Targeting cytoplasmic SET oncoprotein by sphingolipid analog drugs for the induction of necroptosis to treat lung cancer - SUMMARY Non-small cell lung cancer (NSCLC) remains the deadliest cancer, and novel therapeutic strategies are needed to improve treatment outcomes of patients with this disease. FTY720 (Fingolimod/Gilenya, Novartis) is a sphingosine analog drug approved for the treatment of refractory multiple sclerosis due to its immune suppressor function. Immune suppression by FTY720 requires its phosphorylation (P-FTY720) mainly by sphingosine kinase 2 (SphK2). However, pro-drug FTY720, without its phosphorylation, exhibits anti-cancer functions in various models, including NSCLC. Our published and unpublished preliminary studies demonstrated that FTY720, and not P-FTY720, directly binds and targets cytoplasmic SET oncoprotein, which is overexpressed in majority of NSCLC cells and tumors. Cytoplasmic SET is a known inhibitor of protein phosphatase 2A (PP2A). FTY720-SET binding prevents inhibitory SET-PP2A association, leading to the activation of tumor suppressor PP2A. Mechanistically, activation of PP2A by FTY720 induces necroptosis and cell death in NSCLC cells in culture and in animal models through the formation of RIPK1-C16-ceramide-non- muscle myosin IIA (NMIIA) enriched pores (we coined the name “ceramidosomes”) at the plasma membrane. Thus, we designed this application to test the novel hypothesis that FTY720 and its functional mimetics mediate NSCLC cell death and tumor suppression, at least in part, by directly binding and targeting SET oncoprotein, resulting in the generation of plasma membrane pores and necroptosis. There are three Specific Aims proposed in this application: Aim 1) Define the mechanism by which FTY720 mediates necroptosis via induction of ceramidosomes. Aim 2) Identify how FTY720-induced ceramidosomes are activated by PP2A to mediate necroptosis. Aim 3) Determine the therapeutic and clinical significance of targeting SET by FTY720 and its functional mimetics, like A7-2, to mediate PP2A-dependent ceramidosome activation and NSCLC tumor suppression. These studies will help define how targeting SET induces necroptosis using molecular, genetic, pharmacologic, and structural tools, including X-ray crystallography and cryo-EM. Moreover, data obtained from this proposal will help predict which of the NSCLC patients are likely to benefit from the treatment with FTY720 and its analog drugs. We will also utilize functional mimetics of FTY720, such as A7-2, which is a recently developed analog that inhibits SET and induces necroptosis at nanomolar concentrations, in the absence/presence of mutant (G12C)-K-Ras inhibitors, such as sotorasib or adagrasib, to suppress NSCLC tumor growth/progression. Thus, these studies will provide first-in-class treatment options and precision therapy for the treatment of patients with refractory NSCLC.
