THERAPEUTIC MODULATION OF CHOLESTEROL HOMEOSTASIS IN DIPG THROUGH MASSIVE GENERATION OF 24,25-EPOXYCHOLESTEROL

Diffuse intrinsic pontine glioma (DIPG) remains a uniformly fatal childhood tumor for which novel pharmacological treatments are desperately needed. In recent work, we performed a small molecule screen in a pre-clinical DIPG model, identifying MI-2 as the top ‘hit’, showing anti-tumor activity in vitro and in vivo. MI-2 was developed as an inhibitor of the protein menin, an epigenetic regulator which has oncogenic activity in acute leukemia. Here, we characterize the mechanism of action of MI-2 in DIPG. We generated menin knockout patient-derived DIPG cell-lines using CRISPR/Cas9 and observed that they retained sensitivity to MI-2 at nanomolar concentrations (comparable to wild-type parental cells), suggesting a menin-independent mechanism of action in DIPG. Transcriptome analysis of DIPG cells after MI-2 treatment revealed significant upregulation of gene targets of LXR (liver X receptor), a ligand-activated transcription factor which promotes cholesterol efflux to reduce cellular cholesterol. Consistent with a cholesterol depleting activity of MI-2, we showed markedly reduced cholesterol in DIPG cells following MI-2 treatment, and rescue of MI-2 induced cell-death by provision of exogenous cholesterol. To investigate how LXR is activated, we performed LC-MS analysis of major sterols species in MI-2 treated cells, and observed massive upregulation of 24,25-epoxycholesterol, an oxysterol known to be a potent endogenous activator of LXR. These findings characterize MI-2 as a novel cholesterol depleting agent, acting via an epoxycholesterol-LXR axis to promote cholesterol efflux from tumor cells. Our work suggests DIPG may be uniquely sensitive to perturbations in cholesterol homeostasis and implicates LXR activation as a potential therapeutic strategy in DIPG.