Elizabeth Peterson-Roth

Mismatch Repair Proteins Are Activators of Toxic Responses to Chromium-DNA Damage

ABSTRACT Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of γ-H2AX foci in G2 phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of γ-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G2 arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers.

Rapid DNA Double-Strand Breaks Resulting from Processing of Cr-DNA Cross-Links by Both MutS Dimers

Mismatch repair (MMR) strongly enhances cyto- and genotoxicity of several chemotherapeutic agents and environmental carcinogens. DNA double-strand breaks (DSB) formed after two replication cycles play a major role in MMR-dependent cell death by DNA alkylating drugs. Here, we examined DNA damage detection and the mechanisms of the unusually rapid induction of DSB by MMR proteins in response to carcinogenic chromium(VI). We found that MSH2-MSH6 (MutSalpha) dimer effectively bound DNA probes containing ascorbate-Cr-DNA and cysteine-Cr-DNA cross-links. Binary Cr-DNA adducts, the most abundant form of Cr-DNA damage, were poor substrates for MSH2-MSH6, and their toxicity in cells was weak and MMR independent. Although not involved in the initial recognition of Cr-DNA damage, MSH2-MSH3 (MutSbeta) complex was essential for the induction of DSB, micronuclei, and apoptosis in human cells by chromate. In situ fractionation of Cr-treated cells revealed MSH6 and MSH3 chromatin foci that originated in late S phase and did not require replication of damaged DNA. Formation of MSH3 foci was MSH6 and MLH1 dependent, whereas MSH6 foci were unaffected by MSH3 status. DSB production was associated with progression of cells from S into G(2) phase and was completely blocked by the DNA synthesis inhibitor aphidicolin. Interestingly, chromosome 3 transfer into MSH3-null HCT116 cells activated an alternative, MSH3-like activity that restored dinucleotide repeat stability and sensitivity to chromate. Thus, sequential recruitment and unprecedented cooperation of MutSalpha and MutSbeta branches of MMR in processing of Cr-DNA cross-links is the main cause of DSB and chromosomal breakage at low and moderate Cr(VI) doses.

Targeting Cancer with a Lupus Autoantibody

A cell-penetrating lupus anti-DNA antibody inhibits DNA repair, sensitizes cancer cells to DNA-damaging therapy in vitro and in vivo, and is synthetically lethal to BRCA2-deficient human cancer cells. Taming the Big Bad Wolf Just like the wolves for which lupus is named, the antibodies involved in its pathogenesis can attack almost any part of a patient, causing widespread damage. Now, Hansen et al. show that these biological wolves can sometimes be tamed and their ferociousness put to use in treating another deadly disease. Lupus is an autoimmune disease associated with antibodies that target host DNA, wreaking havoc on patients’ cells throughout the body. Recently, cancer researchers have tried to co-opt some of these antibodies, particularly those that can penetrate human cells, for use as vehicles for therapeutic agents. While using lupus antibodies to deliver proteins to protect normal cells from therapeutic ionizing radiation delivered to a tumor, researchers discovered that one antibody, 3E10, could itself sensitize cancer cells to radiation treatment. The authors then characterized this observed effect in malignant cells and determined its mechanism. They found that 3E10 bound single-stranded DNA and interfered with its repair, making the cells more susceptible to DNA-damaging agents such as doxorubicin and radiation. In addition, 3E10 alone was toxic to cancer cells with deficient DNA repair pathways, such as those that harbor BRCA2 mutations. Further research is necessary to identify other pathways that make tumor cells susceptible to 3E10 and to analyze the pharmacokinetics and other characteristics of this treatment. However, 3E10 was already shown to be safe in a previous phase 1 trial in lupus patients and should now be able to transition into clinical trials for cancer patients as well. Although researchers have not yet discovered a cure for lupus, the big bad wolf’s offspring may potentially tame another life-threatening illness. Systemic lupus erythematosus (SLE) is distinct among autoimmune diseases because of its association with circulating autoantibodies reactive against host DNA. The precise role that anti-DNA antibodies play in SLE pathophysiology remains to be elucidated, and potential applications of lupus autoantibodies in cancer therapy have not previously been explored. We report the unexpected finding that a cell-penetrating lupus autoantibody, 3E10, has potential as a targeted therapy for DNA repair–deficient malignancies. We find that 3E10 preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes cultured tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we demonstrate that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low-dose doxorubicin. Our results establish an approach to cancer therapy that we expect will be particularly applicable to BRCA2-related malignancies such as breast, ovarian, and prostate cancers. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the intrinsic deficiencies in DNA repair and the unexpectedly low rates of breast, ovarian, and prostate cancers observed in SLE patients. In summary, this study provides the basis for the potential use of a lupus anti-DNA antibody in cancer therapy and identifies lupus autoantibodies as a potentially rich source of therapeutic agents.

Src-Induced Cisplatin Resistance Mediated by Cell-to-Cell Communication

Cisplatin-induced cell death can be triggered by cell-to-cell communication through gap junctions. Here, we show that activated src produces tyrosine phosphorylation of the gap junction protein connexin 43, decreases gap junction communication, and increases cell survival in response to cisplatin. Experiments with mixed cell populations show that src activity in one cell can confer increased cisplatin survival on neighboring cells, even when the neighboring cells lack such src activity. This work is the first demonstration that expression of an oncogene in one cell can affect the survival of a neighboring cell not expressing the oncogene in response to a chemotherapeutic drug. The trans-acting effect of activated src on neighboring cells can be blocked by inhibitors of src kinase or by siRNA-mediated knockdown of src expression, and it can be counteracted by forced up-regulation of connexin 43, via either gene transfer or proteasome inhibition. These results identify a novel pathway of cisplatin resistance that may be amenable to therapeutic intervention.

Killing of chromium-damaged cells by mismatch repair and its relevance to carcinogenesis.

Hexavalent chromium compounds are widespread environmental contaminants that are well recognized as human carcinogens and potent respiratory toxicants. Intracellular metabolism of chromium(VI) leads to the production of numerous chromium-DNA adducts that are primarily formed at the phosphate groups. The mechanism of toxicity of these DNA modifications in human cells has been uncertain for a long time because chromium and other phosphate-based adducts did not block DNA replication with purified polymerases. Our recent studies identified mismatch repair proteins as activators of toxic responses to chromium-DNA damage, which resolved an apparent discrepancy in genotoxic activity of chromium adducts in cells and in vitro. The discovered mechanism of toxicity provided the basis for a novel model of chromium carcinogenesis based on the selection of resistant clones that lack mismatch repair and progress to cancer due to high levels of spontaneous mutagenesis.

YU238259 Is a Novel Inhibitor of Homology- Dependent DNA Repair That Exhibits Synthetic Lethality and Radiosensitization in Repair- Deficient Tumors

Radiotherapy and DNA-damaging chemotherapy are frequently utilized in the treatment of solid tumors. Innate or acquired resistance to these therapies remains a major clinical challenge in oncology. The development of small molecules that sensitize cancers to established therapies represents an attractive approach to extending survival and quality of life in patients. Here, we demonstrate that YU238259, a member of a novel class of DNA double-strand break repair inhibitors, exhibits potent synthetic lethality in the setting of DNA damage response and DNA repair defects. YU238259 specifically inhibits homology-dependent DNA repair, but not non-homologous end-joining, in cell-based GFP reporter assays. Treatment with YU238259 is not only synergistic with ionizing radiation, etoposide, and PARP inhibition, but this synergism is heightened by BRCA2 deficiency. Further, growth of BRCA2-deficient human tumor xenografts in nude mice is significantly delayed by YU238259 treatment even in the absence of concomitant DNA-damaging therapy. The cytotoxicity of these small molecules in repair-deficient cells results from an accumulation of unresolved DNA double-strand breaks. These findings suggest that YU238259 or related small molecules may have clinical benefit to patients with advanced BRCA2-negative tumors, either as a monotherapy or as an adjuvant to radiotherapy and certain chemotherapies. Implications: We have identified a novel series of compounds that demonstrate synthetic lethality in DNA repair–deficient cell and animal models and have strong potential for clinical translation. Mol Cancer Res; 13(10); 1389–97. ©2015 AACR.

Abstract 4319: Lupus antibody-based cancer therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC A subset of autoantibodies produced by patients with systemic lupus erythematosus (SLE) penetrates into cell nuclei and binds DNA, and we believe that such antibodies may have applications in cancer therapy. We have discovered that the cell-penetrating, nuclear-localizing anti-DNA lupus antibody 3E10 inhibits key steps in DNA single- and double-strand break repair and has potential for development as a targeted therapy for tumors harboring deficiencies in DNA repair. 3E10 preferentially binds DNA substrates with free single-strand tails and interferes with both base excision repair and homology-directed repair (HDR) in vitro, and HDR efficiency is reduced in cells treated with 3E10 as measured in the chromosome-based DR-GFP fluorescent reporter assay. The binding of 3E10 to DNA can be directly visualized under electron microscopy (EM), and EM studies confirmed that 3E10 interferes with RAD51 filament formation, which is a critical step in HDR. The 3E10 single chain variable fragment penetrates into human tumor xenografts in nude mice, and 3E10 sensitizes cancer cells and tumors to DNA-damaging therapy. In addition, 3E10, by itself, is toxic to BRCA2-deficient cancer cells but not to repair-proficient cells, and when combined with a DNA-damaging agent, 3E10 has a very large cytotoxic effect on BRCA2-deficient cancer cells. The synthetically lethal effect of 3E10 on BRCA2-deficient cancer cells is consistent with our finding that 3E10 inhibits DNA repair, and it suggests that 3E10 has potential as a targeted therapy for tumors harboring deficiencies in DNA repair, such as certain breast, ovarian, and prostate cancers. Of note, patients with SLE have lower than expected incidence rates of breast, ovarian, and prostate cancers, and it is tempting to speculate that the circulating cell-penetrating anti-DNA autoantibodies provide patients with SLE some protection against the development of DNA repair-deficient tumors. In summary, our work with the 3E10 antibody has provided proof of principle for the development of a lupus antibody as a cancer therapy and opened up new avenues for exploration into the biology of lupus antibodies. Citation Format: James E. Hansen, Grace Chan, Yanfeng Liu, Denise C. Hegan, Shibani Dalal, Eloise Dray, Youngho Kwon, Yuanyuan Xu, Xiaohua Xu, Elizabeth Peterson-Roth, Erik Geiger, Yilun Liu, Joseph Gera, Joann B. Sweasy, Patrick Sung, Sara Rockwell, Robert N. Nishimura, Richard H. Weisbart, Peter M. Glazer. Lupus antibody-based cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4319. doi:10.1158/1538-7445.AM2013-4319

Abstract 2185: Identification of novel compounds that preferentially kill repair deficient cells using high-throughput screening

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Loss of functional DNA repair pathways is a common occurrence in cancer cells and discovery of novel drugs that selectively kill repair deficient tumors cells could identify new cancer therapies. In this work we describe the use of high-throughput screening to determine chemicals that sensitize cells deficient in the fanconi anemia protein, FANCD2. Cell growth of human fibroblasts proficient (PD20+D2) and deficient (PD20) for FANCD2 were screened using two libraries containing over 1000 bioactive compounds and kinase inhibitors. The cells were treated with compounds continuously for 3 days and cell growth was assessed using a luciferase-based assay measuring ATP levels. The initial screen identified numerous compounds inducing differential survival in cells with and without FANCD2 and follow-up testing will be required to confirm these potential hits. However, it is promising that, in addition to novel compounds, several crosslinking agents, including mitomycinC, which are known to sensitize fanconi anemia cells to cell death are among the positive hits. Our results validate chemical screening as a tool to discover potential new cancer therapies targeting repair deficient cells lines. In addition, other than their role in crosslink repair, there is still not much known about the fanconi anemia protein family and the discovery of novel compounds involved in FANCD2 dependent cell survival could lead to the discovery of new functions of this protein family. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2185.