DDDR-28. SPHINGOSINE AND N,N-DIMETHYLSPHINGOSINE MODULATE CHOLESTEROL HOMEOSTASIS IN IDH1MUT GLIOMAS, CAUSING PRO-APOPTOTIC MEMBRANE DEGRADATION

Abstract BACKGROUND Previous work in our team revealed a therapeutic potential for metabolic reprogramming along the sphingolipid pathway within IDH1mut gliomas. The dosing of various IDH1mut glioma subtypes with sphingosine and sphingosine kinase inhibitor (SphKi), N,N-dimethylsphingosine (NDMS) led to the enrichment of pro-apoptotic ceramides and sphingosines while suspending the proliferation of the IDH1mut neurospheres. However, the mechanism of drug action (MOA) was not well understood. We postulated that suppressing the production of S1P production with SphKi treatment combined with the global accumulation of sphingosine and certain ceramides triggers apoptotic membrane stress in highly susceptible IDH1mut gliomas. METHODS We cultured and treated IDH1mut (TS603, BT142 & NCH1681) and IDH1wt (GSC923) glioma cell lines with a combination of NDMS and C17-sphingosine. We used a comprehensive multi-omics approach involving LC-MS lipidomic, RNA sequencing, immunoblotting, flow cytometry, and fluorescent microscopy. RESULTS Following combination treatment, a global increase in sphingosines, ceramides, and their derivatives was accompanied by a decline in cholesterol levels. The transcriptomic expression was elevated for NR1H3, MYLIP, ABCA1, and ABCG1, which regulate the trafficking and biosynthesis of membrane-associated cholesterol. In contrast, total gene expression for catalytic enzymes involved in cholesterol (and isoprenoid) biosynthesis was negatively impacted. In addition, early onset changes included an elevation in the expression of ROCK (a marker for apoptotic membrane blebbing). Moreover, fluorescent microscopy revealed that sphingosine and cholesterol tend to become associated within the cellular membranes. This interaction then saturates the plasma membrane (PM), causing apparent vesiculation, permeation, and disruption of membrane integrity. CONCLUSION The MOA involved concomitant attenuation of cholesterol metabolism and influx in response to membrane saturation with sphingosine-associated cholesterol. As a result, PM stress was incurred, leading to PM disintegration in a blebbing fashion. Our study revealed how reprogramming sphingolipid metabolism increases the susceptibility of IDHmut gliomas to sphingosine-associated cholesterol-induced blebbing and resulting cell death.