Richard D. Kennedy

DNA Repair Pathways in Clinical Practice: Lessons From Pediatric Cancer Susceptibility Syndromes

Human cancers exhibit genomic instability and an increased mutation rate due to underlying defects in DNA repair. Cancer cells are often defective in one of six major DNA repair pathways, namely: mismatch repair, base excision repair, nucleotide excision repair, homologous recombination, nonhomologous endjoining and translesion synthesis. The specific DNA repair pathway affected is predictive of the kinds of mutations, the tumor drug sensitivity, and the treatment outcome. The study of rare inherited DNA repair disorders, such as Fanconi anemia, has yielded new insights to drug sensitivity and treatment of sporadic cancers, such as breast or ovarian epithelial tumors, in the general population. The Fanconi anemia pathway is an example of how DNA repair pathways can be deregulated in cancer cells and how biomarkers of the integrity of these pathways could be useful as a guide to cancer management and may be used in the development of novel therapeutic agents.

RNA expression analysis from formalin fixed paraffin embedded tissues

Formalin fixation and paraffin embedding (FFPE) is the most commonly used method worldwide for tissue storage. This method preserves the tissue integrity but causes extensive damage to nucleic acids stored within the tissue. As methods for measuring gene expression such as RT-PCR and microarray are adopted into clinical practice there is an increasing necessity to access the wealth of information locked in the Formalin fixation and paraffin embedding archives. This paper reviews the progress in this field and discusses the unique opportunities that exist for the application of these techniques in the development of personalized medicine.

Chemosensitization to cisplatin by inhibitors of the Fanconi anemia/BRCA pathway

Cisplatin resistance occurs, at least in part, through the function of the Fanconi anemia (FA)/BRCA pathway, a DNA-damage response pathway required for repair of cisplatin cross-links. In the current study, we designed a cell-based screening strategy to identify small-molecule inhibitors of the FA/BRCA pathway with the hypothesis that such molecules could restore sensitivity to platinum agents. We identified four inhibitors, including three protein kinase inhibitors (wortmannin, H-9, and alsterpaullone) and one natural compound (curcumin) that inhibit the FA/BRCA pathway. We show that curcumin, a compound that is generally regarded as safe, inhibits the monoubiquitination of the FANCD2 protein as predicted by the screen and consequently sensitizes ovarian and breast tumor cell lines to cisplatin through apoptotic cell death. We believe that this study shows an efficient, high-throughput method for identifying new compounds that may sensitize cancer cells to DNA-damaging chemotherapy. [Mol Cancer Ther 2006;5(4):952–61]

Fanconi anemia pathway-deficient tumor cells are hypersensitive to inhibition of ataxia telangiectasia mutated

The Fanconi anemia (FA) pathway maintains genomic stability in replicating cells. Some sporadic breast, ovarian, pancreatic, and hematological tumors are deficient in FA pathway function, resulting in sensitivity to DNA-damaging agents. FA pathway dysfunction in these tumors may result in hyperdependence on alternative DNA repair pathways that could be targeted as a treatment strategy. We used a high-throughput siRNA screening approach that identified ataxia telangiectasia mutated (ATM) as a critical kinase for FA pathway-deficient human fibroblasts. Human fibroblasts and murine embryonic fibroblasts deficient for the FA pathway were observed to have constitutive ATM activation and Fancg(-/-)Atm(-/-) mice were found to be nonviable. Abrogation of ATM function in FA pathway-deficient cells resulted in DNA breakage, cell cycle arrest, and apoptotic cell death. Moreover, Fanconi anemia complementation group G- (FANCG-) and FANCC-deficient pancreatic tumor lines were more sensitive to the ATM inhibitor KU-55933 than isogenic corrected lines. These data suggest that ATM and FA genes function in parallel and compensatory roles to maintain genomic integrity and cell viability. Pharmaceutical inhibition of ATM may have a role in the treatment of FA pathway-deficient human cancers.

Chk1-Mediated Phosphorylation of FANCE Is Required for the Fanconi Anemia/BRCA Pathway

ABSTRACT The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.

Development and Independent Validation of a Prognostic Assay for Stage II Colon Cancer Using Formalin-Fixed Paraffin-Embedded Tissue

PURPOSE Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. PATIENTS AND METHODS A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. RESULTS The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P < .001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P < .001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P < .001). CONCLUSION This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples.

BRCA1: mechanisms of inactivation and implications for management of patients.

The BRCA1 gene was cloned in 1994 as one of the genes that conferred genetic predisposition to early-onset breast and ovarian cancer. Since then, a genetic test for identification of high-risk individuals has been developed. Despite being implicated in many important cellular pathways, including DNA repair and regulation of transcription, the exact mechanism by which inactivation of BRCA1 might lead to malignant transformation of cells remains unknown. We examine the mechanisms that underlie inactivation of BRCA1 and assess how they affect management of patients, in terms of both primary and secondary cancer prevention strategies. Furthermore, we look at the potential usefulness of BRCA1 as a prognostic tool and as a predictive marker of response to different classes of drugs. Finally, throughout this review, we draw links between the functional consequences of BRCA1 inactivation, in terms of key cellular signalling pathways, and how they might explain specific clinical observations in individuals who carry mutations in the gene.

CHK1 inhibition as a strategy for targeting Fanconi Anemia (FA) DNA repair pathway deficient tumors.

BackgroundDNA repair deficient tumor cells have been shown to accumulate high levels of DNA damage. Consequently, these cells become hyper-dependent on DNA damage response pathways, including the CHK1-kinase-mediated response. These observations suggest that DNA repair deficient tumors should exhibit increased sensitivity to CHK1 inhibition. Here we offer experimental evidence in support of this hypothesis.ResultsUsing isogenic pairs of cell lines differing only in the Fanconi Anemia (FA) DNA repair pathway, we showed that FA deficient cell lines were hypersensitive to CHK1 silencing by independent siRNAs as well as CHK1 pharmacologic inhibition by Gö6976 and UCN-01. In parallel, an siRNA screen designed to identify gene silencings synthetically lethal with CHK1 inhibition identified genes required for FA pathway function. To confirm these findings in vivo, we demonstrated that whole zebrafish embryos, depleted for FANCD2 by a morpholino approach, were hypersensitive to Gö6976. Silencing of FA genes led to hyper-activation of CHK1 and vice versa. Furthermore, inactivation of CHK1 in FA deficient cell lines caused increased accumulation of DNA strand and chromosomal breakages. These results suggest that the functions subserved by CHK1 and the FA pathway mutually compensate in maintaining genome integrity. As CHK1 inhibition has been under clinical trial in combination with cisplatin, we showed that the FA specific tumoricidal effect of CHK1 inhibition and cisplatin was synergistic.ConclusionTaken together, these results suggest CHK1 inhibition as a strategy for targeting FA deficient tumors.

BRCA1 and c-Myc Associate to Transcriptionally Repress Psoriasin, a DNA Damage–Inducible Gene

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease-associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIalpha poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

BRCA1 and implications for response to chemotherapy in ovarian cancer

OBJECTIVES Treatment of epithelial ovarian cancer (EOC) remains a challenge, despite advances in surgery and chemotherapy. Hereditary ovarian cancer is primarily due to germline mutations in the BRCA1 tumour suppressor gene. In addition, sporadic EOC tumours display significant of loss of BRCA1 function due to epigenetic inactivation of the BRCA1 gene. This article reviews the preclinical and clinical evidence to support a role for BRCA1 as a potential predictive biomarker of response to both platinum and taxane based chemotherapy in EOC. METHODS We conducted a Medline and Pubmed search for reports between 1990 and 2008 using the search terms: BRCA1 and hereditary ovarian cancer, BRCA1 and sporadic ovarian cancer, ovarian cancer and chemotherapy, ovarian cancer and taxanes, ovarian cancer and platinums, ovarian cancer and clinical response, BRCA1 and DNA damage, BRCA1 and DNA repair, BRCA1 and mitotic checkpoint. If reports identified by these criteria referred to other papers not in the initial search, then these were also reviewed if relevant to BRCA1 and ovarian cancer. RESULTS The BRCA1 pathway plays a significant role in the development of both hereditary and sporadic EOC. Evidence suggests that BRCA1 is a potential biomarker of response to platinum chemotherapy in EOC with BRCA1 deficiency predicting for enhanced response. In contrast, initial evidence suggests that loss of BRCA1 function results in reduced response to antimicrotubule-based chemotherapy. The ability of BRCA1 to differentially modulate response to these agents involves loss of BRCA1 mediated DNA repair and mitotic checkpoint control, respectively. CONCLUSIONS Standard first line treatment of EOC consists of a combination of platinum and taxane chemotherapy, however clinically useful biomarkers for predicting response to these agents have yet to be established. BRCA1 may prove useful as a biomarker in EOC for assigning chemotherapy treatments based on the presence or absence of BRCA1 function.