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

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

Plasmids.

Patient sample information.

Antibodies.

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.

All supplemental figures and legends.

Next generation sequencing for 52 myeloid malignancies-related genes.

Oligonucleotide sequences.

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.

Supplementary Figure 4

Supplementary Methods, Legends

Supplementary Table 1

Supplementary Figure 1

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Supplementary Figure 2

Purpose: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.

Supplementary Figure 5

Supplementary Table 2

Supplementary Table 3-8

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.

DNA-encoded chemical libraries (DECLs) interrogate the interactions of a target of interest with vast numbers of molecules. DECLs hence provide abundant information about the chemical ligand space for therapeutic targets, and there is considerable interest in methods for exploiting DECL screening data to predict novel ligands. Here we introduce one such approach and demonstrate its feasibility using the cancer-related poly-(ADP-ribose)transferase tankyrase 1 (TNKS1) as a model target. First, DECL affinity selections resulted in structurally diverse TNKS1 inhibitors with high potency including compound 2 with an IC

Purpose: 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.

Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates 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.

Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm with five-year overall survival rates To identify survival-critical AML genes irrespective of mutational status, 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), the only known enzyme with lysine desuccinylase, demalonylase, or deglutarylase activity, was amongst the top candidates. SIRT5 is implicated in regulating metabolic pathways, including energy metabolism. We first confirmed growth inhibition upon SIRT5 knockdown (KD) in cryopreserved AML cells from the original screen. Next, we transduced a panel of 25 AML cell lines with doxycycline (dox) - inducible shSIRT5 (dox-shSIRT5). SIRT5 KD strongly inhibited growth, reduced colony formation and increased apoptosis in 18/25 lines (SIRT5-dependent cell lines). Seven cell lines showed 80% reduction of SIRT5 protein and were considered SIRT5-independent. SIRT5 dependence was neither correlated with specific mutations nor basal SIRT5 expression. To control for off-target effects we tested three unique shSIRT5s targeting different sequences in three SIRT5-dependent and three SIRT5-independent cell lines, and confirmed the differential sensitivity for all lines and all constructs. 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 a profound reduction of oxidative phosphorylation and glycolysis in SIRT5-dependent, but not SIRT5-independent cell lines. Mitochondrial reactive oxygen species (ROS) in SIRT5-dependent cell lines were strongly increased upon SIRT5 KD, and this increase preceded apoptosis. Ectopic expression of superoxide dismutase 2 (SOD2, mitochondrial) abrogated the increase in ROS and rescued cells from apoptosis. Metabolomics and RNAseq suggest that SIRT5-dependent cells exhibit profound metabolic disruption, including reduced TCA cycle activity, and recurrent transcriptional changes, while SIRT5 KD in SIRT5-independent cells is inconsequential. We next injected mice with CMK-1 cells (SIRT5-dependent) expressing dox-shSIRT5 and luciferase. Mice were randomized to dox-supplemented (dox-water) or control water 18 hours post injection, and monitored by luminescence imaging. Control mice showed abundant luminescence at week 3, and died before week 5, while mice receiving dox-water survived throughout the 13-week experiment 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 with extensive leukemic involvement despite downregulation of SIRT5 in leukemia cells. We are currently testing the requirement of SIRT5 in MLL-AF9-mediated AML using a retroviral BM transplant mouse model. Preliminary data suggest that absence of SIRT5 may prolong survival. Our data suggest that SIRT5 KD preferentially targets AML cells over normal cells. 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. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.