Oncogene https://doi.org/10.1038/s41388-018-0578-4 CORRESPONDENCE RASA2 and NF1; two-negative regulators of Ras with complementary functions in melanoma Rand Arafeh 1 ● Antonella Di Pizio 2 ● Abdel G. Elkahloun 3 ● Orly Dym 1 ● Masha Y. Niv 2 ● Yardena Samuels 1 Received: 10 January 2018 / Revised: 7 August 2018 / Accepted: 17 October 2018 © Springer Nature Limited 2018 RASA2 has previously been shown to be a functional RasGAP in melanoma cells [1]. Mutation or loss of RASA2 promotes RAS activation in melanoma [1]. Our genetic analysis of RASA2 mutations identified that RASA2 and NRAS mutations are mutually exclusive (p = 0.002, Fisher’s exact test), and that NF1 mutations [2, 3] significantly co- occur with RASA2 mutations (p = 0.000011, Fisher’s exact test) in BRAF and NRAS wild-type melanomas, suggesting that loss of RASA2 and NF1 have complementary pro- tumorigenic functions (Fig. 1A). Maertens et al. [3] showed that NF1 is specific for KRAS and HRAS in melanoma. To validate this observation in our cells, we overexpressed vector alone (EV) or wild-type NF1 in melanoma cells that harbor RASA2 and NF1 mutations (CO84) and found that wild-type NF1 substantially sup- pressed KRAS-GTP and HRAS-GTP levels compared to NRAS-GTP levels (Fig. 1B). As NF1 and RASA2 are RasGAPs and their alterations co- occur, and co-occurring gene alterations in the same tumor are often held to entail synergy, we hypothesized that RASA2 and NF1 may synergize by acting on different RAS isoforms. Indeed, we found that RNAi-mediated suppression of RASA2 led to activation of NRAS-GTP, but not HRAS or KRAS (Fig. 1C and Supplementary Fig. 1A). Conversely, over- expression of wild-type RASA2 substantially suppressed NRAS-GTP, but not KRAS or HRAS, whereas RASA2 mutants failed to do so (Supplementary Fig. 1B, C). Impor- tantly, re-introduction of wild-type RASA2 into melanoma cells harboring RASA2 mutations inhibited NRAS activation (Fig. 1D and Supplementary Table. 1). To gain insight into this specificity, we predicted the binding interface of RASA2 and NF1 with RAS proteins. RASA2, compared to NF1, has a different length and amino acid composition in its L6c loop 555 SKSKSSFKE- T 564 RASA2 (Fig. 1E and Supplementary Fig. 4) and in the fragment 345 SAAYILSEICRDK 357 RASA2. The interactions established by the latter may be specific for NRAS helix 3 as they are not observed in RASA2-HRAS nor in RASA2- KRAS. In RASA2-NRAS complex, RASA2 R355 establishes an H-bond with NRAS D92, while in both RASA2-HRAS and RASA2-KRAS, R355 interacts via intra-molecular H-bond with D356, preventing the interac- tion with D92, which, in turn, is involved in intra- mole- cular H-bonds with Q95 and H95 in HRAS and KRAS, respectively. The same region, in NF1-HRAS and NF1- KRAS involves H-bond between C1233 and E91 and intra- molecular H-bonds between D92 and K88 and Q(H)95, while in NF1-NRAS complex, only H-bond between C1233 and D92 is observed. These differences might explain the different specificity of RASA2 and NF1 toward the RAS proteins. To assess the effects of RASA2 and NF1 knockdown on the MAPK pathway, we established stable pooled clones in which we knocked-down the expression of RASA2 and NF1 individually or in combination in A375 cells and checked the effect on MAPK pathway activation. We observed that the phosphorylation of ERK is enhanced when both RASA2 and NF1 are knocked-down compared to when each is knocked- down individually (Supplementary Figure 5A). Additionally, knockdown of both RASA2 and NF1 potently increases the growth of xenografts in immunocompromised mice compared to their individual knockdowns (Supplementary Figure 5B). Therefore, loss of both GAPs, RASA2 and NF1 activates the MAPK pathway and enhances tumor formation in vivo. * Yardena Samuels Yardena.Samuels@weizmann.ac.il 1 Weizmann Institute of Science, Rehovot, Israel 2 Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, Israel 3 National Human Genome Research Institute, US National Institutes of Health, Bethesda, MD, USA Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41388-018-0578-4) contains supplementary material, which is available to authorized users. 1234567890();,: 1234567890();,: