Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.

Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA-damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radiosensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules that selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits that potently inhibit DSB repair, and we have validated their functional activity in a comprehensive panel of orthogonal secondary assays. A selection of these inhibitors was found to radiosensitize cancer cell lines in vitro, which suggests that they may be useful as novel chemo- and radio sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker mibefradil dihydrochloride, that demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a phase I clinical trial testing mibefradil as a glioma radiosensitizer.

DNA repair and synthetic lethality.

Tumors often have DNA repair defects, suggesting additional inhibition of other DNA repair pathways in tumors may lead to synthetic lethality. Accumulating data demonstrate that DNA repair-defective tumors, in particular homologous recombination (HR), are highly sensitive to DNA-damaging agents. Thus, HR-defective tumors exhibit potential vulnerability to the synthetic lethality approach, which may lead to new therapeutic strategies. It is well known that poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) inhibitors show the synthetically lethal effect in tumors defective in BRCA1 or BRCA2 genes encoded proteins that are required for efficient HR. In this review, we summarize the strategies of targeting DNA repair pathways and other DNA metabolic functions to cause synthetic lethality in HR-defective tumor cells.

The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma.

Malignant pleural mesotheliomas (MPMs) often show CDKN2A and NF2 inactivation, but other highly recurrent mutations have not been described. To identify additional driver genes, we used an integrated genomic analysis of 53 MPM tumor samples to guide a focused sequencing effort that uncovered somatic inactivating mutations in BAP1 in 23% of MPMs. The BAP1 nuclear deubiquitinase is known to target histones (together with ASXL1 as a Polycomb repressor subunit) and the HCF1 transcriptional co-factor, and we show that BAP1 knockdown in MPM cell lines affects E2F and Polycomb target genes. These findings implicate transcriptional deregulation in the pathogenesis of MPM.

Loss of p53 function in colon cancer cells results in increased phosphocholine and total choline.

Mutations in the p53 gene are the most frequently observed genetic lesions in human cancers. Human cancers that contain a p53 mutation are more aggressive, more apt to metastasize, and more often fatal. p53 controls numerous downstream targets that can influence various outcomes such as apoptosis, growth arrest, and DNA repair. Based on previous observations using (1)H magnetic resonance spectroscopy (MRS), we have identified choline phospholipid metabolite intensities typical of increased malignancy. Here we have used (1)H MRS to characterize the choline phospholipid metabolite levels of p53(+/ +) and p53(-/-) cells, and demonstrated that loss of p53 function results in increased phosphocholine and total choline. These data suggest that the increased malignancy of cancer cells resulting from loss of p53 may be mediated, in part, through the choline phospholipid pathway.

Mitochondrial impairment is accompanied by impaired oxidative DNA repair in the nucleus.

Depletion of the mitochondrial genome is involved in several human diseases, as well as in mitochondrial diseases induced by drug therapies used in the treatment of cancer and human immunodeficiency virus. In order to identify the molecular changes underlying the pathogenesis of mitochondrial diseases, we determined the oxidative status of a human cell line following depletion of the mitochondrial genome (denoted rho0 cells). Our analysis revealed that rho0 cells contained approximately 10-fold lower levels of superoxide than parental cells (rho+), as detected by oxidation of dihydroethidium. No concurrent decrease in oxidation of hydrogen peroxide, detected using the dye dichloroflorescein diacetate, was observed in rho0 cells. Depletion of the mitochondrial genome did not affect either the expression of superoxide dismutase or its activity. However, catalase expression and its activity decreased in rho0 cells. In addition, glutathione peroxidase activity was higher in rho0 cells compared with rho+. rho0 cells showed increased lipid peroxidation, increased oxidative damage to the nuclear genome and impaired DNA repair. Our data illustrate the importance of the mitochondrial genome and its function to the cellular oxidative environment and nuclear genome instability. It also provides insights into the development of mitochondrial disease as a consequence of cancer therapy.

Nuclear genes involved in mitochondria-to-nucleus communication in breast cancer cells.

BACKGROUND: The interaction of nuclear and mitochondrial genes is an essential feature in maintenance of normal cellular function. Of 82 structural subunits that make up the oxidative phosphorylation system in the mitochondria, mitochondrial DNA (mtDNA) encodes 13 subunits and rest of the subunits are encoded by nuclear DNA. Mutations in mitochondrial genes encoding the 13 subunits have been reported in a variety of cancers. However, little is known about the nuclear response to impairment of mitochondrial function in human cells. RESULTS: We isolated a Rho0 (devoid of mtDNA) derivative of a breast cancer cell line. Our study suggests that depletion of mtDNA results in oxidative stress, causing increased lipid peroxidation in breast cancer cells. Using a cDNA microarray we compared differences in the nuclear gene expression profile between a breast cancer cell line (parental Rho+) and its Rho0 derivative impaired in mitochondrial function. Expression of several nuclear genes involved in cell signaling, cell architecture, energy metabolism, cell growth, apoptosis including general transcription factor TFIIH, v-maf, AML1, was induced in Rho0 cells. Expression of several genes was also down regulated. These include phospholipase C, agouti related protein, PKC gamma, protein tyrosine phosphatase C, phosphodiestarase 1A (cell signaling), PIBF1, cytochrome p450, (metabolism) and cyclin dependent kinase inhibitor p19, and GAP43 (cell growth and differentiation). CONCLUSIONS: Mitochondrial impairment in breast cancer cells results in altered expression of nuclear genes involved in signaling, cellular architecture, metabolism, cell growth and differentiation, and apoptosis. These genes may mediate the cross talk between mitochondria and the nucleus.