Abstract 419: Elevated Akt/mTOR signaling drives therapy resistance in ALM

Abstract

Abstract Metastatic acral lentiginous melanoma (ALM) is the most lethal subtype of cutaneous melanoma and effective therapies for patients have not been established. Studies revealed a link between the dysregulation of CDK4/6 and ALM mortality, however CDK4/6 inhibitors (CDK4i/6i) have proved ineffective in patients with advanced ALM. The tumor suppressor Rb was recently shown to have a novel suppressive role towards mTORC2. Here, we elucidate the role of a non-canonical tumor suppressor function of Rb in negative regulation of mTOR in CDK4i/6i-resistance. We hypothesize that the loss of Rb induced by CDK4i/6i triggers phosphorylation of Akt and downstream mTORC1 signaling pathways. We characterized baseline activity of the PI3K/AKT/mTOR pathway in a panel of ALM cell lines, finding heterogeneity across signaling. We immunoblotted for key proteins involved in the signal reprogramming hypothesized: pRb, pAkt, pS6 to define activation status and found the loss of Rb phosphorylation preceded the enrichment of pAKT, supporting the non-canonical role of Rb tumor suppression and the Rb-mTORC2 signaling axis as intrinsic CDK4i/6i mechanism. In parallel, we assessed the essential protein-protein interactions required for mechanistic mTORC1 activation—mTOR and Rheb GTPase. The enrichment of these interactions shortly after introducing CDK4i/6i supported our hypothesized mechanism. Translational utility of CDK4i/6i and mTORi dual inhibition was determined via long-term Colony Formation Assays (CFAs) using Palbociclib, rapamycin and sapanisertib (TAK228), a catalytic mTOR inhibitor. We generated Palbociclib (acquired)-resistant ALM cell lines and investigated the pharmacological inhibition of PI3K signaling pathways through immunoblot and colony-formation assays. Concurrent mTORi and CDK4i/6i inhibition showed effective reduction in pRb, pS6, Cyclin D and increases in Cleaved PARP protein, suggesting apoptotic cell death was elevated with combined CDK4i/6i+mTORi treatment. Similarly, CFAs revealed dual inhibition elicited superior antiproliferative effect over the monotherapy treatments. CFAs with acquired-resistance cell lines also showed that combination inhibition decreased colony formation, showing the potential utility of concurrent CDK4i/6i and mTORi as an approach against acquired resistance. Our results show the efficacy of combined inhibition as an anti-tumor strategy. Citation Format: Jeremy A. Bravo Narula, Vito W. Rebecca. Elevated Akt/mTOR signaling drives therapy resistance in ALM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 419.