Abstract 2854: Reconstruction of BRAFV600E driven colorectal carcinogenesis and identification of novel drug combinations involving BRAF and RTK inhibitors
Authors: Reischmann Nadine , Herr Ricarda , Halbach Sebastian , Heizmann Miriam , Busch Hauke , Boerries Melanie , Brummer Tilman 
Published: August 2018

Abstract
BRAF mutations are found in approximately 10% of colorectal cancers (CRC) and are associated with an aggressive, less-differentiated and therapy-resistant phenotype. Inhibitors targeting BRAFV600E elicit only limited survival benefits when used as single agents. This unresponsiveness was mechanistically attributed to the relief of negative feedbacks on the epidermal growth factor receptor (EGFR) and initiated dual and triple combinatorial trials. These trials often involve the combination of BRAF inhibitors (BRAFi) and/or MEK inhibitors with anti-EGFR antibodies such as cetuximab or panitumumab. Although first results of these dual or triple therapies demonstrated improved efficacy, the response rates still were heterogeneous. Here, we show that BRAFi upregulate a variety of receptor tyrosine kinases (RTK) in CRC cell lines, including not only the EGFR, but also human epidermal growth factor receptor (HER) 2 and HER3. Importantly, combination of BRAFi (vemurafenib, dabrafenib or encorafenib) with inhibitors dually targeting the EGFR and HER2 (lapatinib, canertinib or afatinib) significantly reduced the metabolic activity and proliferation of CRC cells. Similarly, genetic depletion of HER2 and HER3 re-sensitized CRC cells to BRAF inhibition. Interestingly, BRAF inhibition also led to increased levels of the GRB2-associated binders (Gab) 1 and Gab2, two important mediators of RTK signaling. The Gab2 upregulation was directly dependent on the loss of BRAFV600E signaling and not caused by “off-target” effects, as demonstrated by allele-specific shRNA mediated BRAFV600E knockdown. These findings suggest new escape mechanisms for current treatment regimens and indicate that targeted therapy in BRAF mutant CRC could benefit from broad RTK pathway blockade.
Importantly, the BRAF/HER family inhibitor combination was also more effective in murine intestinal MouseT1 cells (originating from a Vil-Cre;BrafLSL-V637E/+;Tp53LSL-R172H/+ mouse model), which indicates a species-independent phenomenon. These novel and our previous findings that BRAFV600E suppresses features of epithelial differentiation and effector function prompted us to establish crypt organoid cultures from mice carrying conditional BraffloxV600E/+ and/or Tp53LSL-R172H/+ knock-in alleles, either singly or in combination. Using a Vil-CreERT2 transgene, we were able to induce expression of oncogenic BRAF and dominant-negative p53R172H in organoids by 4-hydroxy-tamoxifen mediated Cre activation. We demonstrate that the sudden expression of BRAFV600E and TP53R172H induce marked morphological and molecular changes in small and large intestinal crypts. This organoid model system represents an excellent tool to better understand key characteristics of BRAF mutant CRC such as intrinsic aggressiveness, poor differentiation and resistance to BRAFi.
Citation Format: Nadine Reischmann, Ricarda Herr, Sebastian Halbach, Miriam Heizmann, Hauke Busch, Melanie Boerries, Tilman Brummer. Reconstruction of BRAFV600E driven colorectal carcinogenesis and identification of novel drug combinations involving BRAF and RTK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2854.