Activation Of DNA Damage Response Research Articles

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

Romidepsin enhances the cytotoxicity of fludarabine, clofarabine and busulfan combination in malignant T-cells

Novel approaches to pre-transplant conditioning are needed to improve treatment of advanced T-cell malignancies. We investigated the synergism of fludarabine (Flu), clofarabine (Clo), busulfan (Bu), and romidepsin (Rom) in T-cell lines and patient-derived cell samples. [Flu+Clo+Bu+Rom] had combination indexes of 0.4–0.5 at ∼50% cytotoxicity in PEER and SUPT1 cells, suggesting synergism. Drug exposure resulted in histone modifications, DNA-damage response (DDR), increased reactive oxygen species (ROS), decreased glutathione (GSH) and mitochondrial membrane (MM) potential, and apoptosis. Similar activation of DDR and apoptosis was observed in patient samples. The PI3K-AKT-mTOR, NFκB, Raf-MEK-ERK, JAK-STAT and Wnt/β-catenin pro-survival pathways were inhibited by the 4-drug combination. The SAPK/JNK stress pathway was activated. A novel finding was the down-regulation of the drug transporter MRP1. We propose the following mechanisms of synergism: Flu, Clo and Rom induce histone modifications and chromatin remodeling, exposing DNA to Bu alkylation; the increased production of ROS, due to drug-mediated stress response and decreased GSH, damages the MM causing leakage of pro-apoptotic factors; down-regulation of MRP1 increases intracellular Bu concentration and exacerbates the DDR; and inhibition of multiple survival pathways. Our results provide the basis for a clinical trial to evaluate [Flu+Clo+Bu+Rom] as part of conditioning regimen for refractory T-cell malignancy patients undergoing stem cell transplantation.

Cdc6 contributes to cisplatin-resistance by activation of ATR-Chk1 pathway in bladder cancer cells.

High activation of DNA damage response is implicated in cisplatin (CDDP) resistance which presents as a serious obstacle for bladder cancer treatment. Cdc6 plays an important role in the malignant progression of tumor. Here, we reported that Cdc6 expression is up-regulated in bladder cancer tissues and is positively correlated to high tumor grade. Cdc6 depletion can attenuate the malignant properties of bladder cancer cells, including DNA replication, migration and invasion. Furthermore, higher levels of chromatin-binding Cdc6 and ATR were detected in CDDP-resistant bladder cancer cells than in the parent bladder cancer cells. Intriguingly, down-regulation of Cdc6 can enhance sensitivity to CDDP both in bladder cancer cells and CDDP-resistant bladder cancer cells. Cdc6 depletion abrogates S phase arrest caused by CDDP, leading to aberrant mitosis by inactivating ATR-Chk1-Cdc25C pathway. Our results indicate that Cdc6 may be a promising target for overcoming CDDP resistance in bladder cancer.

Editorial: DNA and RNA Metabolism Meet at Chromatin to Control Genome Stability.

Editorial: DNA and RNA Metabolism Meet at Chromatin to Control Genome Stability.

Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization.

There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including γH2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of aberrant mRNA and protein products encoded by damaged genes.

Efficient Activation of Apoptotic Signaling during Mitotic Arrest with AK301.

Mitotic inhibitors are widely utilized chemotherapeutic agents that take advantage of mitotic defects in cancer cells. We have identified a novel class of piperazine-based mitotic inhibitors, of which AK301 is the most potent derivative identified to date (EC50 < 200 nM). Colon cancer cells arrested in mitosis with AK301 readily underwent a p53-dependent apoptosis following compound withdrawal and arrest release. This apoptotic response was significantly higher for AK301 than for other mitotic inhibitors tested (colchicine, vincristine, and BI 2536). AK301-treated cells exhibited a robust mitosis-associated DNA damage response, including ATM activation, γH2AX phosphorylation and p53 stabilization. The association between mitotic signaling and the DNA damage response was supported by the finding that Aurora B inhibition reduced the level of γH2AX staining. Confocal imaging of AK301-treated cells revealed multiple γ-tubulin microtubule organizing centers attached to microtubules, but with limited centrosome migration, raising the possibility that aberrant microtubule pulling may underlie DNA breakage. AK301 selectively targeted APC-mutant colonocytes and promoted TNF-induced apoptosis in p53-mutant colon cancer cells. Our findings indicate that AK301 induces a mitotic arrest state with a highly active DNA damage response. Together with a reversible arrest state, AK301 is a potent promoter of a mitosis-to-apoptosis transition that can target cancer cells with mitotic defects.

NF-κB-HOTAIR axis links DNA damage response, chemoresistance and cellular senescence in ovarian cancer.

The transcription factor nuclear factor kappa B (NF-κB) and the long non-coding RNA (lncRNA) HOTAIR (HOX transcript antisense RNA) play diverse functional roles in cancer. In this study, we show that upregulation of HOTAIR induced platinum resistance in ovarian cancer, and increased HOTAIR levels were observed in recurrent platinum-resistant ovarian tumors vs. primary ovarian tumors. To investigate the role of HOTAIR during DNA damage induced by platinum, we monitored double-strand breaks and show that HOTAIR expression results in sustained activation of DNA damage response after platinum treatment. We demonstrate that ectopic expression of HOTAIR induces NF-κB activation during DNA damage response and MMP-9 and IL-6 expression, both key NF-κB target genes. We show that HOTAIR regulates activation of NF-κB by decreasing Iκ-Bα (NF-κB inhibitor) and establish that by inducing prolonged NF-κB activation and expression of NF-κB target genes during DNA damage, HOTAIR plays a critical role in cellular senescence and platinum sensitivity. Our findings suggest that a NF-κB-HOTAIR axis drives a positive-feedback loop cascade during DNA damage response and contributes to cellular senescence and chemotherapy resistance in ovarian and other cancers.

Vitamin D/vitamin D receptor axis regulates DNA repair during oncogene-induced senescence.

Oncogenic Ras expression is associated with activation of the DNA damage response (DDR) pathway, as evidenced by elevated DNA damage, primarily DNA double-strand breaks (DSBs), and activation of DNA damage checkpoints, which in primary human cells leads to entry into senescence. DDR activation is viewed as a physiological barrier against uncontrolled proliferation in oncogenic Ras-expressing cells, and arises in response to genotoxic stress due to the production of reactive oxygen species (ROS) that damage DNA, and to hyper-replication stress. Although oncogene-induced senescence (OIS) is considered a tumor suppressor mechanism, the accumulation of DNA damage in senescent cells is thought to cause genomic instability, eventually allowing secondary hits in the genome that promote tumorigenesis. To date, the molecular mechanisms behind DNA repair defects during OIS remain poorly understood. Here, we show that oncogenic Ras expression in human primary cells results in down-regulation of BRCA1 and 53BP1, two key factors in DNA DSBs repair by homologous recombination (HR) and non-homologous end joining (NHEJ), respectively. As a consequence, Ras-induced senescent cells are hindered in their ability to recruit BRCA1 and 53BP1 to DNA damage sites. While BRCA1 is down-regulated at transcripts levels, 53BP1 loss is caused by activation of cathepsin L (CTSL)-mediated degradation of 53BP1 protein. Moreover, we discovered a marked down-regulation of vitamin D receptor (VDR) during OIS, and a role for the vitamin D/VDR axis regulating the levels of these DNA repair factors during OIS. This study reveals a new functional relationship between the oncogene Ras, the vitamin D/VDR axis, and the expression of DNA repair factors, in the context of OIS. The observed deficiencies in DNA repair factors in senescent cells could contribute to the genomic instability that allows senescence bypass and tumorigenesis.

LZ-106, a novel analog of enoxacin, inducing apoptosis via activation of ROS-dependent DNA damage response in NSCLCs

Lung cancer, especially non-small-cell lung cancer (NSCLC), plays the leading role in cancer which is closely related to a myriad of fatal results. Unfortunately, current molecular mechanisms and clinical treatment of NSCLC still remain to be explored despite the fact that intensive investigations have been carried out in the last two decades. Recently, growing attention to finding exploitable sources of anticancer agents is refocused on quinolone compounds, an antibiotic with a long period of clinic application, for their remarkable cell-killing activity against not only bacteria, but eukaryotes as well. In this study, we found LZ-106, an analog of enoxacin, exhibiting potent inhibitory effects on NSCLC in both cultured cells and xenograft mouse model. We identified apoptosis-inducing action of LZ-106 in NSCLC cells through the mitochondrial and endoplasmic reticulum (ER)-stress apoptotic pathways via Annexin-V/PI double-staining assay, membrane potential detection, calcium level detection and the expression analysis of the key apoptotic proteins. Through comet assay, reactive oxygen species (ROS) detection, the expression analysis of DNA damage response (DDR) marker γ-H2AX and other DDR-related proteins, we also demonstrated that LZ-106 notably induced ROS overproduction and DDR. Interestingly, additional evidence in our findings revealed that DDR and apoptosis could be alleviated in the presence of ROS scavenger N-acetyl-cysteine (NAC), indicating ROS-dependent DDR involvement in LZ-106-induced apoptosis. Thus our data not only offered a new therapeutic candidate for NSCLC, but also put new insights into the pharmacological research of quinolones.

Magnification of Inter-individual Variation in Biological Responses After Low Doses and Dose-Rates of Ionizing Radiation

Biological responses of human cells and tissues to ionizing radiation (IR) are strongly influenced by dose and dose-rate. Unlike the robust activation of cellular DNA damage responses (DDR) seen after high IR doses, the efficiency of activation of DNA damage repair and signaling pathways after much lower doses and dose-rates varies appreciably among different individuals. Genomic and functional assays measuring low dose and dose-rate IR responses repeatedly show increased inter-individual variability when cells and tissues experience DNA damage levels comparable to those experienced endogenously (due to aerobic metabolism, diet, lifestyle, etc). Complicating matters for risk assessment are recent observations of dose-response non-linearity (hyper-linearity) in the low dose range. With both physical and biological factors strongly influencing individual responses to IR at low doses and dose-rates, further radiobiological research is required to assist regulatory agencies in determining appropriate radiological protection standards for such exposures.

Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage

Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2- and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs.

MASTL(Greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation

The G2 DNA damage checkpoint is one of the most important mechanisms controlling G2–mitosis transition. The kinase Greatwall (MASTL in human) promotes normal G2–mitosis transition by inhibiting PP2A via ARPP19 and ENSA. In this study, we demonstrate that MASTL is critical for maintaining genome integrity after DNA damage. Although MASTL did not affect the activation of DNA damage responses and subsequent repair, it determined the timing of entry into mitosis and the subsequent fate of the recovering cells. Constitutively active MASTL promoted dephosphorylation of CDK1Tyr15 and accelerated mitotic entry after DNA damage. Conversely, downregulation of MASTL or ARPP19/ENSA delayed mitotic entry. Remarkably, APC/C was activated precociously, resulting in the damaged cells progressing from G2 directly to G1 and skipping mitosis all together. Collectively, these results established that precise control of MASTL is essential to couple DNA damage to mitosis through the rate of mitotic entry and APC/C activation.

The dark side of circulating nucleic acids.

SummaryFree circulating or cell‐free DNA (cfDNA), possibly from dying cells that release their contents into the blood as they break down, have become of major interest as a source for noninvasive diagnostics. Recent work demonstrated the uptake of human cfDNA in mouse cells in vitro and in vivo, accompanied by the activation of a cellular DNA damage response (DDR) and the appearance of apoptotic proteins in the host cells. By acting as a source of mobile genetic elements, cfDNA could be a continuous source of DNA mutagenesis of healthy cells in the body throughout life, promoting progressive cellular aging in vivo. As such, cfDNA may causally contribute to multiple aging‐related diseases, such as cancer, diabetes, and Alzheimer's disease.

A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells.

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.

Robust DNA Damage Response and Elevated Reactive Oxygen Species in TINF2-Mutated Dyskeratosis Congenita Cells

Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients’ cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism whereby telomerase deficiency and subsequent shortened telomeres initiate a DDR and create a pro-oxidant environment, especially in cells carrying the TINF2 mutations. Finally, the ameliorative effects of antioxidants in vitro suggest this could translate to therapeutic benefits in DC patients.

MLL1 is essential for the senescence-associated secretory phenotype.

Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces "SASP-like" inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS- or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression.

Abstract A47: Chromatin remodeling factor SMARCA4 as a predictive biomarker of cisplatin therapy in non-small cell lung cancer

Abstract Adjuvant cisplatin-based chemotherapy is recommended for patients with completely resected non-small cell lung cancer (NSCLC). However, due to limited efficacy of this therapy, studies are required to identify biomarkers to select patients who would derive the most benefit of the therapy. SMARCA4 (also known as BRG1) and SMARCA2 (also known as BRM) are two important mutually exclusive catalytic subunits with ATPase activity of the mammalian chromatin remodeling complex SWItch/Sucrose NonFermentable (SWI/SNF). These subunits are required for mammalian development and are altered in a variety of malignancies including lung, prostate, pancreatic, breast, and colon. Several studies have reported that both SMARCA4 and SMARCA2 are required for the full activation of DNA damage response by ATM-mediated activation of γ-H2AX. As SMARCA4 is frequently deleted in many primary tumors including NSCLC, we hypothesized that the loss of SMARCA4 may enhance the sensitivity of tumor cells to chemotherapeutic agents causing DNA damage and may serve as predictive biomarkers of sensitivity for these agents. To that end, in the present study we evaluated the association between SMARCA4 and/or SMARCA2 alterations and the outcome of DNA-damaging chemotherapeutic cisplatin in NSCLC. We used a gene expression profiling microarray (n=133) from both control and treatment arms of the North American Intergroup phase III trial of adjuvant cisplatin plus vinorelbine (JBR.10). Kaplan-Meier method and log-rank tests were used to estimate and test the differences of probabilities in overall survival and disease-specific survival between expression groups and treatment arms. Multivariate Cox regression models were used while adjusting for other baseline clinical covariates. Improved five-year disease-specific survival was detected only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine compared with the observational arm (HR=0.1, 95% CI: 0.0.0·5, P=0.001 (low); HR 1.1, 95% CI: 0·5-2.4, P=0·76 [high]). The interaction test was statistically significant (P=0.007). In contrast, no significant survival benefits were noted in patients with high SMARCA4 or high SMARCA2 expression. Taken together, these results suggest that decreased expression of chromatin remodeling factors SMARCA4 may serve as putative predictive biomarkers of platinum-based therapy (cisplatin) in NSCLC and requires further validation. Citation Format: Arup R. Chakraborty, Erica Bell, Mo Xiaokui, Ziyan Liu, Konstantin Shilo, Simon Kirste, Petra Stegmaier, Maureen McNulty, Niki Karachaliou, Rafael Rosell, Gerold Bepler, David P. Carbone, Arnab Chakravarti. Chromatin remodeling factor SMARCA4 as a predictive biomarker of cisplatin therapy in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A47.

Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells.

Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells.

Enhanced efficacy of combined HDAC and PARP targeting in glioblastoma

Recent clinical trials have demonstrated that targeting chromatin remodeling factors is as a promising strategy for the treatment of glioblastoma (GBM). We and others have shown constitutive activation of DNA damage response (DDR) pathways in gliomas and suggested that targeting the DDR may improve the currently grim prognosis for patients. Based on our previous findings that inhibition of poly(ADP-ribose) polymerase (PARP) increases radio-sensitivity of the notoriously radio-resistant GBM cells, we hypothesized that epigenetic down-regulation of the DDR responses and induction of oxidative stress via HDAC inhibition would contribute to more efficient targeting of this deadly disease. Our data show that SAHA, an HDAC class I + II inhibitor, in combination with olaparib (PARP inhibitor): i) enhanced inhibition of GBM cell survival, ii) induced apoptosis, and iii) impaired cell cycle progression. These results provide a pre-clinical rationale for combined administration of SAHA and olaparib, which are already individually in clinical trials.

P19(Arf) is required for the cellular response to chronic DNA damage.

The p53 tumor suppressor is a stress sensor, driving cell-cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19Arf tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19Arf-independent. Accordingly, p19Arf-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19Arf is required for replicative senescence, a condition associated with an activated DNA damage response, as p19Arf−/− MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19Arf is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19Arf has a specific role. We therefore examined the role of p19Arf in cellular responses to chronic, low-dose DNA damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5 Gy γ-irradiation daily or 150μM hydroxyurea, a replication stress-inducer. In contrast to their response to acute DNA damage, p19Arf−/− MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19Arf in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19Arf−/− MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19Arf in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA damaging agent treatment is insufficient to promote senescence in p19Arf−/− MEFs, suggesting that the role of p19Arf in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19Arf for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19Arf serves as a tumor suppressor. Moreover, our study helps reconcile reports suggesting crucial roles for both p19Arf and DNA damage signaling pathways in tumor suppression.