Jennifer T. Fox

High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death.

Human ATAD5 is a biomarker for identifying genotoxic compounds because ATAD5 protein levels increase posttranscriptionally in response to DNA damage. We screened over 4,000 compounds with a cell-based quantitative high-throughput ATAD5-luciferase assay detecting genotoxic compounds. We identified 22 antioxidants, including resveratrol, genistein, and baicalein, that are currently used or investigated for the treatment of cardiovascular disease, type 2 diabetes, osteopenia, osteoporosis, and chronic hepatitis, as well as for antiaging. Treatment of dividing cells with these compounds induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anticancer drugs. Furthermore, resveratrol and genistein killed multidrug-resistant cancer cells. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents.

Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

Dynamic regulation of PCNA ubiquitylation/deubiquitylation

Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation plays a crucial role in maintaining genomic stability during DNA replication. DNA damage stalling the DNA replication fork induces PCNA ubiquitylation that activates DNA damage bypass to prevent the collapse of DNA replication forks that could potentially produce double‐strand breaks and chromosomal rearrangements. PCNA ubiquitylation dictates the mode of bypass depending on the level of ubiquitylation; monoubiquitylation and polyubiquitylation activate error‐prone translesion synthesis and error‐free template switching, respectively. Due to the error‐prone nature of DNA damage bypass, PCNA ubiquitylation needs to be tightly regulated. Here, we review the molecular mechanisms to remove ubiquitin from PCNA including the emerging role of USP1 and ELG1 in this fascinating process.

Werner syndrome protein associates with γH2AX in a manner that depends upon Nbs1

The WRN protein is mutated in the chromosomally unstable Werner syndrome (WS) and the Nbs1 protein is mutated in Nijmegen breakage syndrome (NBS). The Nbs1 protein is an integral component of the M/R/N complex. Although WRN is known to interact with this complex in response to γ‐irradiation, the mechanism of action is unclear. Here, we show that WRN co‐localizes and associates with γH2AX, a marker protein of DNA double strand breaks (DSBs), after cellular exposure to γ‐irradiation. While the DNA damage‐inducible Nbs1 foci formation is normal in WS cells, WRN focus formation is defective in NBS cells. Consistent with this, γH2AX colocalizes with Nbs1 in WS cells but not with WRN in NBS cells. The defective WRN‐γH2AX association in NBS cells can be complemented with wild‐type Nbs1, but not with an Nbs1 S343A point mutant that lacks an ATM phosphorylation site. WRN associates with H2AX in a manner dependent upon the M/R/N complex. Our results suggest a novel pathway in which Nbs1 may recruit WRN to the site of DNA DSBs in an ATM‐dependent manner.

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

Histone Deacetylase Inhibitors Selectively Target Homology Dependent DNA Repair Defective Cells and Elevate Non-Homologous Endjoining Activity

Background We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity. Results HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency. Conclusions HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival.

ATAD5 Deficiency Decreases B Cell Division and Igh Recombination

Mammalian ATPase family AAA domain–containing protein 5 (ATAD5) and its yeast homolog enhanced level of genomic instability 1 are responsible for unloading proliferating cell nuclear antigen from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound proliferating cell nuclear antigen, slowed cell division, and increased genomic instability. In this study, B cells from heterozygous (Atad5+/m) mice were used to examine the effects of decreased cell proliferation on Ab diversity. ATAD5 haploinsufficiency did not change the frequency or spectrum of somatic hypermutation in Ab genes, indicating that DNA repair and error-prone DNA polymerase η usage were unaffected. However, immunized Atad5+/m mice had decreased serum IgG1 Abs, demonstrating a functional effect on class switch recombination. The mechanism of this altered immune response was then examined following ex vivo stimulation of splenic B cells, where Atad5+/m cells accumulated in the S phase of the cell cycle and had reduced proliferation compared with wild-type cells. These haploinsufficient cells underwent a significant decline in activation-induced deaminase expression, resulting in decreased switch region DNA double-strand breaks and interchromosomal translocations in the Igh locus. Class switch recombination to several isotypes was also reduced in Atad5+/m cells, although the types of end-joining pathways were not affected. These results describe a defect in DNA replication that affects Igh recombination via reduced cell division.

A novel chemotherapeutic agent to treat tumors with DNA mismatch repair deficiencies

Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a MMR-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S-phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS-induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency. Cancer Res; 76(14); 4183-91. ©2016 AACR.

Reply to Kojo: Mechanisms of antioxidant-induced DNA damage

There are several different mechanisms by which antioxidants induce DNA damage in cultured cells. In a Letter to the Editor, Kojo (1) suggests that one of these mechanisms, the generation of hydrogen peroxide (H2O2) resulting from the exposure of antioxidants to high levels of oxygen in cell culture media, could be …

Abstract 2164: S100B affects MAPK signaling in malignant melanoma via a direct interaction with RSK

Establishing the mechanism by which S100B affects ERK and its downstream signaling provided insight into how S100B affects the progression of malignant melanoma and could aid in developing new pharmacological drugs. S100B is a 21.5 kDa symmetric homodimer that is highly conserved and expressed in a number of tissues and cell lines, including melanocytes. Generally, low levels of S100B have trophic effects, while higher levels can have dire consequences, as is the case in human malignant melanoma. S100B is an effective and widely used prognostic marker for malignant melanoma, with its increased level in serum being predictive of disease stage, increased recurrence, and low overall survival. More recently, S100B has been investigated as a potential contributor to cancer progression, which may be related to how it impacts cell signaling, including the MAPK pathway (BRAF-MEK-ERK). To further evaluate its significance, S100B knock-down clones were created from the WM115 melanoma cell line, and a positive correlation was found between S100B expression and cell viability, as measured by MTT assays. It was also discovered that cells with suppressed S100B expression showed significantly lower levels of ERK phosphorylation. Likewise, over-expression of S100B in the human melanoma cell line, 501-MEL, showed the reciprocal effect, with the introduction of high levels of S100B leading to increased cell viability and ERK phosphorylation. However, the phosphorylation status of ERK does not translate to all of its downstream targets. For example, increased RSK phosphorylation was observed in the S100B knock-down clones, and correspondingly, RSK phosphorylation was decreased with over-expression of S100B. Additionally, over-expression of a mutant S100B construct (E31A + E72A) that was incapable of binding calcium yielded neither effect, indicating that the effect of S100B on RSK phosphorylation was calcium-dependent. To determine if S100B interacted directly with RSK, pull-down assays were performed next. Consistent with the calcium-mutant data, RSK was detected in S100B pull-downs in the presence of calcium, but not in the presence of the calcium chelator EDTA. Changes in RSK localization was also observed, where RSK was enriched in the nucleus of WM115 cells when S100B was knocked down and diffuse in control cells. Together these data are consistent with a mechanism in which elevated S100B binds directly to RSK, preventing its phosphorylation by ERK and its subsequent translocation to the nucleus. Thus, the calcium-binding protein S100B affects MAPK signaling by increasing levels of phosphorylated ERK while simultaneously decreasing phosphorylated RSK. Together, these two effects of S100B on MAPK signaling could impact cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2164. doi:1538-7445.AM2012-2164