Cholesterol biosynthesis inhibition synergizes with AKT inhibitors in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer deaths due to its molecular heterogeneity, high recurrence rate and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. We performed a genome-wide negative selection CRISPR/Cas9 screen with PI3K? and AKT inhibitors to identify targetable synthetic lethalities in TNBC. We found that cholesterol homeostasis is a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro, in mouse TNBC xenografts and in patient-derived organoids of estrogen receptor (ER)-negative breast cancer. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBCs show dysregulated SREBP-2 activation in response to single agent or combination AKT inhibitor and pitavastatin, and this was rescued by inhibition of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss promoted lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. This work motivates combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. Overall design: We performed comparative mRNA sequencing (RNAseq) in TNBC (MDA-MB-468) and ER+ breast cancer cells (T47D), exposed to the catalytic AKT inhibitor AZD5363, pitavastatin, or the combination of AZD5363 and pitavastatin for 24 or 48 h. To limit the confounding effect of differential potency, we determined the doses of AZD5363 that would result in comparable suppression of downstream signaling in each cell line at these time points (MDA-MB-468: 10 µM, T47D: 0.25 µM). As a control, cells were treated with DMSO. The data are from three independent experiments performed in growth factor-complete medium.