The 1,287 genes targeted by the shRNA of the leukemia library.

Next generation sequencing for 52 myeloid malignancies-related genes.

Patient sample information.

Ranked list of 1,287 genes from shRNA library screen in primary AML cells. The 1,287 genes assessed with an shRNA library screen were sorted by the second highest percentile fold change present in 2 shRNA and across 2 samples, with the 34 genes showing a fold change in the top 2 percent in more than 2 samples listed first.

Standard hematologic parameters in SIRT5-/- mice and SIRT5+/+ littermates.

All supplemental figures and legends.

AbstractPurpose: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. Experimental Design: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear–cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). Results: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. Conclusions: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Abstract We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells, but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase. Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor, but promotes endocytosis of common β chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhance leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and blast phase CML. Promoting MS4A3 re-expression or delivery of ectopic MS4A3 may help eliminating LSPCs in vivo.

Abstract 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.

AbstractPurpose: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. Experimental Design: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear–cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). Results: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. Conclusions: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.