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Thomas, Egan et al. report that hexokinase 2 localizes to the nucleus of leukaemic and normal haematopoietic cells to maintain stemness by interacting with nuclear proteins and modulating chromatin accessibility independently of its kinase activity.Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.

Supplementary Figure Legends 1-4 from Selective Inhibition of Histone Deacetylases Sensitizes Malignant Cells to Death Receptor Ligands

Supplementary Figure 2 from Selective Inhibition of Histone Deacetylases Sensitizes Malignant Cells to Death Receptor Ligands

Supplementary Figure 3 from Selective Inhibition of Histone Deacetylases Sensitizes Malignant Cells to Death Receptor Ligands

Supplementary Figure 4 from Selective Inhibition of Histone Deacetylases Sensitizes Malignant Cells to Death Receptor Ligands

Supplementary Figures S1-S9. Avocatin B is the most active avocado lipid analogue (S1); Kinetics of avocatin B-induced death (S2); Cell cycle analysis of avocatin B treated TEX cells (S3); Avocatin B increases DCFH-DA and DHE (S4); Avocatin B's activity is neutralized by co-incubation with PEG-SOD (S5); Jurkat T cells cultured in ethidium bromide medium have reduced mitochondria with decreased function (S6); Avocatin B is absent in mitochondria and cytosolic fractions of vehicle control treated TEX cells (S7); Avocatin B calibration curves and estimated concentraiton in cytosolic and mitochondrial fractions (S8); Avocatin B decreases levels of NADH and NADPH and increases ROS in OCI-AML2 cells (S9).

Legend for Supplementary Figures S1-S9.

Normal epithelial cells undergo apoptosis upon detachment from the extracellular matrix, a process termed “anoikis.” However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. Molecules that sensitize resistant cells to anoikis will be useful chemical probes to understand this pathway. To identify novel anoikis sensitizers in anoikis-resistant PPC-1 prostate adenocarcinoma cells, a library of 2,000 off-patent drugs and natural products was screened for their ability to preferentially induce cell death in suspension over adherent culture conditions. This screen identified five members of the family of cardiac glycosides as anoikis sensitizers, including ouabain, peruvoside, digoxin, digitoxin, and strophanthidin. We conducted further studies with ouabain to discern the mechanism of cardiac glycoside-induced anoikis sensitization. Ouabain initiated anoikis through the mitochondrial pathway of caspase activation. In addition, ouabain sensitized cells to anoikis by inhibiting its known target, the Na+/K+ ATPase pump, and inducing hypoosmotic stress. Resistance to anoikis permits cancer cells to survive in the circulation and facilitates their metastasis to distant organs, so we tested the effects of Na+/K+ ATPase inhibition on distant tumor formation in mouse models. In these mouse models, ouabain inhibited tumor metastases but did not alter the growth of subcutaneous tumors. Thus, we have identified a novel mechanism to sensitize resistant cells to anoikis and decrease tumor metastasis. These results suggest a potential mechanism for the observed clinical reduction in metastasis and relapse in breast cancer patients who have undergone treatments with cardiac glycosides. [Cancer Res 2009;69(7):2739–47]

Supplementary Table 1 from Identification of Small Molecules that Sensitize Resistant Tumor Cells to Tumor Necrosis Factor-Family Death Receptors

Supplementary Figure 3 from Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

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Supplementary Tables S1-S3. Summary of EC50 values for avocatin B in a panel of AML cell lines (S1); AML patient sample details used for annexin/PI (S2); AML patient sample details used for colony formation assays (S3).

Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/β-catenin signaling, we identified a series of acyl hydrazones that act downstream of the β-catenin destruction complex to inhibit both Wnt-induced and cancer-associated constitutive Wnt signaling via destabilization of β-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans. Cancer Res; 71(24); 7628–39. ©2011 AACR.

Supplementary Figure 1 from Identification of Small Molecules that Sensitize Resistant Tumor Cells to Tumor Necrosis Factor-Family Death Receptors

Supplementary Figure 2 from Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

Supplementary Figure 1 from Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

Two major pathways for apoptosis have been identified, involving either mitochondria (intrinsic) or tumor necrosis factor (TNF)-family death receptors (extrinsic) as initiators of caspase protease activation and cell death. Because tumor resistance to TNF-family death receptor ligands is a common problem, helping malignant cells evade host immune defenses, we sought to identify compounds that selectively sensitize resistant tumor cells to death receptor ligands. We screened a 50,000-compound library for agents that enhanced anti-FAS antibody–mediated killing of FAS-resistant PPC-1 prostate cancer cell, then did additional analysis of the resulting hits to arrive at eight compounds that selectively sensitized PPC-1 cells to anti-FAS antibody (extrinsic pathway agonist) without altering sensitivity to staurosporine and etoposide (VP-16; intrinsic pathway agonists). These eight compounds did not increase Fas surface levels and also sensitized PPC-1 cells to apoptosis induced by TNF-family member TNF-related apoptosis-inducing ligand, consistent with a post-receptor mechanism. Of these, two reduced expression of c-FLIP, an intracellular antagonist of the extrinsic pathway. Characterization of the effects of the eight compounds on a panel of 10 solid tumor cell lines revealed two structurally distinct compounds that frequently sensitize to extrinsic pathway agonists. Structure-activity relation studies of one of these compounds revealed a pharmacophore from which it should be possible to generate analogues with improved potency. Altogether, these findings show the feasibility of identifying compounds that selectively enhance apoptosis via the extrinsic pathway, thus providing research tools for uncovering resistance mechanisms and a starting point for novel therapeutics aimed at restoring sensitivity of tumor cells to immune effector mechanisms. (Cancer Res 2006; 66(4): 2367-75)

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin β family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.

Eukaryotic cells have two separate genomes; nuclear chromosomal DNA and circular mitochondrial DNA. Mitochondrial DNA lacks introns, and encodes 2 rRNAs, 22 t-RNAs and 13 of the 90 proteins that constitute the mitochondrial respiratory chain. To maintain homeostasis, mitochondrial RNA degradation machinery regulates RNA turnover. ATP-dependent helicase, SUV3 (gene SUPV3L1), and exonuclease PNPase (gene PNPT1) function as a complex to degrade mitochondrial dsRNA. By immunoblotting, PNPase and SUV3 proteins were increased in 7/7 AML patient samples and 13/13 of AML cell lines, compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall and event free survival. Genetic knockdown or knockout using shRNA or sgRNA against PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML 8227 cells. Furthermore, SUPV3L1 & PNPT1 ranked in the top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (depmap.org). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells and significantly reduced engraftment of TEX cells into the marrow of immune deficient mice, demonstrating the functional importance on leukemia initiating cells in vivo. SUPV3L1 knockdown in primary AML cells reduced engraftment in marrow of immune deficient mice. Bioinformatics analysis to detect processes associated with PNPT1 and SUPV3L1, we identified associations with Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies. Consistent with this, we observed knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) suggesting an interferon response. As PNPT1 and SUPV3L1 degrade mitochondrial dsRNA, we measured levels of dsRNA after knockdown of these genes. Knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic/mitochondrial dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased dsRNA in 143B cells, but not Rho (0) 143B cells that lack mitochondrial DNA. Upregulation of inflammatory genes leads to viral mimicry and can increase sensitivity to immune mediated killing. We observed enhanced sensitivity to Double Negative T (DNT) cells mediated killing in PNPT1 and SUPV3L1 knockdown OCI-AML2 cells compared to control cells. In summary, RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML, and are essential for the survival of AML cells and AML stem/progenitors. These enzymes regulate levels of mitochondrial dsRNA, and their inhibition leads to increased cytoplasmic dsRNA triggering a viral mimicry response and enhanced sensitivity to immune-mediated killing. Citation Format: Geethu Emily Thomas, Kazem Nouri, Jong Bok Lee, Rose Hurren, Yongran Yan, Neil MacLean, Yulia Jitkova, Li Ma, Xiao Ming Wang, Chaitra Sarathy, Andrea Arruda, Mark D. Minden, Li Zhang, Vito Spadavecchio, Aaron Schimmer. Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4833.

In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit., Molecular heterogeneity of acute myeloid leukaemia (AML) across patients is a major challenge for prognosis and therapy. Here, the authors show that NPM1 mutated AML is a heterogeneous class, consisting of two subtypes which exhibit distinct molecular characteristics, differentiation state, patient survival and drug response.

Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine in vivo, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.

The combination of venetoclax and the hypomethylating agent azacytidine induces a high rate of complete responses in treatment-naïve older patients with acute myeloid leukemia (AML). Lee and colleagues elucidate a novel mechanism of action for the synergy of these agents, demonstrating that venetoclax enhances reactive oxygen species (ROS) generation and enhances T-cell effector function while azacytidine renders AML cells more susceptible to T cell–mediated cytotoxicity., Key Points A Bcl-2 inhibitor, venetoclax, increases the effector activity of antileukemic T cells without inducing T-cell apoptosis.Venetoclax increases T-cell effector function in a reactive oxygen species–dependent manner., Visual Abstract, Venetoclax, a Bcl-2 inhibitor, in combination with the hypomethylating agent azacytidine, achieves complete remission with or without count recovery in ∼70% of treatment-naive elderly patients unfit for conventional intensive chemotherapy. However, the mechanism of action of this drug combination is not fully understood. We discovered that venetoclax directly activated T cells to increase their cytotoxicity against acute myeloid leukemia (AML) in vitro and in vivo. Venetoclax enhanced T-cell effector function by increasing reactive oxygen species generation through inhibition of respiratory chain supercomplexes formation. In addition, azacytidine induced a viral mimicry response in AML cells by activating the STING/cGAS pathway, thereby rendering the AML cells more susceptible to T cell–mediated cytotoxicity. Similar findings were seen in patients treated with venetoclax, as this treatment increased reactive oxygen species generation and activated T cells. Collectively, this study presents a new immune-mediated mechanism of action for venetoclax and azacytidine in the treatment of AML and highlights a potential combination of venetoclax and adoptive cell therapy for patients with AML.

TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 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 confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP 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.

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.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.

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.

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.

Human mitochondrial DNA (mtDNA) is replicated by the mitochondrial DNA polymerase gamma (POLG). Using proximity dependent biotin labelling (BioID), we characterized the POLG interactome and identified new interaction partners involved in mtDNA maintenance, transcription, translation and protein quality control. We also identified interaction with the nuclear AAA+ ATPase Ruvbl2, suggesting mitochondrial localization for this protein. Ruvbl2 was detected in mitochondria-enriched fractions in leukemic cells. Additionally, transgenic overexpression of Ruvbl2 from an alternative translation initiation site resulted in mitochondrial co-localization. Overall, POLG interactome mapping identifies novel proteins which support mitochondrial biogenesis and a potential novel mitochondrial isoform of Ruvbl2.

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 low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. 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. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 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 (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of 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 total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. 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. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .