This study investigates the mechanism by which avicins—plant-derived triterpenes with potent anticancer activity—alter lysosomal functions. The anticancer properties of avicins have been known for over two decades, yet the precise mechanism of action of avicins remains unknown. To elucidate their mechanism of action, we conducted a pooled CRISPR interference (CRISPRi) genetic screen in Jurkat T-cell leukemia cells treated with avicin D. Our screen revealed that the depletion of genes encoding lysosomal components protected cells from the drug's cytotoxic effects. Consistent with this finding, blocking lysosomal functions with chloroquine protected cells from avicins. Moreover, blocking the lysosomal cholesterol transporter NPC1 abolished avicin cytotoxicity, hinting that avicins—which resemble steroidogenic molecules—use the same cellular factors required for cholesterol transport. Together, these results suggest a causal relationship between a requirement for lysosomal functions and avicin cytotoxicity. The top hit whose depletion sensitized cells to avicin D was the endoplasmic reticulum (ER) stress sensor ATF6, which is a membrane-tethered transcription factor capable of detecting sphingolipid lipotoxicity in the ER membrane. Consistent with this result, recent observations indicate that avicins alter the cell's sphingolipid and cholesterol balance (1), suggesting that avicins induce ER stress by disrupting ER membrane homeostasis. Together, our results support a potential mechanism of action wherein avicins are pro-drugs activated in lysosomes and induce organelle stress by altering the cell's lipid balance. We are currently working on elucidating the molecular mechanisms by which avicins and their putative metabolic products disrupt organelle homeostasis. We anticipate that our study will reveal the molecular circuitry that controls the cell's response to avicins and may open new avenues for therapeutic intervention in cancer.

1. Garrido CM, Henkels KM, Rehl KM, Liang H, Zhou Y, Gutterman JU, Cho K-J. Avicin G is a potent sphingomyelinase inhibitor and blocks oncogenic K- and H-Ras signaling. PMC7272413

Citation Format: Friederike Braig, Jordan U. Gutterman, Diego Acosta-Alvear. The anticancer compound avicin D targets lysosomal functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1033.