Abstract 301: Identification of NRAS isoform 2 overexpression as a novel mechanism facilitating BRAF inhibitor resistance in malignant melanoma (MM)
Authors: Duggan Megan , Stiff Andrew , Olaverria Salavaggione Gonzalo , Bainazar Maryam , Latchana Nicholas , Markowitz Joseph , de la Chapelle Albert , Eisfeld Ann-Kathrin , Carson William 
Published: July 2016

Abstract
Activating mutations in BRAF are found in 50% of melanomas and are associated with poor outcomes due to constitutive activation of the mitogen activated protein kinase (MAPK) pathway. Treatment with BRAF inhibitors (BRAFi), such as vemurafenib, is effective but resistance can develop. A major mechanism of resistance involves feedback loop-mediated increased expression of the NRAS gene. Recently, we discovered the NRAS transcript is differentially spliced to give 5 novel isoforms (isos), which differ in their expression, cellular localization and downstream effects. We hypothesized that differential expression of one or more of the isos may contribute to the development of BRAFi resistance. We assessed iso-specific NRAS expression in 14 MM cell lines and melanocytes, and noted a curious expression profile of iso2: while undetectable in melanocytes and BRAFwt MM cells, its abundance was increased in BRAFmut cells, and it was the only NRAS iso highly overexpressed in 3 BRAFi resistant cell lines (p&lt;0.01). Iso2 is characterized by an additional exon (exon 3b) compared to canonical NRAS iso1. To explore the possible role of NRAS iso2 in MM, BRAFmut A375 cells were engineered to overexpress NRAS isos 1, 2 and empty vector (EV). Iso2 overexpression led to significantly increased MM cell proliferation in vitro, and surprisingly also increased proliferation during treatment with BRAFi (p&lt;0.001). In a xenograft model, athymic nude mice with A375 tumors stably overexpressing iso1, iso2 or EV were treated with oral BRAFi (50 mg/kg) or PBS for 21 days. Iso 2 overexpressing tumors did not respond to in vivo treatment with BRAFi and grew faster than their iso 1 overexpressing and EV counterparts, as quantified by IVIS imaging, which resulted in a significantly shortened survival of both BRAFi and PBS treated iso2 mice. In contrast, overexpression of canonical iso1 did not affect BRAFi sensitivity. Strikingly, shRNA-mediated knockdown of iso2 in BRAFi resistant cells resulted in decreased survival compared to iso1 or scramble knockdowns. Downstream signaling analysis utilizing immunoblots and confocal microscopy indicated a decrease in MAPK signaling and an increase in PI3K signaling in iso2 overexpressing cells compared to iso1 overexpressing cells. Protein complex immunoprecipitation of iso2 in COS-7 cells validated a binding affinity of NRAS iso2 to both PI3K and BRAF/RAF1. Since MAPK signaling is strongly reduced in iso2 expressing cells, this may suggest an allosteric inhibition mechanism, which prevents binding and activation of BRAF by canonical NRAS iso1.
This study indicates for the first time that NRAS iso2 may play a role in BRAFi resistance by facilitating alternative survival signaling through the PI3K pathway in the presence of MAPK pathway inhibition. Targeting NRAS iso2 may be a beneficial treatment strategy in the prevention and management of BRAFi resistance in melanoma.
Citation Format: Megan Duggan, Andrew Stiff, Gonzalo Olaverria Salavaggione, Maryam Bainazar, Nicholas Latchana, Joseph Markowitz, Albert de la Chapelle, Ann-Kathrin Eisfeld, William Carson. Identification of NRAS isoform 2 overexpression as a novel mechanism facilitating BRAF inhibitor resistance in malignant melanoma (MM). [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 301.