Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates <30%. We sought to identify genes critical to AML cells, irrespective of mutational status, and performed an shRNA screen targeting 1,287 genes on 12 AML patient samples. This screen identified Sirtuin 5 (SIRT5) as a top candidate. SIRT5 is the only known enzyme with desuccinylase, demalonylase, and/or deglutarylase activity and we are the first to report the dependence of AML cells on SIRT5. Next, we stably transduced a panel of AML cell lines with doxycycline (dox)-inducible shSIRT5 (dox-shSIRT5). SIRT5 knockdown (KD) strongly inhibited cell growth, colony formation and increased apoptosis in 15/22 lines (SIRT5-dependent), while 7/22 lines were SIRT5-independent. SIRT5 dependence did not correlate with AML-related mutations nor basal SIRT5 expression. SIRT5 KD in primary AML samples (N=25) revealed a therapeutic window (~50% reduction), with no effect in CB samples (N=5). We examined the requirement of SIRT5 in vivo using three mouse models of leukemia. In a xenograft model with AML cell lines, SIRT5 KD indefinitely prolonged survival of mice injected with SIRT5-dependent cells with no sign of leukemia. Bone marrow transplant with transduced (MLL-AF9 or BCR-ABL1) SIRT5 null cells showed reduced leukemia cell burden and splenomegaly, and significantly prolonged survival. FLT3-ITD-driven disease was also blunted by the absence of SIRT5 in a genetic knockout mouse model. Mechanically, SIRT5 KD profoundly reduced oxidative phosphorylation (OXPHOS) and glycolysis. Additionally, SIRT5 KD increased mitochondrial superoxide selectively in annexin V-negative, SIRT5-dependent cells. Concomitant, ectopic expression of SOD2 abrogated the increase in superoxide, rescued cells from apoptosis, and rescued the colony formation deficit. Untargeted metabolomics revealed RNA charging and alanine and serine metabolism as top metabolic pathways regulated by SIRT5, with glutaminase (GLS) and α-ketoglutarate identified as potential upstream regulators. Metabolic tracing experiments with [13C5,15N2]-glutamine confirmed disrupted glutamine metabolism in SIRT5-dependent cells. Together, these results indicate that SIRT5 is required to regulate glutamine flux to sustain redox homeostasis and/or anabolism. NRD167, a novel SIRT5 inhibitor, was used to target SIRT5 in AML. NRD167 reduced cell proliferation, induced apoptosis, and reduced OXPHOS in SIRT5-dependent but not SIRT5-independent cells. NRD167 inhibited colony formation from AML patient samples, but not in CB samples. An AML patient-derived xenograft model trended toward prolonged survival following ex vivo treatment with NRD167. Our data suggest that the majority of AML samples are dependent on SIRT5 and that inhibition preferentially targets AML cells, implicating SIRT5 as a therapy target in AML.
Citation Format: Dongqing Yan, Anca Franzini, Anthony D. Pomicter, Brayden J. Halverson, Orlando Antelope, Clinton C. Mason, Jonathan M. Ahmann, Anna V. Senina, Courtney L. L. Jones, Matthew S. Zabriskie, Hein Than, Michael J. Xiao, Alexandria van Scoyk, Ami B. Patel, William L. L. Heaton, Shawn C. Owen, Joshua L. Andersen, Christina M. Egbert, Julie A. Reisz, Angelo D'Alessandro, James E. Cox, Kevin C. Gantz, Hannah M. Redwine, Siddharth M. Iyer, Jamshid S. Khorashad, Nima Rajabi, Christian A. Olsen, Thomas O'Hare, Michael W. Deininger. A critical role for SIRT5 in acute myeloid leukemia metabolism [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 LB109.