Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile.

Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

Succinate Accumulation Links Mitochondrial MnSOD Depletion to Aberrant Nuclear DNA Methylation and Altered Cell Fate.

Previous studies showed that human cell line HEK293 lacking mitochondrial superoxide dismutase (MnSOD) exhibited decreased succinate dehydrogenase (SDH) activity, and mice lacking MnSOD displayed significant reductions in SDH and aconitase activities. Since MnSOD has significant effects on SDH activity, and succinate is a key regulator of TET enzymes needed for proper differentiation, we hypothesized that SOD2 loss would lead to succinate accumulation, inhibition of TET activity, and impaired erythroid precursor differentiation. To test this hypothesis, we genetically disrupted the SOD2 gene using the CRISPR/Cas9 genetic strategy in a human erythroleukemia cell line (HEL 92.1.7) capable of induced differentiation toward an erythroid phenotype. Cells obtained in this manner displayed significant inhibition of SDH activity and ~10-fold increases in cellular succinate levels compared to their parent cell controls. Furthermore, SOD2 -/- cells exhibited significantly reduced TET enzyme activity concomitant with decreases in genomic 5-hmC and corresponding increases in 5-mC. Finally, when stimulated with δ-aminolevulonic acid (δ-ALA), SOD2 -/- HEL cells failed to properly differentiate toward an erythroid phenotype, likely due to failure to complete the necessary global DNA demethylation program required for erythroid maturation. Together, our findings support the model of an SDH/succinate/TET axis and a role for succinate as a retrograde signaling molecule of mitochondrial origin that significantly perturbs nuclear epigenetic reprogramming and introduce MnSOD as a governor of the SDH/succinate/TET axis.

Nicotinamide phosphoribosyltransferase expression and clinical outcome of resected stage I/II pancreatic ductal adenocarcinoma.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) plays a key role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), which is a vital cofactor in redox reactions and a substrate for NAD+ consuming enzymes including CD38, PARPs and sirtuins. NAMPT over-expression has been shown in various cancers and its inhibition decreases cancer cell growth, making it an attractive therapeutic target. Here we examine the NAMPT expression in a large cohort of resected stage I/II pancreatic ductal adenocarcinomas (PDAs) and correlate its expression with clinical outcomes and pathologic features. METHODS: A retrospective review of patients with PDAs was conducted at a single institution. Tissue microarrays (TMAs) containing primary PDAs and their metastatic lymph nodes (mLNs) were constructed and stained for NAMPT expression. Each TMA core was evaluated for staining intensity of cancer cells (0 = no staining, 1+ = weak, 2+ = moderate, 3+ = strong) and a mean score was calculated for each case with at least two evaluable cores. NAMPT expression was correlated with clinicopathological variables using chi-squared or Fisher's exact test, and t-tests for categorical and continuous variables, respectively. Survival probabilities were estimated and plotted using the Kaplan-Meier method. Cox proportional hazards regression was used to assess the effects of NAMPT staining values on recurrence-free survival (RFS) and overall survival (OS). This study was conducted under an approved IRB protocol. RESULTS: 173 primary PDAs had at least 2 TMA cores with identifiable cancer cells. The mean IHC score was 0.55 (range: 0 to 2.33). The mean IHC score of mLNs was 0.39 (range: 0-2), which was not significantly different from their primary tumors (mean IHC score = 0.47, P = 0.38). Sixty-four percent (111/173) of PDAs were positive for NAMPT staining. Stage II tumors were more likely to be positive (68% of 151 vs 41% of 22; P = 0.01). Non-obese non-diabetic patients were more likely to have NAMPT+ tumors (43.7% vs. 27.9%, P = 0.04). While RFS and OS were not statistically different between NAMPT+ vs. NAMPT- PDAs, patients with NAMPT- tumors tended to have a longer median OS (26.0 vs. 20.4 months, P = 0.34). CONCLUSION: NAMPT expression was detected in 64% of stage I/II PDAs and up to 72% in non-obese non-diabetic patients. Frequency of NAMPT expression correlated with pathological stage, consistent with published literature regarding its role in cancer progression. While RFS and OS were not statistically significantly different, patients with NAMPT+ PDAs tended to have a shorter survival. Thus, NAMPT inhibition may prove beneficial in clinical trials.

O2⋠- and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate.

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells.

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

Identification of a Small Molecule Inhibitor of RAD52 by Structure-Based Selection.

It has been reported that inhibition of RAD52 either by specific shRNA or a small peptide aptamer induced synthetic lethality in tumor cell lines carrying BRCA1 and BRCA2 inactivating mutations. Molecular docking was used to screen two chemical libraries: 1) 1,217 FDA approved drugs, and 2) 139,735 drug-like compounds to identify candidates for interacting with DNA binding domain of human RAD52. Thirty six lead candidate compounds were identified that were predicted to interfere with RAD52 -DNA binding. Further biological testing confirmed that 9 of 36 candidate compounds were able to inhibit the binding of RAD52 to single-stranded DNA in vitro. Based on molecular binding combined with functional assays, we propose a model in which the active compounds bind to a critical "hotspot" in RAD52 DNA binding domain 1. In addition, one of the 9 active compounds, adenosine 5'-monophosphate (A5MP), and also its mimic 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) 5' phosphate (ZMP) inhibited RAD52 activity in vivo and exerted synthetic lethality against BRCA1 and BRCA2-mutated carcinomas. These data suggest that active, inhibitory RAD52 binding compounds could be further refined for efficacy and safety to develop drugs inducing synthetic lethality in tumors displaying deficiencies in BRCA1/2-mediated homologous recombination.

SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD.

To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells.

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma.

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.