Abstract 308: Interrogating Goldilocks: Searching for mediators of oncogene overdose in engineered BRAFV600E inhibitor resistant melanoma cells
Authors: Pringle Daphne R. , McMahon. Martin 
Published: July 2016

Abstract
The phenomenon of oncogene overdose has been exhibited in patient-derived melanoma xenografts, and clinical trials are underway to determine if intermittent BRAFV600E inhibitor (BRAFi) treatment may take advantage of this phenomenon to benefit melanoma patient outcomes. While oncogene overdose has been described in many settings, molecules that mediate this phenomenon of are poorly characterized. This abstract describes the generation and initial testing of a melanoma cell system to identify the mediators downstream of BRAFV600E hyperactivity that mediate oncogene overdose.
BRAFi resistant cell lines were generated from YUMM1.1 (BRAFV600E, PTENNull, INK4A-ARFNull) mouse melanoma cells. To generate BRAFi resistant cell lines, retroviruses harboring known human BRAFi resistance alleles were used to infect YUMM1.1 cells. Infected cells were selected directly in the appropriate antibiotic and 1 μM LGX818, a BRAFV600E specific inhibitor currently in clinical trials. Resulting drug resistant populations were single cell cloned and expression of transduced proteins verified. To probe the ability of resistance alleles to lead to oncogene overdose and growth arrest, colony forming assays, cell counting assays, cell cycle analyses, BrdU incorporation assays and immunoblots of relevant cellular pathways were performed.
Results indicate that expression of alleles that confer resistance to LGX818, as well as to inhibition of other nodes in the MAPK pathway, does not lead to cross-resistance to inhibitors of other pathways such as the PI3K pathway. Cells expressing some resistance alleles (e.g. BRAFV600E), but not others (e.g. NRASQ61K), demonstrate oncogene overdose when LGX818 is withdrawn. Data from cell cycle analyses suggest that, unlike the G1 cell cycle arrest seen in parental cells exposed to BRAFi, cells exhibiting oncogene overdose show no aberrations in their cell cycle state, despite reduced DNA synthesis as measured by BrdU incorporation, suggesting a distinct mechanism of arrest. Immunoblot analyses of critical signaling pathways show that mTORC signaling to the protein synthesis machinery is activated upon oncogene overdose, suggesting a potential role for this pathway, and its amino acid sensing function, in oncogene overdose induced arrest. The role of protein synthesis is under further investigation.
These data suggest that not all mechanisms of BRAFi resistance are equivalent in their ability to elicit oncogene overdose. It has become clear that oncogene overdose induced growth arrest is inherently more complicated than G1 cell cycle arrest seen in drug naïve melanoma cells or in primary human or mouse cells. Further elucidation of mediators of the ‘Goldilocks effect’ of BRAFV600E signaling in melanoma and in other cellular contexts has the potential to shed light on alternative mechanisms to induce melanoma cell growth arrest, even after the development of resistance.
Citation Format: Daphne R. Pringle, Martin McMahon. Interrogating Goldilocks: Searching for mediators of oncogene overdose in engineered BRAFV600E inhibitor resistant melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 308.