Simultaneous Sessions I: Acute myeloid leukemia - Biology & translational research - Genes and regulation
Acute myeloid leukemia (AML) is a genetically heterogeneous hematopoietic neoplasm with five-year overall survival rates <30% on standard of care therapy. While recent progress with inhibitors of FLT-ITD and mutated IDH1/2 validates the concept of targeting driver mutants, the efficacy of the venetoclax/5-azacytidine combination is evidence that critical vulnerabilities can encompass multiple AML genotypes.
Identify novel therapy targets in AML in a genotype-agnostic manner.
We performed an shRNA screen on patient samples (N = 12) using a barcoded lentiviral shRNA library. Transduced cells were cultured for 9 days on HS-5 stromal cells (to mimic the BM microenvironment) and subjected to NGS for barcode quantification. Sirtuin 5 (SIRT5), a lysine desuccinylase and metabolic regulator, was amongst the top candidates.
We transduced 25 genotypically heterogeneous 25 AML cell lines with doxycycline (dox) - inducible shSIRT5 (dox-shSIRT5). SIRT5 KD strongly inhibited proliferation, reduced colony formation and increased apoptosis in 18/25 lines (SIRT5-dependent cell lines). SIRT5 dependence did not correlate with specific genotype or SIRT5 expression. Data were validated using three shSIRT5 s targeting different unique in 3 SIRT5-dependent and 3 SIRT5-independent cell lines, with consistent results. We next transduced CD34+ cells from AML patients (n = 15) or cord blood (CB, n = 5) with dox-shSIRT5 and plated cells in colony assays. SIRT5 KD (˜40% in AML and CB) reduced AML colonies by ˜50%, with no effect on CB. Colony formation by SIRT5 null mouse bone marrow infected with FLT3-ITD or MLL-AF9 retrovirus was reduced by 50-60% compared to wild type controls.
Metabolic profiling showed that SIRT5 KD induced reduced oxidative phosphorylation in SIRT5-dependent, but not SIRT5-independent cell lines, with a steep increase in mitochondrial reactive oxygen species (ROS) that preceded apoptosis and was abrogated by ectopic expression of superoxide dismutase 2 (SOD2, mitochondrial). Further SIRT5 KD disrupted glutamine/glutamate metabolism in SIRT5-dependent but not SIRT5-independent cells, while SIRT5 KD in SIRT5-independent cells was inconsequential.
We injected mice with CMK-1 cells (SIRT5-dependent) expressing dox-shSIRT5 and luciferase. Mice were randomized to dox-supplemented (dox-water) or control water, and monitored by luminescence imaging. Control mice showed abundant luminescence at week 3, and died before week 5, while mice receiving dox-water survived without evidence of leukemia. When mice with established leukemia were switched to dox-water following week 3, luminescence rapidly decreased and the mice survived without evidence of leukemia until termination of the experiment (week 13). Dox-water had no effect on mice engrafted with OCI-AML3 cells (SIRT5-independent), all of whom died from leukemia despite downregulation of SIRT5 in leukemia cells. We next assessed SIRT5 function in a retroviral transduction/transplantation model, using MLL-AF9 and BCR-ABL1 in a SIRT5 null or SIRT5 wild type background. Absence of SIRT5 attenuated leukemia in both cases, with significant prolongation of survival. We also functionally characterized hematopoietic stem and progenitor subsets from young and old SIRT5 wild type and null mice, but identified no significant differences.
AML, but not normal hematopoietic cells are dependent on SIRT5. As SIRT5 null mice are viable with only minor metabolic abnormalities at steady state, these data implicate SIRT5 as a potential therapy target in AML and support the development of clinical SIRT5 inhibitors.