Comprehensive Failure Of Intracellular Protein Homeostasis Kills Myeloma And Solid Cancer Cells Following Vcp/P97 Inhibition

Introduction: Intracellular protein homeostasis requires a well-controlled protein degradation machinery to clear damaged and old proteins and to replenish intracellular amino acid pools. Cancer cells may be particularly susceptible to agents that disrupt protein degradation because of unbalanced protein levels related to gain or loss of genetic material, high protein turnover, or high-level production of specific proteins such as Ig in multiple myeloma (MM). The AAA ATPase VCP (p97) is a master regulator of protein degradation that has been implicated in oncogenesis. Small molecule VCP inhibition (VCP-i) rapidly activates caspases in cancer cells, induces endoplasmic reticulum (ER) stress, and has anti-tumor activity in murine xenograft models. A phase 1 trial of VCP-i in relapsed MM has recently opened. Methods: We investigated the cellular mechanisms that govern VCP-i mediated cancer cell death in MM cell lines including bortezomib-adapted AMO1 cells, MM cells from patients with bortezomib-resistant MM, as well as lung cancer (A549) and osteosarcoma (Saos2) cells. Results: The ATP-competitive inhibitor DBeQ and the allosteric inhibitor NMS873 induced cell line as well as primary MM cell death at similar low micromolar concentrations independently of bortezomib sensitivity. DBeQ and NMS873 caused phosphorylation of eIF2alpha in a time- and dose-dependent manner and resulted in strong transcriptional and translational up-regulation of ATF4, CHOP and GADD34. VCP-i also increased expression of ER chaperones BiP and p58IPK. Inhibition of eIF2alpha de-phosphorylation with guanabenz reduced S6 phosphorylation, a marker of protein translation, and increased A549 and OPM2 cell survival early (8h) after VCP-i. Direct inhibition of protein translation with cycloheximide also decreased early VCP-i mediated cell death. Using MEFs deficient in eIF2alpha kinases we show that eIF2alpha phosphorylation following VCP-i depends on both the unfolded protein response mediator PERK and the nutrient sensor GCN2. We found that DBeQ induces GCN2 phosphorylation in parallel with loss of mTORC1 signalling, induction of the key autophagy factor p62, and accumulation of LC3-II. DBeQ also induced a rapid decrease in free intracellular L-amino acids. Depletion of selected amino acids in the cell culture medium increased cell death and mRNA levels of CHOP and GADD34 following VCP-i with DBeQ or NMS873, but not following induction of ER stress with tunicamycin. Conclusion: Collectively, these data show that both ATP-competitive and allosteric VCP-i effectively kills cancer cells independently of bortezomib sensitivity. Disrupting VCP function induces early cell death via inappropriate eIF2alpha-regulated protein translation downstream of GADD34. VCP-i also depletes the intracellular free amino acid pool, resulting in cell death despite compensatory catabolic processes. Citation Format: Katarzyna Parzych, Sandra Loaiza, Tamara M. Chinn, Philippa May, Florentina Porsch, Anastasios Karadimitris, Christoph Driessen, Heather P. Harding, David Ron, Holger W. Auner. Comprehensive failure of intracellular protein homeostasis kills myeloma and solid cancer cells following VCP/p97 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2015-1261