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Summary We describe a method to silence genes in primary acute myeloid leukemia cells by transducing them with shRNA in lentiviral vectors. The transduction of primary non-adherent cells is particularly challenging. The protocol will aid in performing such experiments and is particularly helpful to prepare cells for in vivo engraftment studies. Use of a special medium supplemented with cytokines preserves the viability of the leukemic stem cells and their ability to engraft the marrow of immune-deficient mice. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020)., Graphical Abstract, Highlights • An optimized protocol to silence genes in primary AML cells • Transduces primary AML cells with shRNA in lentiviral vectors • Preserves viability of the primary cells • Suitable for downstream assays including cell viability and in vivo engraftment, We describe a method to silence genes in primary acute myeloid leukemia cells by transducing them with shRNA in lentiviral vectors. The transduction of primary non-adherent cells is particularly challenging. The protocol will aid in performing such experiments and is particularly helpful to prepare cells for in vivo engraftment studies. Use of a special medium supplemented with cytokines preserves the viability of the leukemic stem cells and their ability to engraft the marrow of immune-deficient mice.

While most patients with acute myeloid leukemia (AML) achieve remission with initial therapy, the majority relapse leading to poor overall survival. Relapse is frequently driven by a rare subset of leukemic stem cells (LSC). Understanding biological mechanisms that maintain LSCs will help identify new therapeutic strategies for this disease. To identify such vulnerabilities, we overlaid the results of a genome-wide CRISPR screen with the expression of genes enriched in functionally defined LSCs. Through our CRISPR screen, we identified 570 genes whose knockout reduced the growth and viability of OCI-AML2 cells. Essential genes for growth and viability by our CRIPSR screen were enriched in the LSC+ population. By overlaying the hits from our CRISPR screen with genes upregulated in LSCs, we identified IPO11, as a top hit, with 7.5-fold increase in the LSC+ fraction compared to the LSC- fraction. IPO11 is a member of the importin-β family of proteins and facilitates the import of protein cargo into the nucleus. Further analysis showed that IPO11 was upregulated in LSC+ (engrafting) vs. LSC- (non-engrafting) primary AML samples, CD34+ vs CD34- AML samples, undifferentiated progenitor vs. myeloid cluster AML samples, and relapse vs de novo AML. IPO11 was increased in AML cells compared to normal hematopoietic cells and increased IPO11 expression was associated with decreased overall survival in AML. By immunoblotting, IPO11 protein was increased in primary AML (n=4) compared to normal hematopoietic cells (n=4). To determine whether IPO11 is necessary for AML growth and viability, we knocked down IPO11 in OCI-AML2, TEX and NB4 leukemia cells with shRNA in lentiviral vectors. Knockdown of IPO11 reduced AML growth and viability by 80-90%. In contrast, knockdown of another importin-β family member, IPO5, that was not a hit in our CRIPSR screen, did not reduce AML growth and viability. Knockdown of IPO11 increased differentiation of AML cells as evidenced by the changes in gene expression, decreased chromatin accessibility, increased CD11b expression and increased non-specific esterase staining. Finally, knockdown of IPO11 reduced the engraftment of TEX cells and the low passage primary AML 8227 cells into immune deficient mice by over 90%. Importantly, IPO11 knockdown reduced engraftment of primary AML cells into mouse marrow. To identify novel cargos of IPO11, we performed proximity-dependent biotin labeling (BioID) coupled with mass spectrometry and identified proteins that interacted with IPO11. Among the top hits were BZW1 and BZW 2 (Basic leucine zipper and W2 domains 1 and 2). BZW1 and BZW2 are members of the bZIP super family of transcription factors. Knockdown of IPO11 reduced levels of BZW1 in the nucleus detected by immunoblotting and confocal microscopy. Commercial antibodies could not detect BZW2. To determine if the nuclear import of BZW1 and 2 were functionally important for the effects of IPO11 on AML stem cell function and differentiation, we knocked down BZW1 and BZW2. Dual knockdown of BZW1 and BZW2 (but not individual) mimicked the effects of IPO11 inhibition and decreased the growth and viability of AML cells. Changes in gene expression after BZW1/2 knockdown were similar to IPO11 knockdown with enrichment in myeloid-differentiated genes. By pathway analysis, we identified that IPO11 knockdown, as well as BZW1/2 knockdown decreased expression of MYC target genes, suggesting a mechanism by which these proteins regulate AML stem cell function. Thus, in summary, we identified IPO11 as an essential gene for the viability of AML cells and stem cells. This work highlights a previously unappreciated role of the protein import pathway in regulating AML stem cell function and highlights a potential new therapeutic target for AML. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a primary AML culture model arranged in a hierarchy with defined stem cells. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. In contrast, other metabolic enzymes including PFK1, FH, PKM2, GPI1, ENO1, CS, ACO2, and SDHA1, 2 were not detected in the nucleus of these cells. We also detected HK2 in the nucleus of AML cell lines as well as 7 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals in 8227 and NB4 leukemia cells. This increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Nuclear HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with 3'UTR shRNA. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation with ATRA, and decreased CD34+CD38- 8227 stem cells without changing basal proliferation. Nuclear HK2 was also higher in normal human hematopoietic stem cells and multipotent progenitor fractions and declined as the cells matured. Over-expression of nuclear HK2 in normal cord blood increased the primary and secondary engraftment into mice. Transgenic mice over-expressing nuclear HK2 driven by a Vav promoter had increased hematopoietic stem cells in the marrow and decreased monocytes and lymphocytes in the peripheral blood. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2 (D209A D657A) and observed increased clonogenic growth and inhibited differentiation on ATRA treatment, nuclear HK2 function is independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry identified proteins related to chromatin organization and regulation to interact with nuclear HK2. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Citation Format: Geethu Emily Thomas, Grace Egan, Laura Gracia Prat, Aaron Botham, Veronique Voisin, Elias Orouji, Jordan Chin, Boaz Nachmias, Kerstin B. Kaufmann, Neil Maclean, Rose Hurren, Marcela Gronda, Xiaoming Wang, Dilshad H. Khan, Rashim P. Singh, Andrea Arruda, Mark Minden, Gary D. Bader, John E. Dick, Aaron D. Schimmer. The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness [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 3099.

Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML Relapse of acute myeloid leukemia (AML) is common and the main cause of mortality in this disease. Leukemia stem cells (LSCs) contribute to relapse however the mechanisms underpinning chemoresistance are not well understood. Here, we explored the DNA damage response in AML LSCs to investigate if enhanced DNA repair contributes to relapse and chemoresistance. We discovered that expression of DNA repair pathway genes were up-regulated in functionally defined LSC vs bulk AML cells (n= 227, accession number GSE76008), NES = 1.74, FDR = 0.057, and undifferentiated (n= 7) vs committed AML samples (n = 4, Princess Margaret Cancer Centre cohort), NES = 2.041, FDR = 0.000. Next, we investigated the expression of DNA damage repair genes and response to DNA damage in FACs sorted stem and bulk fractions of 8227 cells. 8227 cells are low passage primary AML cells that maintain a hierarchical organization with functionally defined stem cells in the CD34+CD38- fraction. Compared to bulk cells, the stem cell fraction of 8227 cells had increased expression of genes associated with DNA repair through homologous recombination (RAD51, XRCC2, XRCC3) and non-homologous end joining (XRCC4, XRCC5, PRKDC). Sorted 8227 cells were treated with daunorubicin, an intercalating anthracycline that causes double stranded breaks. DNA damage and repair were evaluated by measuring foci of 53BP1, RAD51 and γH2AX by fluorescent microscopy and quantified using image J. Compared to bulk cells, 8227 stem cells demonstrated increased DNA damage repair with increased foci of 53BP1 and RAD51 and decreased γH2AX foci. We recently discovered that the metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus to maintain stem cell number and function. Therefore, to understand how stemness impacts DNA damage repair, we selectively over-expressed HK2 in the nucleus of 8227 and NB4 cells by tagging HK2 with a nuclear localizing sequence. Over-expressing nuclear HK2 increased stem cell function as shown by clonogenic growth assays and engraftment into mouse marrow. We then treated these cells with daunorubicin and measured DNA damage repair. Increasing stem cell number and function by over-expressing nuclear HK2, increased 53BP1 and RAD51 foci and decreased γH2AX foci, similar to the phenotype observed in LSCs. As measured by the COMET assay, these cells had decreased levels of double strand DNA breaks under basal conditions and increased repair of DNA breaks after treatment with daunorubicin treatment. Over-expression of nuclear HK2 also conferred resistance to daunorubicin as measured by clonogenic growth assays. In summary, LSCs have increased levels of DNA repair genes and increased rates of DNA damage repair after exposure to chemotherapy. The accelerated DNA damage repair seen in LSCs may contribute to chemoresistance and subsequent disease relapse. Citation Format: Grace Egan, G.E. Thomas, Parasvi Patel, Veronique Voisin, Rose Hurren, Neil MacLean, Razq Hakem, Aaron D. Schimmer. Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML [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 3098.

While most patients with AML achieve remission with standard induction chemotherapy, the majority ultimately relapse. Relapsed AML is due, at least in part, to the persistence of chemoresistant leukemia stem cells (LSCs). The mechanisms of chemoresistance in LSCs are not fully understood. Here, we explored DNA damage repair in LSCs. 8227 cells are low passage primary AML cells that maintain a hierarchical organization with functionally defined stem cells in the CD34+CD38- fraction. We FACS sorted 8227 cells into stem and bulk fractions and measured expression of DNA repair genes. LSCs were primed for DNA repair with increased expression of genes associated with homologous recombination (RAD51, XRCC2, XRCC3) and non-homologous end joining (XRCC4, XRCC5, PRKDC). Next, we treated the cell fractions with daunorubicin, an intercalating anthracycline that causes double stranded breaks. DNA damage and repair were evaluated by measuring foci of 53BP1, RAD51 and γH2AX by fluorescent microscopy and quantified using image J. Compared to bulk cells, 8227 stem cells demonstrated enhanced DNA damage repair with increased foci of 53BP1 and RAD51 and decreased γH2AX foci, compared to their basal levels. Similar findings were noted after exposing the stem and bulk cells to radiation. We recently discovered that the metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus to maintain stem cell number and function. Therefore, we selectively over-expressed HK2 in the nucleus of 8227 and NB4 cells by tagging HK2 with a nuclear localizing sequence (PAAKRVKLD). We confirmed selective over-expression of HK2 in the nucleus by immunoblotting and confocal microscopy. Over-expressing HK2 increased stem cell function as shown by clonogenic growth assays and engraftment into mouse marrow. We then treated these cells with daunorubicin and measured DNA damage repair. Over-expression of nuclear HK2 increased 53BP1 and RAD51 foci with decreased γH2AX foci, similar to the phenotype observed in LSCs. In addition, over-expression of nuclear HK2 conferred resistance to daunorubicin as measured by clonogenic growth assays. In summary, LSCs appear to be primed for DNA repair with increased levels of DNA damage repair genes. After exposure to chemotherapy and radiation, LSCs have increased repair of double strand DNA breaks compared to more differentiated blasts. This accelerated DNA damage repair may partly explain the increased chemoresistance seen in LSCs. Disclosures Schimmer: Takeda:Honoraria, Research Funding;Novartis:Honoraria;Jazz:Honoraria;AbbVie Pharmaceuticals:Other: owns stock ;Otsuka:Honoraria;Medivir AB:Research Funding.

Leukemia stem cells are responsible for the initiation and recurrence of AML. Cell differentiation requires the coordination of metabolic state and gene expression programs. It is known that metabolic intermediates can serve as cofactors in epigenetic modifications in the nucleus. Whether metabolic enzymes can directly localize to the nucleus, influence gene expression and regulate stemness in AML is unknown. To identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells we analyzed stem and bulk fractions of 8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells in the CD34+CD38- fraction. Hexokinase 2 (HK2) was detected in the nuclear fraction of 8227 cells and nuclear HK2 was increased in 8227 stem cells compared to bulk cells. Conversely, the metabolic enzymes aconitase, citrate synthesase, enolase, fumarase, GAPDH, glucose-6-phosphate isomerase, phosphofructokinase, pyruvate kinase and succinate dehydrogenase A were not detectable in the nuclear fraction of either stem or bulk cells. We confirmed that HK2 was present in the nuclear fraction of OCI-AML2, NB4, HL60 and TEX AML cell lines and 7/9 primary patient samples. To determine whether nuclear HK2 was functionally important for AML stem cells, we overexpressed HK2 tagged with a nuclear localizing sequence (PAAKRVKLD). Over-expression of nuclear localized HK2 enhanced clonogenic growth and engraftment into immune deficient mice. Likewise, over-expression of nuclear localized HK2 blocked differentiation after treatment with all-trans retinoic acid (ATRA). Next, we investigated whether the kinase activity of HK2 was necessary for its nuclear effects on AML stemness and differentiation. We generated a kinase dead mutant of HK2 (D209A, D657A) tagged with a nuclear localizing signal (PAAKRVKLD). Over-expression of nuclear-localized kinase dead HK2 exerted a similar phenotype to overexpressed nuclear HK2 without manipulation of the kinase domain as it enhanced clonogenic growth and blocked cell differentiation after ATRA treatment. To understand the mechanism by which nuclear HK2 influenced AML stemness, we identified proteins that interacted with nuclear HK2 using proximity-dependent biotin labeling (BioID) and mass spectrometry. Proteins involved in DNA damage repair and chromatin remodeling were identified, including exonuclease 3′-5′ domain-containing 2 (EXD2), sirtuin 1 (SIRT1), E3 ubiquitin-protein ligase (URB5) and tyrosyl-DNA phosphodiesterase 2 (TDP2). Thus, our data suggest that nuclear HK2 influences DNA repair, which has been linked to stemness and differentiation. In support of this hypothesis, we identified a PAR binding motif in HK2 and over-expression of nuclear HK2 conferred resistance to the PARP inhibitor, olaparib. In summary, HK2 localizes to the nucleus in AML stem cells and maintains stemness. These effects are independent of its kinase activity. Rather, HK2 may regulate DNA damage repair through a PAR dependent mechanism. Thus, we have identified a new role for metabolic enzymes in the regulation of stemness and differentiation. Citation Format: Grace Egan, Geethu E. Thomas, Parasvi S. Patel, Jordan Chin, Aaron Botham, Rose Hurren, Neil MacLean, Razq Hakem, Aaron D. Schimmer. The metabolic enzyme Hexokinase 2 localizes to the nucleus and regulates stemness in AML through a kinase independent mechanism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3784.

TAK-243 (MLN7243) is a first-in-class inhibitor of the ubiquitin-activating enzyme (UBA1) that catalyzes the first step in the ubiquitylation cascade whereby proteins are tagged with mono- or poly-ubiquitin to induce their degradation or modify their functions. Based on its preclinical efficacy and tolerability, TAK-243 has entered phase 1 clinical trials in advanced malignancies. However, the determinants of sensitivity to TAK-243 remain largely unknown. Therefore, we conducted a positive-selection, genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with lethal TAK-243 concentrations to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout conferred resistance to TAK-243 (FDR = 0.0012). BEND3-targeting gRNAs were enriched up to 10,000-fold after selection with the drug. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells and confirmed the resistance phenotype. In vivo, tumors of BEND3-knockout cells were resistant to TAK243 (20 mg/kg twice weekly) as opposed to control tumors that showed dramatic reductions in tumor growth rate. As assessed by immunoblotting, BEND3 knockout dampened TAK-243 effects on ubiquitylation, proteotoxic stress and DNA damage response. Mechanistically, BEND3 knockout upregulated the ABC efflux transporter breast cancer resistance protein (BCRP; ABCG2), and decreased intracellular levels of TAK-243. It also conferred partial cross-resistance to pevonedistat and TAK-981–related selective inhibitors of the NEDD8-activating enzyme (NAE) and the SUMO-activating enzyme (SAE), respectively, as well as known substrates of BCRP (mitoxantrone and doxorubicin). Finally, TAK-243 sensitivity strongly correlated with BCRP expression in a panel of 30 cancer cell lines of different origin, and chemical inhibition of BCRP but not P-gp sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity. Citation Format: Samir H. Barghout, Ahmed Aman, Zachary Blatman, Karen Arevalo, Geethu Thomas, Neil MacLean, Xiaoming Wang, Rose Hurren, Troy Ketela, Moustafa Abohawya, Taira Kiyota, Rima Al-Awar, Aaron D. Schimmer. BEND3 modulates sensitivity to the UBA1 inhibitor TAK-243 by regulating expression of the multidrug transporter BCRP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6321.

Summary Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

Leukemic stem cells (LSCs) rely on oxidative metabolism and are differentially sensitive to targeting mitochondrial pathways, which spares normal hematopoietic cells. A subset of mitochondrial proteins is folded in the intermembrane space via the mitochondrial intermembrane assembly (MIA) pathway. We found increased mRNA expression of MIA pathway substrates in acute myeloid leukemia (AML) stem cells. Therefore, we evaluated the effects of inhibiting this pathway in AML. Genetic and chemical inhibition of ALR reduces AML growth and viability, disrupts LSC self-renewal, and induces their differentiation. ALR inhibition preferentially decreases its substrate COX17, a mitochondrial copper chaperone, and knockdown of COX17 phenocopies ALR loss. Inhibiting ALR and COX17 increases mitochondrial copper levels which in turn inhibit S-adenosylhomocysteine hydrolase (SAHH) and lower levels of S-adenosylmethionine (SAM), DNA methylation, and chromatin accessibility to lower LSC viability. These results provide insight into mechanisms through which mitochondrial copper controls epigenetic status and viability of LSCs.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the accumulation of malignant myeloid cells that have arrested maturation. Most therapeutic regimens approved or under development are cytotoxics. An alternate, but less explored therapeutic approach, is to induce terminal differentiation of AML cells. Upon differentiation, AML cells cease to proliferate or die. Phosphatidylserine decarboxylase (PISD) is a mitochondrial enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE). Here, we explored the effects of inhibiting PISD on AML growth, stemness and differentiation. Knockout of PISD by CRISPR reduced the growth and clonogenic growth of OCI-AML2 cells. The reported chemical PISD inhibitor, 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine (aka: MMV007285), reduced growth and viability of OCI-AML2 cells (IC50 = 4.741 μM) and TEX cells (IC50 = 4.868 μM). Using the 8227 primary AML cell culture model, we showed that inhibiting PISD induced cell death in the functionally defined stem cell fraction (CD34+CD38-). MMV007285 also preferentially inhibited the clonogenic growth of primary AML cells (n = 7) over normal hematopoietic cells (n= 3). Moreover, MMV007285 induced AML cell differentiation as evidenced by increased CD11b expression and staining for non-specific esterase. Using high-performance thin layer chromatography (HPTLC), we found that inhibition of PISD with MMV007285 increased intracellular PS. To determine whether increased PS was functionally important, OCI-AML2 cells were treated with PS, resulting in reduced growth and clonogenic growth. Furthermore, PS supplementation targeted AML progenitor cells as it decreased engraftment of TEX cells in mice. Mechanistically, inhibiting PISD induced differentiation and decreased stemness in AML by activating Toll-like receptor (TLR) signaling. Specifically, inhibiting PISD upregulated TLR4 and 8 expression and increased expression of cytokines downstream of TLR activation. We also showed that TLR activation was functionally important to induce AML differentiation. Finally, we evaluated the effects of PISD inhibition in AML mouse models. MMV007285 (300 mg/kg/5 of 7 days orally for 10 days) decreased the growth of OCI-AML2 cells in SCID mice. Moreover, MMV007285 (150 mg/kg/5 of 7 days orally for 5 weeks) impeded the leukemic engraftment of primary AML cell in NOD/SCID mice without toxicity. Using secondary transplants, we showed that MMV007285 also targeted the leukemic stem cells. Taken together, inhibition of PISD altered phospholipid metabolism, inhibited growth and stemness, and increased differentiation in AML cells. Our findings reveal a previously undescribed link between mitochondrial phospholipid metabolism and AML stemness and differentiation, highlighting a potential new therapeutic strategy for AML. Citation Format: Mingjing Xu, Ayesh Seneviratne, Val A. Fajardo, Geethu E. Thomas, G. Wei Xu, Rose Hurren, S. Kim, Neil MacLean, Xiaoming Wang, Marcela Gronda, Danny Jeyaraju, Yulia Jitkova, David Sharon, Ahmed Aman, Rima Al-awar, Steven Chan, Mark D. Minden, Paul LeBlanc, Aaron D. Schimmer. Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3003.

Mode of reproduction is generally considered to have long-range evolutionary implications on population survival. Because sexual reproduction produces genetically diverse genotypes, this mode of reproduction is predicted to positively influence the success potential of offspring in evolutionary arms race with parasites (Red queen) whereas, without segregation and recombination, the obligate asexual multiplication may push a species into extinction due to the steady accumulation of deleterious mutations (Muller’s ratchet). However, the extent of linearity between reproductive strategies, genetic diversity and population fitness, and the contributions of different breeding strategies to population fitness are yet to be understood clearly. Genus Zingiber belonging to the pan-tropic family Zingiberaceae represents a good system to study contributions of different breeding behavior on genetic diversity and population fitness, as this genus comprises species with contrasting breeding systems. In this study, we analyzed breeding behavior, amplified fragment length polymorphism diversity and response to the soft-rot pathogen Pythium aphanidermatum in 18 natural populations of three wild Zingiber spp.: Z. neesanum, Z. nimmonii, and Z. zerumbet, together with the obligately asexual cultivated congener, ginger (Z. officinale). Ginger showed an exceptionally narrow genetic base, and adding to this, all the tested cultivars were uniformly susceptible to soft-rot. Concordant with the postulates of Muller’s ratchet, the background selection may be continuously pushing ginger into the ancestral state, rendering it inefficient in host-pathogen coevolution. Z. neesanum and Z. nimmonii populations were sexual and genetically diverse; however, contrary to Red Queen expectations, the populations were highly susceptible to soft-rot. Z. zerumbet showed a hemiclonal breeding behavior. The populations inhabiting forest understory were large and continuous, sexual and genetically diverse, but were susceptible, whereas populations inhabiting the revenue land were fragmented and monoclonal, but were resistant. It may be possible that, when genetic recombination becomes at a premium due to the genetic constraints imparted by habitat fragmentation or pathogen pressure, Z. zerumbet

UBA1 is the major ubiquitin-activating enzyme that initiates the ubiquitylation cascade whereby proteins are tagged with mono- or polyubiquitin to mark them for proteasomal degradation or modify their functions. Despite having equal levels of UBA1 protein, AML cell lines and primary AML cells are more dependent on UBA1 activity compared to normal hematopoietic cells, rendering them more vulnerable to UBA1 inhibition. Recently, we demonstrated that inhibiting UBA1 with the small-molecule inhibitor TAK-243 was selectively cytotoxic to a subset of AML cells and stem cells in vitro and in vivo through a mechanism at least partly dependent on inducing ER stress (Leukemia, 2018). To identify potential determinants of sensitivity/resistance to TAK-243 (Millennium Pharmaceuticals, Takeda) in AML, we conducted a genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with cytotoxic TAK-243 concentrations corresponding to the IC90 and IC99. By next-generation sequencing and enrichment analysis, we then identified genes whose knockout renders AML cells resistant to TAK-243. We identified 34 hits in the IC90 and 11 hits in the IC99 arms of the screen (cut off FDR < 0.2). These hits are involved in signaling pathways including transcriptional regulation, histone methylation, ubiquitin conjugation, cell cycle progression, mTOR and NF-κB signaling pathways, consistent with the broad range of pathways regulated by UBA1-mediated ubiquitylation. We focused our investigation on BEN domain-containing protein 3 (BEND3) that ranked as a top hit in both arms (FDR = 0.0012). Compared to control, all 6 BEND3-targeting gRNAs were enriched up to 10,000 times. BEND3 is a transcriptional repressor that regulates heterochromatin organization. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells using the 4 top performing gRNAs in the screen. We confirmed target knockout by immunoblotting. BEND3 knockout did not alter the basal proliferation rate of the cells. However, knockout of BEND3 rendered OCI-AML2 cells resistant to TAK-243 with up to a 4-fold increase in the IC50 by the MTS assay. Resistance to TAK-243 was confirmed by Annexin V/PI staining, PARP cleavage, and colony-forming assays. Cells were cross resistant to the NEDD8-activating enzyme inhibitor pevonedistat (2-fold IC50 increase), but not bortezomib or daunorubicin. As assessed by immunoblotting, BEND3 knockout did not change expression of UBA1, or the related enzymes UBA2, UBA3, or UBA6. BEND3 knockout was associated with reduced induction of ER stress as assessed by levels of CHOP and ATF4 after TAK-243 treatment. Conclusions Through a genome-wide CRISPR screen, we identified BEND3 as a determinant of sensitivity to TAK-243 in AML. Mechanistically, lack of BEND3 expression dampens the ER stress response to UBA1 inhibition. Thus, these results may highlight a new mechanism of sensitivity to TAK-243. Disclosures Schimmer: Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Hematopoietic cells are arranged in a hierarchy where stem and progenitor cells differentiate into mature blood cells. Likewise, AML (Acute Myeloid Leukemia) is also hierarchical with leukemic stem and progenitor cells giving rise to more mature and differentiated blasts. Recent studies have shown that mitochondrial enzymes such as IDH2 can regulate AML stemness by altering metabolites that affect epigenetic marks. However, it is unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we show that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. We sought to identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells, by evaluating the stem and bulk fractions from 8227 leukemia cells. 8227 leukemia cells are arranged in a hierarchy with functionally defined stem cells present in the CD34+CD38- fraction. We separated 8227 cells into CD34+CD38- and CD34-CD38+ populations by FACS sorting and prepared lysates of the nuclear and cytoplasmic fractions from each population. Using immunoblotting, we measured levels of mitochondrial enzymes in the subcellular fractions of each population. We discovered that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Aconitase 2 and Succinate Dehydrogenase B were not detected in the nuclear fractions. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, thereby initiating glycolysis and the entry of glucose metabolites into the TCA cycle in the mitochondria. The nuclear localization of HK2 in mammalian cells has not been previous reported. We confirmed that 8227 cells have nuclear HK2 by confocal fluorescent microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K562, and MV411) and primary AML samples. We also FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (B cells, T cells, NK cells, Monocytes and Granulocytes) cells. The localization of HK2 in these cell fractions was measured by immunofluorescence and quantified by Metamorph and Imaris. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as the cells matured (Fig 1A). The mitochondrial localization of HK2 is dependent on AKT-mediated phosphorylation of Thr-473 and inhibited by dephosphorylation by the phosphatase PHLPP1. We asked whether phosphorylation of HK2 regulates the nuclear abundance of HK2. Using AML2 cells, we showed that knockdown of PHLPP1 decreased the abundance of nuclear HK2, while inhibition of AKT increased HK2 in the nucleus. Finally, we tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed the selective over-expression of HK2 in the nucleus of these cells by immunoblotting and immunofluorescence. Increasing nuclear HK2 did not alter the proliferation of the cells under basal conditions. However, increasing nuclear HK2 enhanced clonogenic growth and blocked retinoic acid-mediated cell differentiation. In summary, we discovered that the unphosphorylated form of the metabolic enzyme HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells and is functionally important to maintain stem/progenitor state. Thus, we define a new role for mitochondrial enzymes in the regulation of stemness and differentiation. Disclosures Schimmer: Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy.