Approximately 10% of patients with colorectal cancer (CRC) harbor the BRAF V600E driver mutation. Unlike melanoma, the response rate of BRAF-mutant CRC to the combination of BRAF and MEK inhibitors is limited. In order to target the MAPK signaling pathway more effectively by blocking EGFR-mediated re-activation of the pathway, triple combination trials of BRAF, MEK and EGFR inhibitors are on-going, but the response is underwhelming.
To find alternative combination strategies that could deepen therapeutic responses driven by a BRAFi and MEKi combination, we performed pooled shRNA screens under the treatment pressure of the dual combination of the BRAF inhibitor dabrafenib and MEK inhibitor trametinib. In some of the BRAF-mutant CRC models, we observed marked discrepancies in the therapeutic responses between in vitro and in vivo conditions. Therefore, shRNA screens were conducted in cancer cell lines grown both in vitro (i.e. 2D and 3D culture conditions) and in vivo in xenograft tumor models. The aim of the study was to identify novel targets to combine with BRAFi/MEKi, and to compare the results of the screens preformed in vitro and in vivo.
The biggest technical challenge for an in vivo pooled screening approach is achieving adequate library representation after the bottleneck of cell implantation and engraftment in mice. Our in vivo screen had an additional bottleneck due to the dabrafenib/trametinib combination treatment. Therefore, by performing a pilot screen with the BRAF-mutant cell line model HT29 we aimed to address two questions: 1) whether the in vivo screen under treatment pressure would be technically feasible and 2) if novel combination partners to dabrafenib/trametinib would be identified to potentially improve efficacy beyond that observed with the triple combination with EGFR inhibitors.
We were able to achieve comparable intra-group variability and repeatability between in vitro and in vivo conditions, whereby gene level analysis revealed several differential hits between the two conditions, which were both sensitizers and activators to the dabrafenib/trametinib combination treatment. We identified targets specific for the in vivo condition that had not been identified in vitro and vice versa. Thus, in vivo screening may identify powerful hits that would not be realized by in vitro investigations. With success of this pilot effort, the screen is currently being expanded into additional BRAF-mutant CRC models.
Citation Format: Hyo-eun C. Bhang, Matthew T. DiMare, David P. Kodack, Lujian Tan, Grainne Kerr, Viveksagar Krishnamurthy Radhakrishna, Javad Golji, David A. Ruddy, Tina Yuan, Matthew J. Niederst, Joshua M. Korn, Diana Graus Porta, Peter S. Hammerman, Jeffrey A. Engelman, Tinya Abrams, Juliet Williams. In vivo shRNA screens under treatment pressure by BRAF and MEK inhibitors to identify novel combination treatment strategies for BRAF-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 394.