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The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of “apoptosis-associated speck-like protein containing a CARD” (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.

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.

Summary Cockayne syndrome (CS) is a severe neurodevelopmental disorder characterized by growth abnormalities, premature aging, and photosensitivity. Mutation of Cockayne syndrome B (CSB) affects neuronal gene expression and differentiation, so we attempted to bypass its function by expressing downstream target genes. Intriguingly, ectopic expression of Synaptotagmin 9 (SYT9), a key component of the machinery controlling neurotrophin release, bypasses the need for CSB in neuritogenesis. Importantly, brain-derived neurotrophic factor (BDNF), a neurotrophin implicated in neuronal differentiation and synaptic modulation, and pharmacological mimics such as 7,8-dihydroxyflavone and amitriptyline can compensate for CSB deficiency in cell models of neuronal differentiation as well. SYT9 and BDNF are downregulated in CS patient brain tissue, further indicating that sub-optimal neurotrophin signaling underlies neurological defects in CS. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for pharmacological intervention, these data suggest an important role for SYT9 in neuronal differentiation., Graphical Abstract, Highlights • Neuritogenesis defects in CS cell lines can be overcome by overexpression of SYT9 • SYT9 is crucial for neuritogenesis and involved in neurotrophin (BDNF) signaling • Neuritogenesis defects in CS cell lines can be overcome by BDNF treatment • They can also be overcome by treatment with amitriptyline, an FDA-approved BDNF mimic, Wang et al. show that Cockayne syndrome cell lines have defects in gene regulatory loops, resulting in sub-optimal neurotrophin signaling and explaining their defects in neurogenesis. These defects can be overcome by Synaptotagmin 9 overexpression or by treatment with NTRK2 (TrkB) agonists, pointing to future disease intervention.

Dysregulated metabolism is a hallmark of cancer and a substantial subset of many cancers rely on oxidative phosphorylation (OXPHOS) for growth and viability, including AML, breast cancer, lung cancer, glioblastoma, ovarian and pancreatic cancer. The electron transport chain (ETC) in mitochondria contains both nuclear DNA- and mitochondrial DNA-encoded subunits. Mitochondrial DNA-encoded subunits are essential for ETC function and are transcribed by the mitochondrial RNA polymerase POLRMT. We have previously validated POLRMT as an anticancer target in acute myeloid leukemia models. The challenge of inhibiting POLRMT with small molecules will arise from both a) selectivity to off targets and b) transport of the drug to the mitochondria. There exist several small nucleoside / nucleotide analogues that are known to inhibit RNA polymerase. However, this target space is rife with the opportunity for off-target activities and toxicity. Therefore, we targeted a novel pocket with the aim of finding novel chemical matter. Using AtomNet, an artificial intelligence platform that utilizes deep convolutional neural networks to predict small molecule recognition, a virtual library of 8 million compounds was screened against POLRMT. The top 72 hit compounds were tested in OCI-AML2 human acute myeloid leukemia cells for a potential decrease of mitochondrial RNA levels (MT-ND1) using reverse transcriptase polymerase chain reaction (RT-PCR). OCI-AML2 cells were treated with compounds at 0.1, 1 or 10 micromolar for 72 hours, 3 days or 6 days or with the positive control POLRMT ribonucleoside analogue inhibitor, 4'-azidocytidine. Gene expression was normalized to 18S. Twelve compounds demonstrated a time-dependent decrease in mitochondrial gene expression. In summary, these data indicate that functional inhibitors of human POLRMT can be identified rapidly via deep convolutional neural networks and machine learning techniques. Financial relationships: RL received an Artificial Intelligence Molecular Screening Award from Atomwise, and funding from the Centre for Collaborative Drug Research, Toronto, Canada. JW is an employee of Atomwise. Citation Format: Noor-Ul-Ain Khalid, Jeffrey M. Warrington, Rebecca Rachelle Laposa. Identification of mitochondrial RNA polymerase inhibitors via deep convolutional neural networks: A therapeutic strategy for cancer metabolism [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 6361.

Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3- d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3- d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2',4,5]pyrrolo[2,3- d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

Replication and maintenance of mitochondrial DNA (mtDNA) is essential for cellular function, yet few DNA polymerases are known to function in mitochondria. Here, we conclusively demonstrate that DNA polymerase θ (Polθ) localizes to mitochondria and explore whether this protein is overexpressed in patient-derived cells and tumors. Polθ appears to play an important role in facilitating mtDNA replication under conditions of oxidative stress, and this error-prone polymerase was found to introduce mutations into mtDNA. In patient-derived cells bearing a pathogenic mtDNA mutation, Polθ expression levels were increased, indicating that the oxidative conditions in these cells promote higher expression levels for Polθ. Heightened Polθ expression levels were also associated with elevated mtDNA mutation rates in a selected panel of human tumor tissues, suggesting that this protein can influence mutational frequencies in tumors. The results reported indicate that the mitochondrial function of Polθ may have relevance to human disease.

Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.

Mitochondria contain multiple copies of their own 16.6 kb circular genome. To explore the impact of mitochondrial DNA (mtDNA) damage on mitochondrial (mt) function and viability of AML cells, we screened a panel of DNA damaging chemotherapeutic agents to identify drugs that could damage mtDNA. We identified bleomycin as an agent that damaged mtDNA in AML cells at concentrations that induced cell death. Bleomycin also induced mtDNA damage in primary AML samples. Consistent with the observed mtDNA damage, bleomycin reduced mt mass and basal oxygen consumption in AML cells. We also demonstrated that the observed mtDNA damage was functionally important for bleomycin-induced cell death. Finally, bleomycin delayed tumor growth in xenograft mouse models of AML and anti-leukemic concentrations of the drug induced mtDNA damage in AML cells preferentially over normal lung tissue. Taken together, mtDNA-targeted therapy may be an effective strategy to target AML cells and bleomycin could be useful in the treatment of this disease.

An analogue of the anticancer drug cisplatin (mtPt) was delivered to mitochondria of human cells using a peptide specifically targeting this organelle. mtPt induces apoptosis without damaging nuclear DNA, indicating that mtDNA damage is sufficient to mediate the activity of a platinum-based chemotherapeutic. This study is the first to demonstrate specific delivery of a platinum drug to mitochondria and to investigate the effects of directing this agent outside the nucleus.

Neurodevelopmental defects are observed in the hereditary disorder Cockayne syndrome (CS). The gene most frequently mutated in CS, Cockayne Syndrome B (CSB), is required for the repair of bulky DNA adducts in transcribed genes during transcription-coupled nucleotide excision repair. CSB also plays a role in chromatin remodeling and mitochondrial function. The role of CSB in neural development is poorly understood. Here we report that the abundance of neural progenitors is normal in Csb(-/-) mice and the frequency of apoptotic cells in the neurogenic niche of the adult subependymal zone is similar in Csb(-/-) and wild type mice. Both embryonic and adult Csb(-/-) neural precursors exhibited defective self-renewal in the neurosphere assay. In Csb(-/-) neural precursors, self-renewal progressively decreased in serially passaged neurospheres. The data also indicate that Csb and the nucleotide excision repair protein Xpa preserve embryonic neural stem cell self-renewal after UV DNA damage. Although Csb(-/-) neural precursors do not exhibit altered neuronal lineage commitment after low-dose UV (1J/m(2)) in vitro, neurons differentiated in vitro from Csb(-/-) neural precursors that had been irradiated with 1J/m(2) UV exhibited defective neurite outgrowth. These findings identify a function for Csb in neural precursors.

Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.

Developmental pathologies may result from endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS), which oxidatively damage cellular macromolecules and/or alter signal transduction. This minireview focuses upon several model drugs (phenytoin, thalidomide, methamphetamine), environmental chemicals (benzo[a]pyrene) and gamma irradiation to examine this hypothesis in vivo and in embryo culture using mouse, rat and rabbit models. Embryonic prostaglandin H synthases (PHSs) and lipoxygenases bioactivate xenobiotics to free radical intermediates that initiate ROS formation, resulting in oxidation of proteins, lipids and DNA. Oxidative DNA damage and embryopathies are reduced in PHS knockout mice, and in mice treated with PHS inhibitors, antioxidative enzymes, antioxidants and free radical trapping agents. Thalidomide causes embryonic DNA oxidation in susceptible (rabbit) but not resistant (mouse) species. Embryopathies are increased in mutant mice deficient in the antioxidative enzyme glucose-6-phosphate dehydrogenase (G6PD), or by glutathione (GSH) depletion, or inhibition of GSH peroxidase or GSH reductase. Inducible nitric oxide synthase knockout mice are partially protected. Inhibition of Ras or NF-kB pathways reduces embryopathies, implicating ROS-mediated signal transduction. Atm and p53 knockout mice deficient in DNA damage response/repair are more susceptible to xenobiotic or radiation embryopathies, suggesting a teratological role for DNA damage, consistent with enhanced susceptibility to methamphetamine in ogg1 knockout mice with deficient repair of oxidative DNA damage. Even endogenous embryonic oxidative stress carries a risk, since untreated G6PD- or ATM-deficient mice have increased embryopathies. Thus, embryonic processes regulating the balance of ROS formation, oxidative DNA damage and repair, and ROS-mediated signal transduction may be important determinants of teratological risk.

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 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.

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

ATM (ataxia-telangiectasia mutated) is a genotoxic stress transducer. In this first report of Atm-dependent birth defects, Atm-null embryos were uniquely susceptible to low-dose (0.5 Gy) radiation, exhibiting severe runting, tail anomalies, and lethality, independent of cell cycle arrest or insulin-like growth factor 1. This treatment enhanced levels of p53 protein and central nervous system (CNS) apoptosis in wild-type mice, but not Atm-null mutants, at 6 h postirradiation. At 48 h, however, this pattern was reversed, with Atm-null mice exhibiting high levels of a hybrid form of programmed cell death within the CNS. Even heterozygous Atm-deficient embryos were radiosensitive to a higher radiation dose of 2 Gy. These results show that Atm is a novel teratologic suppressor gene protecting embryos from pathological cell death and teratogenesis initiated by even mild DNA damage.

A subset of AML cells display unique mitochondrial characteristics such as increased mitochondrial DNA (mtDNA), mt mass, and sensitivity to inhibition of mtDNA transcription and protein translation. Given these unique mitochondrial characteristics, we evaluated the effect of inhibiting mtDNA replication by targeting the mtDNA polymerase gamma (POLG). POLG is a nuclear-encoded gene that replicates and repairs mtDNA. POLG mRNA is increased in leukemia cell lines and a subset of AML patients. We evaluated the preclinical efficacy of targeting POLG using the nucleoside analog 2’,3’-dideoxycytidine (ddC), an FDA-approved anti-retroviral drug that cross-reacts with POLG. Treatment with ddC at 500nM depleted mtDNA by >90%, decreased mtDNA-encoded COXI and COXII proteins and basal oxygen consumption, and induced apoptosis in AML cell lines (OCI-AML2, TEX, K562). ddC further decreased cell viability in a subset of primary AML cells, while normal peripheral blood stem cells (PBSC’s) were resistant to ddC in vitro. Next, we assessed the preclinical efficacy and toxicity of ddC in an OCI-AML2 xenograft model of human AML. Treatment with 35mg/kg of i.p. ddC induced tumor regression and decreased tumor mass by >75% compared to vehicle. No toxicity was observed including changes in liver enzymes or organ histology. We also observed reductions in COXI and COXII protein in tumors but not liver excised from treated mice. We next investigated the mechanism for preferential sensitivity to ddC in AML cells. A two-fold greater decrease in mtDNA content was observed in AML cells treated with ddC compared to PBSC’s, in part explaining the heightened sensitivity. However, the increased sensitivity to ddC in AML was not due to increased mtDNA turnover as mtDNA depletion after ethidium bromide treatment was comparable between AML and PBSC’s. Therefore, we investigated the role of ddC uptake and metabolism. ddC is transported by nucleoside transporters (ENT’s, CNT’s) and activated by cytoplasmic nucleoside kinases to ddC-triphosphate (ddC-TP). Analysis of Haferlach et. al. (2010) dataset demonstrated that a subset of AML cells have higher mRNA expression of hENT2 and hCNT1, and the nucleoside diphosphate kinases NME1,2,3,5 compared to normal hematopoietic cells. Using mass spectrometry, we observed higher levels of ddC and ddCTP in sensitive AML cells compared to resistant cells. Lastly, knockdown of the deoxycytidine kinase rescued ddC cytotoxicity in TEX cells. In summary, targeting POLG with ddC displays anti-leukemic activity in both in vitro and in vivo models of AML. The preferential toxicity of ddC is due, in part, to increased rates of uptake and activation in AML along as well as their increased sensitivity to inhibition of oxidative phosphorylation. Thus, POLG inhibitors such as ddC may have clinical utility in a subset of AML. Citation Format: Sanduni Liyanage, Rose Hurren, Xiaoming Wang, Neil Maclean, Rebecca Laposa, Aaron Schimmer. Targeting the mitochondrial DNA polymerase gamma with 2’3’-dideoxycytidine as a novel therapeutic strategy for acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 210.

8-Oxo-deoxyguanosine (8-oxo-dG) is a pervasive oxidative DNA lesion formed by endogenous oxidative stress and enhanced by drugs and environmental chemicals. This lesion results in transcriptional errors and mutations and is linked to neurodegeneration, teratogenesis, cancer, and other pathologies. We demonstrate that the neonatal central nervous system of transgenic mice carrying the tetracycline-regulable DNA repair gene formamidopyrimidine DNA glycosylase (fpg) has a 50% reduction in 8-oxo-dG levels. This enhanced DNA repair is suppressed by treatment with doxycycline. For the first time, this murine model permits the level of a specific DNA oxidation product to be regulated in a temporally and spatially specific manner, allowing its role as a primary or secondary factor in neurodegenerative disease to be determined in vivo.

Current drug screening methods are insufficiently predictive of clinical toxicity and efficacy. Recent advances in stem cell technology have the potential to improve drug screening. For tests of cardiotoxicity and efficacy of cardioactive drugs, cardiomyocytes derived from human embryonic stem cells and/or human induced pluripotent stem cells, collectively termed human pluripotent stem cells (hPSCs), have been utilized as model alternatives to current drug screening platforms. In this review, we report on recent advances in the differentiation of hPSCs to cardiomyocytes and summarize the evidence for pharmacological responses in hPSC-derived cardiomyocytes.

Mitochondria contain their own 16.6kb genome (mtDNA) which encodes 13 protein-coding genes essential for electron transport chain activity. In mammalian cells, it is replicated solely by the nuclear-encoded mitochondrial DNA polymerase gamma (POLG), which exists as a heterotrimer with POLG2 to perform replication and repair of mtDNA. Through bioinformatic analyses, we observed increased POLG expression in a subset of AML cell lines compared to other cancer cell lines. As a subset of AML patients also have unique mitochondrial characteristics, including increased mitochondrial mass and mtDNA content compared to normal hematopoietic stem cells (Skrtic et. al., Cancer Cell, 20:674, 2011), we addressed the effects of targeting POLG as a novel therapeutic strategy for AML. OCI-AML2 and TEX leukemia cells were treated with the anti-retroviral drug 2′,3′-dideoxycytidine(ddC) that exhibits off-target inhibition of POLG catalytic activity. At concentrations as low as 200nM, ddC depleted mtDNA, decreased mRNA expression of mtDNA transcripts, reduced the expression of mitochondrial encoded proteins COX 1 and 2 subunits that form the catalytic core of respiratory chain complex IV, decreased basal oxygen consumption rate (OCR), and reduced the proliferation and viability of AML cells. However, AML cells had large reserves in their mtDNA; as significant changes in mitochondrial proteins, metabolism, proliferation and viability were only observed with a threshold of >95% depletion of mtDNA. In contrast, lesser reductions in mtDNA content with ddC did not significantly affect levels of mitochondrial transcripts, metabolism, or cell viability. Next, we used a genetic approach and explored the impact of knocking down POLG with multiple independent shRNA in AML cells. POLG knockdown in OCI-AML2 cells depleted mtDNA content by 60% compared to controls. Consistent with the findings with ddC, mtDNA depletion to this extent produced only quantitatively minor changes in the protein expression of the mtDNA-encoded COX 1 and 2 and basal OCR. Despite the minimal effect on mitochondrial metabolism, POLG knockdown decreased the growth and viability of OCI-AML2 cells, and increased apoptosis, as measured by Annexin V staining. Results of electron microscopy analyses indicate that POLG knockdown disrupted mitochondrial cristae structure compared to controls. In summary, these results indicate that POLG plays a role in maintaining AML cell viability and mitochondrial structure and this function is independent of mtDNA replication and oxidative metabolism. Citation Format: Sanduni Liyanage, Rose Hurren, Rebecca Laposa, Aaron Schimmer. Targeting mitochondrial DNA polymerase gamma (POLG) as a novel therapeutic strategy for acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3039. doi:10.1158/1538-7445.AM2015-3039

Recent studies have demonstrated a role for telomerase in driving tumor progression, but its mechanism of action remains unclear. Here we show that stable, ribozyme-mediated suppression of mouse telomerase RNA reduced telomerase RNA expression, telomerase activity, and telomere length, which significantly reduced tumor invasion and metastatic potential. Our studies reveal that previously unidentified effects of telomerase may mediate its tumor-promoting effects. First, reducing telomerase activity induced a more dendritic morphology, accompanied by increased melanin content and increased expression of tyrosinase, a key enzyme in melanin biosynthesis. Second, gene expression profiling revealed that telomerase targeting down-regulated expression of several glycolytic pathway genes, with a corresponding decrease in glucose consumption and lactate production. Thus, telomerase activity controls the glycolytic pathway, potentially altering the energy state of tumor cells and thereby modulating tyrosinase activity and melanin production. These studies have important implications for understanding the mechanisms by which telomerase promotes tumor invasion and metastasis.

In order to compare the abilities of arctic and temperate fish to accumulate PCBs we conduct a metabolic analysis to determine how process rates in a mathematical fish contaminant model change with temperature. We evaluate the model by applying the original and adapted models to estimate PCB concentrations in lake trout (Salvelinus namaycush) in Trout Lake, Ontario, Canada, and in arctic char (Salvelinus alphinus) in Lake Øyangen, in the Norwegian high arctic. Modeled concentrations are, for the most part, within 50% of mean measured values and are comparable to the error associated with the fish data. In order to evaluate differences in fish bioaccumulation processes, the model is applied to hypothetical arctic and temperate systems, assuming the same contaminant input values in water and diet. The model predicts that temperate salmonids are able to biomagnify PCBs 6-60% more than arctic salmonids. For all congeners, the lower BMF(MAX) of arctic fish contribute to their lower concentrations. For congeners with log K(ow)6.0, the lower concentrations in arctic fish are also attributed to faster loss due to gill ventilation. Faster growth rates for temperate fish reduce the difference in bioaccumulation for congeners with log K(ow)7.0. These processes are controlled by the influence of lipid in the fish and their diet as well as the dependence of growth on temperature. We suggest that fish models originally calibrated for temperate systems may be directly applied to arctic lakes after accounting for the lipid content of the fish and their diet as well as water temperature.

The lesion-specific DNA polymerase POLH gene is mutated in xeroderma pigmentosum variant (XP-V) patients who exhibit an increased skin cancer incidence from UV exposure. Normal cells in which POLH expression was reduced using short interfering RNAs (siRNAs) were compared with the XP-V cellular phenotype that results from naturally occurring inactivating mutations. Stable clones expressing siRNA had partially reduced POLH protein levels, and intermediate levels of UV sensitivity and S phase checkpoint activation, but similar levels of Mre11 foci as in XP-V cells. Therefore, suppression of POLH expression levels by siRNA recapitulates most of the phenotypes seen in cells from XP-V patients with inactivating mutations in POLH.

Mitochondria contain their own genome and translation machinery, which are required for aerobic energy production through oxidative phosphorylation. A subset of AML patients are sensitive to inhibitors of mitochondrial translation, likely due to increased mitochondrial mass, increased mitochondrial DNA (mtDNA) and reliance on oxidative phosphorylation (Skrtic et. al., Cancer Cell, 20:674, 2011). In the current study we addressed a complementary approach of inhibiting mtDNA replication by targeting the mitochondrial DNA polymerase gamma (POLG). POLG is encoded by the nuclear genome and is the only known DNA polymerase in mammalian mitochondria. POLG indirectly controls electron transport chain (ETC) function since it replicates the mitochondrial genome that encodes 13 proteins essential for ETC activity. We explored the impact of inhibiting POLG in AML cells with the nucleoside analogue reverse transcriptase inhibitor 2′,3′-dideoxycytidine (ddC), an FDA-approved antiviral drug that demonstrates off-target inhibition of POLG. In OCI-AML2 and TEX leukemia cells, treatment with 0.2-2µM ddC reduced levels of mtDNA in a dose- and time-dependent manner, with >90% depletion of mtDNA after 6 days of treatment with 0.2µM ddC (p Importantly, AML cells appear to have a large reserve in their mtDNA content; in both TEX and OCI-AML2 cells, mtDNA depletion down to 5% of control was required for levels of mtDNA-encoded transcripts or proteins to be significantly reduced. ddC treatments (2µM, 6-10 days) that depleted mtDNA beyond the above threshold reduced the proliferation (p In summary, treatment of AML cells with ddC depletes mtDNA, decreases mitochondrial bioenergetics and causes cell kill. However, AML cells have large reserves in their mtDNA content and can withstand depletion of up to 95% of their mtDNA without loss of oxidative metabolism. Citation Format: Sanduni Liyanage, Rose Hurren, Rebecca Laposa, Aaron Schimmer. Inhibiting the mitochondrial DNA polymerase gamma (POLG) with 2′,3′-dideoxycytidine reduces oxidative phosphorylation and increases apoptosis in acute myeloid leukemia (AML) cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4317. doi:10.1158/1538-7445.AM2014-4317

Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) constitute a family of sun-sensitive human diseases (1). They are caused by mutations in the components of the nucleotide excision repair (NER) system and in the postreplication repair system involving the low-fidelity polymerase H (also known as XP variant or XPV). XP was initially recognized as a disease involving a genetic predisposition to extremely high levels of skin carcinogenesis from solar exposure. The relationship between DNA damage from solar UVB exposure and deficiencies in repair, leading to mutagenesis, genetic instability, and eventual carcinogenesis, provides a deceptively simple explanation for the major clinical features of XP and has provided a paradigm for environmentally induced human cancer. These diseases are, however, much more complex at all levels (1, 2), as the new report in this issue by Murai et al. (3) indicates (Fig. 1). NER has two main pathways: GGR and TCR. Global repair involves the genes XPC and XPE and is generally slower and modulated by p53 (4), which also modulates the pol H pathway (5, 6). TCR results in a more rapid repair of the transcribed strand of expressed genes, and involves the two CS genes (CSA, CSB), several of the XP genes (especially XPA, -B, and -D), and the mismatch repair system (1). Some of the genes are involved in both pathways, especially XPA. The XPG endonuclease is also a cofactor for a glycosylase (endonuclease III, encoded by nth) that acts on oxidative damage (7). Mutations in XPB and XPD also can give rise to diseases that combine one or more of the three main disorders, XP, CS, and TTD, depending in part on the precise site of the mutations in the genes.

The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes P450, prostaglandin H synthase (PHS) and lipoxygenases (LPOs) to electrophilic and/or free radical reactive intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the anticonvulsant drug phenytoin, structurally related anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimethadione) and the sedative drug thalidomide, we have examined the potential teratologic relevance of free radical-initiated, reactive oxygen species (ROS)-mediated oxidative molecular target damage, genotoxicity (micronucleus formation) and DNA repair in mouse and rabbit models in vivo and in embryo culture, and in vitro using purified enzymes or cultured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, characterized by electron spin resonance spectrometry, that initiated ROS formation, including hydroxyl radicals, which were characterized by salicylate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyguanosine formation), protein (carbonyl formation), glutathione (GSH) and lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin [HPPH], thalidomide, B[a]P and NNK in vivo and/or in embryo culture, the latter indicating a teratologically critical role for embryonic, as distinct from maternal, processes. DNA oxidation and teratogenicity of phenytoin and thalidomide were reduced by PHS inhibitors. Oxidative macromolecular lesions and teratogenicity also were reduced by the free radical trapping agent phenylbutylnitrone (PBN), and the antioxidants caffeic acid and vitamin E. In embryo culture, addition of superoxide dismutase (SOD) to the medium enhanced embryonic SOD activity, and SOD or catalase blocked the oxidative lesions and embryotoxicity initiated by phenytoin and B[a]P, suggesting a major contribution of ROS, as distinct from covalent binding, to the teratologic mechanism. In in vivo studies, other antioxidative enzymes like GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (G6PD) were similarly protective. Even untreated G6PD-deficient mice had enhanced embryopathies, indicating a teratological role for endogenous oxidative stress. In cultured fibroblasts, B[a]P, NNK, phenytoin and HPPH initiated DNA oxidation and micronucleus formation, which were inhibited by SOD. Oxidation of DNA may be particularly critical, since transgenic mice with +/- or -/- deficiencies in the p53 tumor suppressor gene, which facilitates DNA repair, are more susceptible to phenytoin and B[a]P teratogenicity. Even p53-deficient mice treated only with normal saline showed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These results suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a reactive intermediate, as well in the mechanism of embryopathies occurring in the absence of xenobiotic exposure.

DNA damage may mediate birth defects caused by many drugs and environmental chemicals, therefore p53, a tumour suppressor gene that facilitates DNA repair, may be critically embryoprotective. We have studied the effects of the environmental teratogen, benzo[a]pyrene, on pregnant heterozygous p53–deficient mice. Such mice exhibited between 2– to 4–fold higher embryotoxicity and teratogenicity than normal p53–controls. Fetal resorptions reflecting in utero death were genotyped using the polymerase chain reaction and found to be increased 2.6–fold and 3.6–fold respectively with heterozygous and homozygous p53–deficient embryos. These results provide the first direct evidence that p53 may be an important teratological suppressor gene which protects the embryo from DNA–damaging chemicals and developmental oxidative stress.