective drug combinations in distinct molecular subpopulations and is a resource to guide rational efforts to develop combinatorial drug treatments.

HER2 activating mutations are targets for colorectal cancer treatment.

UNLABELLED: The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. SIGNIFICANCE: HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer.

Sustained Inhibition of HER3 and EGFR Is Necessary to Induce Regression of HER2-Amplified Gastrointestinal Carcinomas.

PURPOSE: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities. EXPERIMENTAL DESIGN: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays. RESULTS: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib-or, importantly, monotherapy with afatinib-resulted in overt tumor shrinkage. CONCLUSIONS: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519-31. ©2015 AACR.

IGF2 is an actionable target that identifies a distinct subpopulation of colorectal cancer patients with marginal response to anti-EGFR therapies.

Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients' outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts ("xenopatients"), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.

Primary and acquired resistance to EGFR-targeted therapies in colorectal cancer: impact on future treatment strategies.

Only approximately 10% of genetically unselected patients with chemorefractory metastatic colorectal cancer experience tumor regression when treated with the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab ("primary" or "de novo" resistance). Moreover, nearly all patients whose tumors initially respond inevitably become refractory ("secondary" or "acquired" resistance). An ever-increasing number of predictors of both primary and acquired resistance to anti-EGFR antibodies have been described, and it is now evident that most of the underlying mechanisms significantly overlap. By trying to extrapolate a unifying perspective out of many idiosyncratic details, here, we discuss the molecular underpinnings of therapeutic resistance, summarize research efforts aimed to improve patient selection, and present alternative therapeutic strategies that are now under development to increase response and combat relapse.