Abstract LB-121: Dissecting MAPK Pathway in BRAFmut Melanoma: Intricacies of ERK1 and ERK2

The MAPK signaling cascade, comprised of the RAS GTPases, the RAF, MEK1/2 and ERK1/2 kinases is frequently deregulated in cancer. ERK1 and ERK2 transmit signals generated by mutant BRAF, Ras or by activated receptor tyrosine kinases to a wide range of nuclear and cytoplasmic substrates, resulting in signal amplification, cell growth, migration and survival. ERK1 and ERK2 have been considered as redundant because of their high homology, large number of overlapping substrates, and ability to substitute for each other in genetically engineered mouse models. Nevertheless, several investigators have identified non-redundant roles for ERK isoforms in oncogenesis; for instance, ERK2, but not ERK1, appears to be responsible for RASmut induced epithelial-to-mesenchymal transformation. Besides, each of the ERK isoforms employs spatially distinct substrate docking domains, DEF (docking site for ERK FXFP) and D (docking domain), to signal to different subsets of substrates and differentially transmit signals downstream. We set out to determine the roles of ERK isoforms as well as DEF- and D-domain dependent signaling in the survival of melanoma tumor cells expressing activating BRAF mutations which are highly sensitive to pharmacological inhibitors of RAF, MEK1/2 and ERK1/2. We designed ERK1 and ERK2 mutants resistant to ATP-competitive ERK1/2 inhibitors and employed auto-activating, MEK-independent, ERK1 and ERK2 mutants to ask if BRAFmut melanoma survival is dependent on either or both ERK isoforms. In addition, we used RNAi and zinc-finger nucleases’ to knockdown or delete each of ERK isoforms. These experimental approaches consistently demonstrated that ERK2, but not ERK1, was the sole driver of cell survival in multiple BRAFmut melanoma cell lines. Moreover, genome-wide gene expression analysis indicated that ERK2, but not ERK1, was largely responsible for transcriptional effects imposed by pharmacological RAF, MEK1/2 or ERK1/2 inhibitors. Thus, in BRAFmut melanoma, functions of ERK1 and 2 are not redundant, and ERK1 cannot substitute for a disabled ERK2. Next, we introduced DEF- and D-substrate docking domain mutations into an ERK inhibitor resistant ERK2 to investigate whether signaling through either domain is sufficient to support melanoma survival. We observed that signaling through D- or DEF- domains of ERK2 had differential effects on gene expression and substrate phosphorylation. Consequently, we have found that a subset of melanoma cell lines was sensitive to elimination of DEF- docking domain interactions, whereas another subset of cell lines tolerated mutations in the DEF-site. Interactions and signaling through ERK D-docking site were dispensable for survival of all melanoma cell lines tested. These data suggest potential novel approaches to target oncogenic MAPK pathway. Citation Format: Tatiana Zavorotinskaya, Upasana Mehra, Yumin Dai, Michel Faure, Ken Crawford, Karen Yu, Jan Marie Cheng, Xiaolei Ma, Jan Xuan, Kelly Yan, Mohammad Hekmat-Nejad, Hanne Merritt, Darrin Stuart, Charles Voliva. Dissecting MAPK pathway in BRAFmut melanoma: Intricacies of ERK1 and ERK2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-121. doi:10.1158/1538-7445.AM2014-LB-121