Mutational activation of RAC1 is detected in similar to 7% of cutaneous melanoma, with the most frequent mutation (RAC1(C85T)) encoding for RAC1(P29S). RAC1(P29S) is a fast-cycling GTPase that leads to accumulation of RAC1(P29S)-GTP, which has potentially pleiotropic regulatory functions in melanoma cell signaling and biology. However, the precise mechanism by which mutationally activated RAC1(P29S) propagates its pro-tumorigenic effects remains unclear. RAC1-GTP is reported to activate the beta isoform of PI3'-kinase (PIK3CB/PI3K beta) leading to downstream activation of PI3'-lipid signaling. Hence, we employed both genetic and isoform-selective pharmacological inhibitors to test if RAC1(P29S) propagates its oncogenic signaling in melanoma through PI3K beta. We observed that RAC1(P29S)-expressing melanoma cells were largely insensitive to inhibitors of PI3K beta. Furthermore, RAC1(P29S) melanoma cell lines showed variable sensitivity to pan-class 1 (alpha/beta/gamma/delta) PI3'-kinase inhibitors, suggesting that RAC1-mutated melanoma cells may not rely on PI3'-lipid signaling for their proliferation. Lastly, we observed that RAC1(P29S)-expressing cell lines also showed variable sensitivity to pharmacological inhibition of the RAC1 -> PAK1 signaling pathway, questioning the relevance of inhibitors of this pathway for the treatment of patients with RAC1-mutated melanoma.

NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.

RASis mutated in a number of cancers, including KRAS-driven colorectal and pancreatic cancer, HRAS-driven bladder cancer, and NRAS-driven melanoma, all of which result in downstream activation of the RAF>MEK>ERK (MAPK) and PI3K>AKT signaling pathways. Most RAS GTPases cannot be targeted directly, and strategies blocking both MAPK and PI3K signaling simultaneously are limited by high toxicity and compensatory signaling mechanisms. Interestingly, oncogene-activated MAPK or PI3K signaling pathways are reasonably well described orchestrators of metabolic transformation through multiple pathways. Our lab has recently shown that autophagy,a conserved metabolic process of self-digestion that recycles intracellular components, is increased in KRAS-driven pancreatic ductal adenocarcinoma (PDA) upon MAPK pathway inhibition. We further showed that co-inhibition of autophagy and MEK1/2, a MAPK component, leads to tumor regression of patient-derived PDA xenografts in mice. Our proposed combination therapy has recently been translated into a phase I/II clinical trial for PDA patients with advanced disease. Our most recent data show that inhibition of RAS>RAF>MEK>ERK signaling also results in induction of autophagy in other RAS-driven cancers, including KRAS-driven colorectal cancer, HRAS-driven bladder cancer and NRAS-driven melanoma. Furthermore, ourin vivodata demonstrate a robust regression of tumors upon combined inhibition of autophagy and MEK1/2 in engrafted KRAS-driven colorectal cancer cells xenografted in mice. Patient data from two KRAS-driven colorectal cancer patients, who were recently treated off-label, indicated clinical responses to the combination treatment co-targeting autophagy and MEK1/2. Altogether, these data suggest that co-inhibition of autophagy and oncogenic signaling may represent a potential new treatment strategy for multiple RAS-driven cancer types. Future experiments aim for a better mechanistic understanding of the combination treatment co-targeting autophagy and oncogenic signaling, with the objective to propose novel therapeutic strategies for cancer patients with RASmutations. Citation Format: Mona Foth, Conan Kinsey, Sophia Schuman, Benjamin Battistone, Emilio Cortes Sanchez, David Kircher, Bryan Welm, Sheri Holmen, Martin McMahon. Co-inhibition of autophagy and MAPK signaling in RAS-driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1225.