EXTH-93. Combination of ALK2 and cholesterol targeting agents exploits linked genetic and metabolic dependencies in diffuse midline glioma

Abstract The discovery that ACVR1-mutations arise in ~25% of DMG H3K27-altered patients has led assessment of both the dependency of ACVR1-mutant cells on ALK2 and the effectiveness of ALK2 inhibitors (ALK2i), however as single-agents these are yet to translate into the clinic. Here, we perform high-throughput screens to identify therapeutic ALK2i combination partners and characterise pre-clinical ALK2 inhibitors of different chemotypes using a multi-omic approach. Combinatorial CRISPR screens identified multiple ALK2i sensitising hit genes which encode for key enzymes in the cholesterol synthesis pathway (EBP/DHCR24/LSS). Drug-combination screens identified clinically well-tolerated statins, including simvastatin/lovastatin which target the rate-limiting cholesterol synthesis enzyme HMG-CoA reductase, and estrogen receptor inhibitors, including Tamoxifen, which exhibits off-target effects on EBP, as sensitisers to ALK2i. Global transcriptomics, proteomics and metabolomics revealed a novel link between ALK2 signalling and cholesterol homeostasis, with ALK2i treatment significantly decreasing the expression cholesterol biosynthesis genes/proteins (SREBF2/HMGCR/EBP) while increasing those associated with cholesterol transport (ABCA1/MYLIP). Metabolomic analysis confirmed these changes were associated with a significant decrease in cholesterol and an increase in the precursor desmosterol. Validation of screening hits revealed strong synergy between ALK2i and cholesterol biosynthesis inhibitors targeting different nodes of the pathway. This was also observed in vivo, the combination treatment significantly increased the median survival compared to vehicle but not to single-agents. Forced differentiation of DMG cells to an astrocyte-like cell state increased cholesterol production and led to a significant decrease in M4K2009 sensitivity and synergy with statins, which was phenocopied when DMG cells were co-cultured with normal astrocytes. This was overcome in vitro, ex vivo and in vivo using a triple combination including a LXR agonist (LXR623) to promote cholesterol export. We identify a previously unappreciated link between ALK2 signalling and cholesterol homeostasis which may be exploited clinically by combining ALK2i with routinely-used statins and BBB-penetrant LXR agonists.