Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer.

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1 Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors.

Penetrance of biallelic SMARCAL1 mutations is associated with environmental and genetic disturbances of gene expression.

Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila and mouse models, we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that, as found in SIOD patients, deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice, although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.

Reactive oxygen species stress increases accumulation of tyrosyl-DNA phsosphodiesterase 1 within mitochondria.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a nuclear and mitochondrial protein that in nuclei and in vitro repairs blocked 3' DNA termini such as 3' phosphotyrosine conjugates resulting from stalling of topoisomerase I-DNA intermediates. Its mutation also causes spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1). Because Tdp1 colocalizes with mitochondria following oxidative stress, we hypothesized that Tdp1 repairs mitochondrial DNA (mtDNA) and that mtDNA damage mediates entry of Tdp1 into the mitochondria. To test this, we used S. cerevisiae mutants, cultured mouse and human cells, and a Tdp1 knockout mouse. H2O2- and rotenone-induced cellular and intramitochondrial reactive oxygen species (ROS) activated oxidant-responsive kinases P38 and ERK1, and the translocation of Tdp1 from the nucleus to the mitochondria via the TIM/TOM complex. This translocation occurred independently of mtDNA. Within the mitochondria, Tdp1 interacted with Ligase III and reduced mtDNA mutations. Tdp1-deficient tissues had impaired mitochondrial respiration and decreased viability. These observations suggest that Tdp1 maintains mtDNA integrity and support the hypothesis that mitochondrial dysfunction contributes to the pathology of SCAN1.

Identification of a putative Tdp1 inhibitor (CD00509) by in vitro and cell-based assays.

Mutations of DNA repair pathways contribute to tumorigenesis and provide a therapeutic target for synthetic lethal interactions in tumor cells. Given that tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs stalled topoisomerase-I DNA complexes, we hypothesized that inhibition of Tdp1 has synthetic lethal effects in some cancers. To test this, we screened tumor arrays for Tdp1 expression and observed that Tdp1 is expressed in many tumors, including more than 90% of human breast tumors. Subsequent chemical screening identified putative Tdp1 inhibitors. Treatment of control human mammary epithelial cells and the breast cancer cell line MCF-7 with compound CD00509 preferentially sensitized MCF-7 cells to camptothecin and decreased cell proliferation 25% more than camptothecin treatment alone. This suggests that CD00509 specifically targeted Tdp1 in vitro, and CD00509 increased the sensitivity of wild-type murine embryonic fibroblasts (MEFs) to camptothecin to a degree comparable to that of Tdp1(-/-) MEFs. In addition, consistent with poly ADP-ribose polymerase-1 (PARP-1) collaborating with Tdp1 in DNA repair, combined Tdp1 and PARP-1 inhibition was more detrimental to MCF-7 cells than either treatment alone, whereas the combination was not additively harmful to control mammary cells. We conclude that targeting Tdp1 in anticancer therapy preferentially enhances the sensitivity of some breast cancer cells to camptothecin and may be an effective adjuvant for breast cancer therapy.

Expression profile and mitochondrial colocalization of Tdp1 in peripheral human tissues.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that processes blocked 3' ends of DNA breaks. Functional loss of Tdp1 causes spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1). Based on the prominent cytoplasmic expression of Tdp1 in the neurons presumably affected in SCAN1, we hypothesized that Tdp1 participates in the repair of mitochondrial DNA. As a step toward testing this hypothesis, we profiled Tdp1 expression in different human tissues by immunohistochemistry and immunofluorescence respectively and determined whether Tdp1 was expressed in the cytoplasm of tissues other than the neurons. We found that Tdp1 was ubiquitously expressed and present in the cytoplasm of many cell types. Within human skeletal muscle and multiple mouse tissues, Tdp1 partially colocalized with the mitochondria. In cultured human dermal fibroblasts, Tdp1 redistributed to the cytoplasm and partially colocalized with mitochondria following oxidative stress. These studies suggest that one role of cytoplasmic Tdp1 is the repair of mitochondrial DNA lesions arising from oxidative stress.

TDP1 and PARP1 deficiency are cytotoxic to rhabdomyosarcoma cells.

UNLABELLED: Rhabdomyosarcoma is the most common soft tissue sarcoma in children. Metastatic rhabdomyosarcoma in children has a 5-year event-free survival rate of <30%, and a recent clinical trial with irinotecan, a topoisomerase I inhibitor, failed to improve outcome. Therefore, it was surmised that failure of irinotecan may be the result of overexpression of the DNA repair enzyme tyrosyl-DNA phosphodiesterase (TDP1), which processes topoisomerase I-DNA complexes resulting from topoisomerase I inhibitor treatment. Using human tissue microarrays and gene expression arrays, a marked overexpression of TDP1 protein and mRNA in RMS tumors was observed. Critically, knockdown of TDP1 or inhibition of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme in the same complex as TDP1, sensitized rhabdomyosarcoma cell lines to analogues of irinotecan. Interestingly, BRCA1/2 mutations or altered expression was not detectable in rhabdomyosarcoma cells; however, TDP1 knockdown and PARP-1 inhibition alone were cytotoxic to a subset of rhabdomyosarcoma cells, suggesting that they harbor genetic lesions in DNA repair components that have synthetic lethal interactions with loss of TDP1 or PARP1 function. Furthermore, culturing embryonal rhabdomyosarcoma cells in serum/nutrient-restricted medium increased cellular cytotoxicity upon PARP-1 inhibition and was intrinsically cytotoxic to alveolar, though not embryonal rhabdomyosarcoma cells. The results of these studies suggest a compensatory role for TDP1 in rhabdomyosarcoma after topoisomerase-I based therapy and further demonstrate that TDP1 knockdown, PARP-1 inhibition, and dietary restriction have therapeutic validity. IMPLICATIONS: Selective targeting of TDP1 and/or PARP-1 in rhabdomyosarcoma induces cytotoxicity and sensitizes to DNA damaging agents.