Abstract B076: Uncoupling of the cell cycle pathway from the MAPK pathway in BRAF/MEK inhibitor resistant melanoma
Authors: Nassar Kelsey W. , Hintzsche Jennifer , Bagby Stacey , Langouet-Astrie Christophe , Samson Jenny , Amato Carol , Gordon Nicholas , Fujita Mayumi , Schweppe Rebecca , Robinson William , Tan Aik-Choon 
Published: March 2020

Abstract
A majority of cutaneous melanomas harboring oncogenic driver BRAF-V600E gene mutations initially demonstrate superior responses to targeted therapy with BRAF inhibitors (BRAFi), MEK inhibitors (MEKi), or the combination of both. Unfortunately, tumor regrowth and acquired resistance to BRAF and MEK inhibitors is inevitable, and new therapies are required to treat these patients. To determine acquired resistance mechanisms, we performed Whole Exome Sequencing (WES) on BRAF-V600 mutant cutaneous melanomas from two patients each at two timepoints; (1) prior to BRAFi/MEKi combination therapy, and (2) upon tumor regrowth following therapy. Following BRAFi/MEKi resistance, both patients acquired an activating mutation in NRAS and loss of the tumor suppressor CDKN2A. We hypothesized the acquired mechanism of resistance was through reactivation of MAPK pathway, via NRAS mutations, and activation of the cell cycle pathway, via loss of CDKN2A. Further, we predicted that these resistant patents samples would be sensitive to the combination of inhibiting the MAPK and cell cycle pathway. Both BRAFi/MEKi resistant patient samples were established as patient-derived xenograft (PDX) models to test the in vivo efficacy of trametinib (MEKi) and palbociclib (CDK4/6i) as single agents and in combination. In both PDX models, the combination of MEKi and CDK4/6i resulted in significant tumor reduction as compared to the vehicle control and single agents. Using immunohistochemistry, we observed that only in the combination treated tumors there was a significant decrease in tumor proliferation, measured by Ki-67, and MAPK pathway activity, measured by phos-ERK. To study the mechanism of response to the combination of MEKi and CDK4/6i in the context of BRAFi acquired resistance, we generated an in vitro resistance model using a panel of BRAF-V600 mutant cutaneous melanoma cell lines constantly treated with the BRAF inhibitor, vemurafenib, to generate a resistant cell population. We observed that parental cells are more sensitive to single agent MEKi, and more resistant to single agent CDK4/6i, as compared to BRAFi resistant cells. In line with this, we found that MEKi in parental cells decreased both phos-ERK and phos-RB, suggesting that the parental cell lines are dependent on the MAPK pathway, and that the cell cycle pathway is under MAPK pathway regulation. However, in BRAFi resistant lines, MEKi only slightly reduced phos-ERK, and did not significantly decrease phos-RB levels. Suggesting that, in BRAFi resistant lines, the cell cycle pathway is not regulated by the MAPK pathway. Decreased phos-ERK and phos-RB levels are only observed with the combination of MEKi and CDK4/6i. Furthermore, we found that the BRAFi resistant cells are more sensitive to a low concentration of CDK4/6i in combination with MEKi, as compared to the parental cells. We found that parental cells required a higher concentrations of CDK4/6i in combination with MEKi to reduce cell proliferation. Whereas, the BRAFi resistance cells are the opposite and require a lower concentration of CDK4/6i, and a higher concentration of MEKi, to have a durable response. Taken together, this data supports the observation that upon BRAFi resistance, the cell cycle pathway is uncoupled from the MAPK pathway, and cell cycle reactivation may represent a common acquired resistance mechanism to MEKi.
Citation Format: Kelsey W. Nassar, Jennifer Hintzsche, Stacey Bagby, Christophe Langouet-Astrie, Jenny Samson, Carol Amato, Nicholas Gordon, Mayumi Fujita, Rebecca Schweppe, William Robinson, Aik-Choon Tan. Uncoupling of the cell cycle pathway from the MAPK pathway in BRAF/MEK inhibitor resistant melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B076. doi:10.1158/1535-7163.TARG-19-B076