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1.
Nat Cell Biol ; 24(6): 872-884, 2022 06.
Article in English | MEDLINE | ID: mdl-35668135

ABSTRACT

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.


Subject(s)
Hexokinase , Leukemia, Myeloid, Acute , Chromatin/metabolism , DNA/metabolism , Hematopoietic Stem Cells/metabolism , Hexokinase/genetics , Hexokinase/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism
2.
Leukemia ; 36(5): 1283-1295, 2022 05.
Article in English | MEDLINE | ID: mdl-35152270

ABSTRACT

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 ß 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.


Subject(s)
Leukemia, Myeloid, Acute , beta Karyopherins , Active Transport, Cell Nucleus , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Humans , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/pathology , Stem Cells/metabolism , beta Karyopherins/genetics , beta Karyopherins/metabolism
3.
Blood ; 138(3): 234-245, 2021 07 22.
Article in English | MEDLINE | ID: mdl-34292323

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.


Subject(s)
Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Sulfonamides/pharmacology , T-Lymphocytes/drug effects , Adult , Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cells, Cultured , Humans , Immunity, Cellular/drug effects , Leukemia, Myeloid, Acute/immunology , Reactive Oxygen Species/immunology , Sulfonamides/therapeutic use , T-Lymphocytes/immunology , Tumor Cells, Cultured
4.
JCI Insight ; 6(5)2021 03 08.
Article in English | MEDLINE | ID: mdl-33476303

ABSTRACT

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.


Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Drug Resistance, Neoplasm , Enzyme Inhibitors , Gene Expression Regulation, Neoplastic , Leukemia, Myeloid, Acute , Neoplasm Proteins/metabolism , Pyrazoles , Pyrimidines , Repressor Proteins/metabolism , Sulfides , Sulfonamides , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , ATP-Binding Cassette Transporters , Animals , CRISPR-Cas Systems , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Genome , Humans , Leukemia, Myeloid, Acute/drug therapy , Male , Mice , Neoplasm Proteins/genetics , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Repressor Proteins/genetics , Sulfides/pharmacology , Sulfides/therapeutic use , Sulfonamides/pharmacology , Sulfonamides/therapeutic use
6.
STAR Protoc ; 1(3): 100163, 2020 12 18.
Article in English | MEDLINE | ID: mdl-33377057

ABSTRACT

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).


Subject(s)
Genetic Vectors/metabolism , Lentivirus/metabolism , Leukemia, Myeloid, Acute/genetics , Neoplasm Transplantation , Transduction, Genetic , Animals , Humans , Mice , Tumor Cells, Cultured
7.
Cell Stem Cell ; 26(6): 926-937.e10, 2020 06 04.
Article in English | MEDLINE | ID: mdl-32416059

ABSTRACT

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.


Subject(s)
Cell Self Renewal , Leukemia, Myeloid, Acute , Cell Differentiation , Copper , Humans , Neoplastic Stem Cells
8.
Blood ; 136(1): 81-92, 2020 07 02.
Article in English | MEDLINE | ID: mdl-32299104

ABSTRACT

Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.


Subject(s)
Leukemia, Myeloid, Acute/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/physiology , Neoplasm Proteins/physiology , Acetylation , Animals , CRISPR-Cas Systems , Cell Differentiation , Cell Line, Tumor , Cell Nucleus/metabolism , Fetal Blood/cytology , Gene Expression Regulation, Leukemic/genetics , Gene Knockdown Techniques , Histones/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Myeloid-Lymphoid Leukemia Protein/physiology , Oncogene Proteins, Fusion/physiology , Protein Processing, Post-Translational , Pyruvic Acid/metabolism , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology
9.
Sci Transl Med ; 12(538)2020 04 08.
Article in English | MEDLINE | ID: mdl-32269163

ABSTRACT

Neurolysin (NLN) is a zinc metallopeptidase whose mitochondrial function is unclear. We found that NLN was overexpressed in almost half of patients with acute myeloid leukemia (AML), and inhibition of NLN was selectively cytotoxic to AML cells and stem cells while sparing normal hematopoietic cells. Mechanistically, NLN interacted with the mitochondrial respiratory chain. Genetic and chemical inhibition of NLN impaired oxidative metabolism and disrupted the formation of respiratory chain supercomplexes (RCS). Furthermore, NLN interacted with the known RCS regulator, LETM1, and inhibition of NLN disrupted LETM1 complex formation. RCS were increased in patients with AML and positively correlated with NLN expression. These findings demonstrate that inhibiting RCS formation selectively targets AML cells and stem cells and highlights the therapeutic potential of pharmacologically targeting NLN in AML.


Subject(s)
Leukemia, Myeloid, Acute , Peptide Hydrolases , Electron Transport , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Metalloendopeptidases , Mitochondria/metabolism , Peptide Hydrolases/metabolism
10.
Proteomics ; 19(24): e1900139, 2019 12.
Article in English | MEDLINE | ID: mdl-31617661

ABSTRACT

A number of unique proteases localize to specific sub-compartments of the mitochondria, but the functions of these enzymes are poorly defined. Here, in vivo proximity-dependent biotinylation (BioID) is used to map the interactomes of seven proteases localized to the mitochondrial intermembrane space (IMS). In total, 802 high confidence proximity interactions with 342 unique proteins are identified. While all seven proteases co-localized with the IMS markers OPA1 and CLPB, 230 of the interacting partners are unique to just one or two protease bait proteins, highlighting the ability of BioID to differentiate unique interactomes within the confined space of the IMS. Notably, high-temperature requirement peptidase 2 (HTRA2) interacts with eight of 13 components of the mitochondrial intermembrane space bridging (MIB) complex, a multiprotein assembly essential for the maintenance of mitochondrial cristae structure. Knockdown of HTRA2 disrupts cristae in HEK 293 and OCI-AML2 cells, and leads to increased intracellular levels of the MIB subunit IMMT. Using a cell-free assay it is demonstrated that HTRA2 can degrade recombinant IMMT but not two other core MIB complex subunits, SAMM50 and CHCHD3. The IMS protease interactome thus represents a rich dataset that can be mined to uncover novel IMS protease biology.


Subject(s)
ATP-Dependent Proteases/metabolism , High-Temperature Requirement A Serine Peptidase 2/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Proteome/metabolism , High-Temperature Requirement A Serine Peptidase 2/antagonists & inhibitors , High-Temperature Requirement A Serine Peptidase 2/genetics , Humans , Membrane Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Protein Interaction Maps , RNA, Small Interfering/genetics
12.
Cancer Cell ; 35(5): 721-737.e9, 2019 05 13.
Article in English | MEDLINE | ID: mdl-31056398

ABSTRACT

The mitochondrial caseinolytic protease P (ClpP) plays a central role in mitochondrial protein quality control by degrading misfolded proteins. Using genetic and chemical approaches, we showed that hyperactivation of the protease selectively kills cancer cells, independently of p53 status, by selective degradation of its respiratory chain protein substrates and disrupts mitochondrial structure and function, while it does not affect non-malignant cells. We identified imipridones as potent activators of ClpP. Through biochemical studies and crystallography, we show that imipridones bind ClpP non-covalently and induce proteolysis by diverse structural changes. Imipridones are presently in clinical trials. Our findings suggest a general concept of inducing cancer cell lethality through activation of mitochondrial proteolysis.


Subject(s)
Endopeptidase Clp/genetics , Endopeptidase Clp/metabolism , Heterocyclic Compounds, 4 or More Rings/administration & dosage , Leukemia, Myeloid, Acute/drug therapy , Mitochondria/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Endopeptidase Clp/chemistry , Female , HCT116 Cells , HEK293 Cells , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Imidazoles , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Models, Molecular , Point Mutation , Protein Conformation/drug effects , Proteolysis , Pyridines , Pyrimidines , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
13.
Cell Stem Cell ; 24(4): 621-636.e16, 2019 04 04.
Article in English | MEDLINE | ID: mdl-30930145

ABSTRACT

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.


Subject(s)
Leukemia, Myeloid, Acute/metabolism , Mitochondria/enzymology , Phospholipids/metabolism , Transcription Factors/metabolism , Acyltransferases , Animals , Cell Line, Tumor , Doxorubicin/pharmacology , Female , Humans , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Signal Transduction/drug effects , Toll-Like Receptors/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/deficiency
14.
Leukemia ; 33(1): 37-51, 2019 01.
Article in English | MEDLINE | ID: mdl-29884901

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Leukemia, Myeloid, Acute/drug therapy , Nucleosides/pharmacology , Sulfonamides/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Xenograft Model Antitumor Assays , Animals , Cell Proliferation/drug effects , Endoplasmic Reticulum Stress/drug effects , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/pathology , Mice , Mice, SCID , Pyrazoles , Pyrimidines , Sulfides , Tumor Cells, Cultured
15.
Haematologica ; 104(5): 963-972, 2019 05.
Article in English | MEDLINE | ID: mdl-30573504

ABSTRACT

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.


Subject(s)
Cell Differentiation/drug effects , DNA Polymerase gamma/antagonists & inhibitors , Dideoxynucleosides/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Mitochondria/pathology , Monocytes/pathology , Oxidative Phosphorylation/drug effects , Animals , Antiviral Agents/pharmacology , Apoptosis , Cell Proliferation , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Mitochondria/drug effects , Mitochondria/metabolism , Monocytes/drug effects , Monocytes/metabolism , Thymidine/chemistry , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
16.
Blood ; 129(19): 2657-2666, 2017 05 11.
Article in English | MEDLINE | ID: mdl-28283480

ABSTRACT

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.


Subject(s)
DNA, Mitochondrial/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Oxidative Phosphorylation , Phosphotransferases/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , DNA Replication , Humans , Mice, SCID , NM23 Nucleoside Diphosphate Kinases/metabolism , Nucleoside-Phosphate Kinase/metabolism , Signal Transduction , Tumor Cells, Cultured , Zalcitabine/metabolism
17.
Mitochondrion ; 32: 31-35, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27845271

ABSTRACT

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.


Subject(s)
Carrier Proteins/analysis , DNA Helicases/analysis , DNA-Directed DNA Polymerase/metabolism , Mitochondria/chemistry , Protein Interaction Mapping , ATPases Associated with Diverse Cellular Activities , DNA Polymerase gamma , Humans
19.
Oncotarget ; 7(31): 49777-49785, 2016 Aug 02.
Article in English | MEDLINE | ID: mdl-27391350

ABSTRACT

The isoflavone ME-344 is a potent anti-cancer agent with preclinical and clinical efficacy in solid tumors. Yet, the mechanism of action of ME-344 has not been fully defined and the preclinical efficacy in leukemia has not been established. Therefore, we investigated the anti-leukemic properties and mechanism of action of ME-344. In a panel of 7 leukemia cell lines, ME-344 was cytotoxic with an IC50 in the range of 70-260 nM. In addition, ME-344 was cytotoxic to primary AML patient samples over normal hematopoietic cells. In an OCI-AML2 xenograft model, ME-344 reduced tumor growth by up to 95% of control without evidence of toxicity. Mechanistically, ME-344 increased mitochondrial ROS generation in leukemic cells. However, antioxidant treatment did not rescue cell death, suggesting that ME-344 had additional targets beyond the mitochondria. We demonstrated that ME-344 inhibited tubulin polymerization by interacting with tubulin near the colchicine-binding site. Furthermore, inhibition of tubulin polymerization was functionally important for ME-344 induced death. Finally, we showed that ME-344 synergizes with vinblastine in leukemia cells. Thus, our study demonstrates that ME-344 displays preclinical efficacy in leukemia through a mechanism at least partly related to targeting tubulin polymerization.


Subject(s)
Cytoskeleton/drug effects , Isoflavones/pharmacology , Leukemia, Myeloid, Acute/metabolism , Animals , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Binding Sites , Cell Proliferation , Cell Survival , Cytoskeleton/metabolism , Gene Expression Regulation, Leukemic , HL-60 Cells , Humans , Inhibitory Concentration 50 , Leukemia, Myeloid, Acute/drug therapy , Male , Mice , Mice, SCID , Microtubules/metabolism , Mitochondria/metabolism , Neoplasm Transplantation , Protein Binding , Reactive Oxygen Species/metabolism , Tubulin/chemistry
20.
Oncotarget ; 7(3): 2765-79, 2016 Jan 19.
Article in English | MEDLINE | ID: mdl-26624983

ABSTRACT

Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease.


Subject(s)
Apoptosis/drug effects , Ethacridine/pharmacology , Glycoside Hydrolases/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/genetics , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Animals , Cell Line, Tumor , Drug Synergism , Drug Therapy, Combination , Humans , Hydrolyzable Tannins/pharmacology , Jurkat Cells , Mice , Mice, SCID , Piperidines , RNA Interference , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism
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