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.
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 cytotoxic nucleoside cytarabine (Ara-C) is a cornerstone of AML induction and consolidation therapies, but drug resistance contributes to disease relapse. Among clinically relevant mechanisms of Ara-C resistance is the over-expression of cytidine deaminase (CDA). CDA degrades Ara-C through deamination into inactive metabolites and its increased expression in AML cells results in drug resistance. Therefore, nucleoside analogues that are not degraded by CDA may be new therapeutic agents for this disease. Through our efforts to develop novel nucleoside analogues that overcome mechanisms of resistance to Ara-C, we identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside. First, we tested whether 5FTRX was a substrate of CDA by evaluating its cytotoxicity in HEK-293 cells over-expressing CDA. Compared to wild type cells, Ara-C was 6-fold less active in cells over-expressing CDA (IC50 wild type HEK293: 3.7µM (95% CI: 3.2-4.2uM) vs IC50 CDA over-expression: 25.6uM (95% CI 21.6-30.3uM), consistent with degradation of Ara-C by CDA. In contrast, cells over-expressing CDA were more sensitive to 5FTRX, compared to wild type cells (IC50 wild type HEK293: 5.5uM (95% CI 4.7-6.3uM) vs IC50 CDA over-expression: 0.4uM (95% CI 0.4-0.5uM), potentially due to depletion of endogenous nucleotide pools by increased CDA. Thus, 5FTRX is not a substrate of CDA. We then treated 6 AML cell lines for 72 hours with increasing concentrations of 5FTRX and then measured cell growth and viability using the MTS assay. 5FTRX reduced the growth of 5 of 6 tested AML cell lines, with mean IC50 values (n= > 3) of 92nM (TEX), 130nM (KG1a), 150nM (MV4-11), 410nM (NB4), and 250nM (OCI-AML2). In contrast, K562 cells that have mutant p53 were resistant with an IC50 >50,000nM. The K562 cells were also resistant to Ara-C. 5FTRX reduced the clonogenic growth of primary AML samples (n=2) with a >90% reduction in growth at 50nM, demonstrating that 5FTRX targets leukemia initiating cells. To test whether 5FTRX induced DNA damage in AML cells, we measured changes in phosphorylated H2AX (pH2AX) after 5FTRX treatment. In the tested cell lines (OCI-AML2, TEX, NB4 and MV4-11 cells), 5FTRX increased pH2AX at concentrations associated with loss of viability. Finally, we evaluated the efficacy and toxicity of 5FTRX in mouse models of AML. 5FTRX displayed robust and dose-dependent inhibition of MV4-11 and OCI-AML2 tumors in mouse xenograft models, with complete tumor regressions and long-lasting tumor growth delays (>20 days at 100mg/kg in MV4-11 and >30 days in OCI-AML2) after a single cycle of 5 days of once-daily drug treatment, with no changes in body weight or behavior. The efficacy of 5FTRX was superior to Ara-C dosed for daily for 10 days at its MTD, 60 mg/kg (35%TGI, no regressions). MV4-11 tumors treated with 5FTRX displayed induction of pH2AX, reduction in proliferation (BrdU incorporation) and induction of necrosis, consistent with the mode of action and dramatic tumor regressions. The anti-leukemic effects of 5FTRX were further evaluated in mice engrafted intrafemorally with primary AML cells. 5FTRX (100 mg/kg i.p, x 5 days) reduced primary AML engraftment >95% compared to controls without toxicity (Fig 1). In summary, 5FTRX was identified as a potent inhibitor of AML cell proliferation in vitro and in vivo. In contrast to Ara-C, 5FTRX was not a substrate for CDA. Further development of analogues of 5FTRX is ongoing using protide prodrugs of the 5FTRX monophosphate to further increase potency and evade additional resistance mechanisms. Taken together, our findings support the further development of 5FTRX-based therapies for the treatment of AML, including AML patients with reduced sensitivity to Ara-C through high CDA expression. #AB and TPS contributed equally to this work. #ADS and MA contributed equally to this work. Disclosures Rizoska: Medivir AB: Employment, Equity Ownership. Rydergård:Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Eneroth:Medivir AB: Employment, Equity Ownership. Pinho:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership. Schimmer:Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership.
The cytotoxic nucleoside cytarabine forms the backbone of AML induction and consolidation therapies, but is associated with severe toxicities that preclude its use in patients unable to tolerate aggressive chemotherapy. Options for patients that do not respond to cytarabine, or relapse post-treatment, are limited. Elderly patients and those with relapsed/refractory AML would particularly benefit from the availability of new agents to develop treatment regimens that provide increased efficacy and tolerability compared to cytarabine, and that have a decreased susceptibility to mechanisms of cytarabine resistance, such as decreased deoxycytidine kinase (dCK) and/or upregulation of cytidine deaminase (CDA). Our preclinical evaluation of potential new anti-proliferative chemotherapeutics identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside, as having promising anti-proliferative activity against AML cell lines, and resistance to degradation by CDA. To understand potential mechanisms of resistance to 5FTRX, we selected a population of THP1 (THP1-R) cells resistant to 5FTRX. THP1-R cells were 66-fold resistant to 5FTRX and cross resistant to cytarabine (35-fold) with CC50 values for both nucleosides >50 μM. We discovered that THP1-R cells had decreased levels of dCK (>95% by Western blot), the kinase responsible for the phosphorylation of cytidine and cytidine analogues such as troxacitabine and cytarabine to their corresponding monophosphates. Confirming the importance of dCK in the activation of 5FTRX and cytarabine, chemical inhibition of dCK also rendered THP1 cells >90-fold resistant to 5FTRX and cytarabine. To develop molecules that overcome resistance to both high CDA and low dCK, we used protide technology to construct nucleotide monophosphate prodrugs of 5FTRX, including one potent example, MV806. MV806 was not dependent upon dCK as it maintained similar efficacy in THP1-R cells with low dCK and against THP1 cells treated with the selective dCK inhibitor. We tested MV806 and 5FTRX in a panel of AML cell lines (n=7). MV806 was more potent than 5FTRX with CC50 values ranging from 0.0020-0.19 μM, compared to 0.057-1.2 μM for 5FTRX. MV806 also demonstrated CC50s <0.1 μM against selected T- and B-cell lymphoma cell lines (e.g. MOLT4 and RAJI). Increased in vitro potency of this prodrug compared to 5FTRX correlated with elevated intracellular triphosphate levels in AML cells; MV806 generated 5-fold more triphosphate than 5FTRX in MV4-11 cells. We also tested MV806 in combination with doxorubicin or azacytidine in two AML cell lines (MV4-11 and THP-1). In both tested cell lines, strong synergy was observed (Bliss independence analysis synergy volumes >100), demonstrating future opportunities for clinical combinations. Finally, we showed that MV806 had DMPK profiles suitable for preclinical and clinical development. Leading protides were highly soluble, had a predicted half-life of >6h in human blood and demonstrated IC50 values >1 μM against major CYP isoforms (2A6, 2C9, 2D6, 3A4) with no evidence of time-dependent inhibition at 1 μM. To conclude, we used protide technology to directly deliver the active monophosphate species of 5FTRX intracellularly and thereby overcome resistance to cytarabine due to down-regulation of dCK and increased CDA expression. Taken together, our findings support the further development of protides of 5FTRX for the treatment of AML, including AML patients with reduced sensitivity to cytarabine through high CDA expression and/or low dCK expression. Disclosures Pinho: Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Rydergård:Medivir AB: Employment, Equity Ownership. Rizoska:Medivir AB: Employment, Equity Ownership. Eneroth:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Schimmer:Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership.
In an initial patient subset (n=80), a 39% discordance rate was identified between BICR and investigator-assessed PD by the sponsor, most commonly due to peritoneal carcinomatosis or fluid collections arising from new non-target lesions. After reviewer intervention, final discordance rate between BICR and investigator improved to 12% and 13% for ITT (N=733) and HRd (n=373) populations, respectively (figure 1). Across the entire study population, median PFS and hazard ratios for the ITT and HRd populations were comparable between BICR and investigator (table 1). Conclusions PRIMA/ENGOT-ov26/GOG-3012 highlights the need to optimize BICR and investigator concordance using early, specialized OC-specific training to maximize trial validity. Funding: GlaxoSmithKline (GSK) study. Editorial support provided by Fishawack Health, funded by GSK.
Mitochondrial DNA (mtDNA) replication requires adequate nucleotide pools from the mitochondria and cytoplasm to support DNA biosynthesis. Gene expression profiling of 542 AML patient samples (GSE13159) demonstrated that 55% of AML patients had upregulated mtDNA biosynthesis pathway expression compared to 73 normal hematopoietic cells (mononuclear cells isolated from peripheral blood and bone marrow). We also identified upregulation of pathways which support mitochondrial nucleotide pools, which include mitochondrial nucleotide transporters and a subset of cytoplasmic nucleotide salvage enzymes, which phosphorylate nucleosides to nucleotides. Upregulation of nucleoside kinases in a subset of primary AML samples compared to normal hematopoietic progenitor cells (normal G-CSF (granulocyte-colony stimulating factor) mobilized peripheral blood stem cells (PBSC's)) was confirmed by immunoblotting. These results suggest that AML cells import cytoplasmic nucleotides to support mitochondrial DNA biogenesis. To determine if cytoplasmic nucleoside kinases regulate mtDNA content, we knocked down nucleoside kinases in AML cells. Partial target knockdown of DCK (deoxycytidine kinase) and CMPK1 (cytidine/uridine monophosphate kinase 1) reduced mtDNA content (60+8%, and 62+13%, respectively compared to controls, 5 and 7 days post-shRNA transduction), indicating a role in mtDNA biogenesis. As expected, knockdown of mtDNA replication factors POLG and TFAM reduced mtDNA content in AML cells. The cytidine nucleoside analog, 2'3'-dideoxycytidine (ddC) is activated by DCK and CMPK1 to produce its triphosphate form, ddC-triphosphate (ddC-TP). To assess nucleoside kinase activity, primary AML and normal hematopoietic cells were treated with ddC and total levels of ddC and ddC-TP were measured by mass spectrometry. Levels of ddC did not differ between AML and normal, but ddC-TP levels was increased in AML samples > 7-fold compared to normal (p< 0.05, one-way ANOVA). Previously we and others demonstrated that AML cells and stem cells have increased mitochondrial biogenesis and reliance on oxidative phosphorylation due to decreased spare reserve capacity and an inability to upregulate glycolysis. ddC-TP inhibits the sole mtDNA polymerase POLG, but not nuclear DNA polymerases. Given the increased activity of nucleoside kinases in AML cells over normal, we examined the effects of ddC treatment on mtDNA content and cellular bioenergetics. AML and normal cells were treated with increasing concentrations of ddC. At increasing times after treatment, ddC depleted mtDNA levels > 85% at 0.5 uM, 3 day treatment in OCI-AML2 and TEX cells as assessed by qPCR. ddC decreased protein expression of mtDNA encoded electron transport chain (ETC) subunits COXI and COX II, but not nuclear encoded subunit COXIV) and reduced basal oxygen consumption. ddC also decreased proliferation of AML cell lines (OCI-AML2, TEX, HL-60, K562) (> 95% reduction at 0.5uM, 10 days). Knockdown of DCK abrogated the effects of ddC on AML cell proliferation. We next examined the effects of ddC in primary human leukemia cells (AML = 7, CML blast crisis = 1, CMML-2 = 1) and normal hematopoietic progenitor cells (n=8). ddC preferentially inhibited mtDNA biosynthesis and reduced viability in a subset of primary cells (6 of 9 AML) compared to normal PBSC's (n=8). Sensitivity to ddC positively correlated with mtDNA depletion. Finally, we evaluated the efficacy and toxicity of ddC in mouse models of human AML. ddC (35 mg/kg daily i.p. x 11 days) caused tumor regression in an OCI-AML2 xenograft model without toxicity (changes body weight, behavior, serum chemistries). In OCI-AML2 cells isolated from treated mice, ddC reduced mtDNA by 95% and mtDNA-encoded ETC proteins by 90%. In addition, ddC (75 mg/kg i.p x 6 weeks) significantly reduced human AML bone marrow engraftment in primary AML (n=2, P<0.0001, n=1,P<0.05, t-test) and secondary AML (n=1, 14 vs 3%, P<0.01, t-test) without toxicity. Thus, AML cells have increased nucleoside kinase activity that is functionally important for mtDNA biogenesis. We leveraged this unique biological vulnerability to preferentially activate ddC, deplete mtDNA and selectively target oxidative phosphorylation in AML. Disclosures Schimmer: Novartis: Honoraria.
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