DNA Checkpoint Research Articles

Insight into the biogenesis of tax nuclear speckles using comparative interactome analysis

HTLV-1 Tax protein displays pleotropic activities that regulate many aspects of virus biology, host-pathogen interaction and disease development. Tax performs these various functions via protein-protein interactions and sub cellular compartmentalization appears to regulate which activities are engaged. We previously identified a novel nuclear body, Tax Speckled Structures (TSS), the biogenesis of which supports the sequestration of cellular DNA Damage Response factors by Tax to chromatin. To identify cellular proteins that drive localization of Tax to chromatin “speckles”, we conducted a Comparative Interactome Analysis of Tax and a Tax mutant missing the TSS localization signal (Tax-TSLS). Tandem affinity tagged Tax and mutant proteins were expressed in eukaryotic cells. Cytoplasmic, nuclear and chromatin fractions were generated and protein interactomes isolated by affinity purification. Those proteins that preferentially bound full-length Tax over Tax-TSLS were defined as the TSLS-specific interactome. Quantitative Ultra-high resolution tandem mass spectrometry was employed to establish the most comprehensive map of Tax-interacting proteins to date. We employed bioinformatics approaches to compare Tax interactomes and identify the functional pathways and interaction networks unique to proteins that bound the TSLS-defined region. The top functional networks for proteins that bound to the TSLS were involved in chromosomal alignment, congression, segregation, remodeling and modification. The overwhelming majority were known components of four chromatin functional super-complexes condensin I, kinesin, SWI/SNF and NURF. We also examined the protein interactions that were shared by full length and mutant Tax. These included significant interaction with DNA damage, cell cycle check point, NFkB signaling, and protein sorting pathways. A comparative analysis between the sub cellular fractions revealed that DNA damage and checkpoint signaling interactions were enriched in the nucleus/chromatin. These results demonstrate that Tax initiates TSS through binding of chromatin complexes and bridges the recruitment of DNA damage response and checkpoint signaling proteins.

Assessment of Oxidative DNA Damage by Alkaline Comet Assay in Human Essential Hypertension.

The objective of the present study was to investigate the antioxidant status and the extent of oxidative DNA damage in lymphocytes and their relation with essential hypertension (EHT). A total of 100 South Indian subjects aged 30-65 were included for the study. Of these 50 were normotensive controls (group-1) with blood pressure ≥120/80mm Hg, 50 were newly diagnosed (group-2) and were not on any antihypertensive drugs, but had systolic blood pressure ranging between 140 and 160mmHg and diastolic blood pressure 95-100mmHg and 50 newly diagnosed essential hypertensive patients underwent drug therapy for 1year was considered as group-3. Enzymatic and non-enzymatic antioxidants significantly decreased and lymphocyte DNA damage was significantly increased in newly diagnosed hypertensive patients compared with control group. The major decrease in DNA damage and significant improvement in enzymatic and non-enzymatic antioxidants were observed after 1year of antihypertensive therapy in treated group compared with newly diagnosed hypertensive patients. Total antioxidant status and lymphocyte DNA damage showed a strong negative correlation in all the three groups. Essential hypertension associated with oxidative stress which in turn causes genotoxic susceptibility to variety of disease including cancer. In the absence of DNA repair process and DNA checkpoint mechanisms, the genomic integrity is susceptible to extensive damage. In our study, increased oxidative DNA damage and decreased antioxidant levels were frequently observed in the newly diagnosed essential hypertensive patients, suggesting that oxidative stress is important in the pathogenesis of EHT. Therefore, the present study has additional clinical implication. Further investigations with large number of patients along with antioxidant supplement are highly warranted.

BMI1, ATM and DDR.

BMI1, ATM and DDR.

Abstract LB-062: XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas

Abstract Mutation and constitutive expression of MYC and BCL2 and/or BCL6 (a.k.a. double and triple-hit lymphomas) defines subsets of diffuse large B-cell lymphoma (DLBCL) pts with particularly poor outcome. Almost 60% of pts with BCL2 and MYC translocations die within six months of diagnosis due to chemorefractory disease, a prognosis that cannot be overcome with intense chemotherapy. A further hindrance to patient survival is that these double and triple-hit lymphomas are frequently found in the elderly who have limited tolerability to aggressive chemotherapeutic regimens. To identify chemoresistant double and triple-hit DLBCL, we screened 38 DLBCL cell lines for mutations in BCL2, MYC and BCL6 by FISH and response to chemotherapy administration in vitro. We found 3 triple-hit and 2 double-hit DLBCL cell lines. Previous work in our laboratory showed that the nuclear export protein, XPO1, is a critical regulator of MYC, BCL2 and BCL6 mRNA transport in DLBCL. We therefore analyzed XPO1 amplification (by PCR) and expression (by immunoblot) in these cell lines and found that all 5 cells expressed XPO1 at higher levels than centroblasts and that, in at least 2 of them, this was result of gene amplification. Inhibition of XPO1 with selinexor, a Selective Inhibitor or Nuclear Export (SINE) compound, increased nuclear localization of MYC, BCL2 and BCL6 transcripts and consequently decreased their protein expression. Moreover, selinexor administration induced chemosensitization in doxorubicin-resistant DLBCL cells through decreasing DNA damage repair mechanisms as demonstrated by comet assays. To determine the potential extent of DLBCL patients that could benefit from such a treatment, we first analyzed 110 DLBCL patient samples by immunohistochemistry and found that XPO1 expression was increased in 85 cases compared to normal tonsils. Within the cohort analyzed, 6 patients had double or triple hit lymphoma (by FISH) and all overexpressed XPO1. Since this population has higher incidence of chemorefractory disease, we decided to develop a pre-clinical model of this disease using primary patient samples. We developed two patient-derived xenograft (PDX) models representing a double and a triple-hit lymphoma. In both cases XPO1 was also amplified. We compared the molecular and pathological characteristics of 12 generations of PDXs with the original pt sample, and found that protein expression and mutations of these genes were stable. We therefore administered selinexor alone, CHOP alone (cyclophosphamide, vincristine, doxorubicin and dexamethasone) and the combination of selinexor with CHOP to these PDXs. We found that selinexor significantly decreased tumor growth, compared to vehicle or CHOP alone. Moreover, combinatorial treatment indicated that selinexor was able to revert the chemorefractoriness of these DLBCLs, without inducing toxicity by clinical chemistry or pathologic examination. Analysis of selinexor vs. vehicle treated PDXs indicated a decrease in protein levels of MYC, BCL6, BCL2 as well as DNA damage and checkpoint regulators such as CHK1. In summary, selinexor has potent anti-proliferative effects in double/triple-hit DLBCL and can be safely combined with CHOP to enhance cancer cell death. These data present a new therapeutic approach for pts with double/triple hit lymphomas, and provide rational support for the study of selinexor/CHOP combination in clinical trials. Citation Format: Rossella Marullo, ShaoNing Yang, Tami Rashal, Yosef Landesman, Robert Carlson, Sharon Shacham, Leandro C. Cerchietti. XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-062. doi:10.1158/1538-7445.AM2015-LB-062

Abstract LB-258: The NAE inhibitor pevonedistat (MLN4924) interacts with the HDAC inhibitor belinostat via disruption of the intra-S checkpoint and both HR and NHEJ DNA repair in AML cells

Abstract Protein degradation via the ubiquitin proteasome system (UPS) is regulated by cullin-RING E3-ligases (CRLs). The first-in-class investigational Nedd8-activating enzyme (NAE) inhibitor MLN4924 (pevonedistat) blocks CRL activity by inhibiting their neddylation, thereby disrupting the turnover of a subset of proteins with broad functions associated with cancer cell growth and survival pathways, including NF-κB, DNA replication, and DNA damage checkpoints, among others. Such findings provide a rationale for combining MLN4924 with HDAC inhibitors (HDACIs e.g., the recently FDA-approved belinostat) that interrupt DNA replication, checkpoints and repair. Notably, both single-agent MLN4924 and HDACIs (e.g., pracinostat) have induced CRs in AML patients. Consequently, interactions between MLN4924 and belinostat were investigated in AML. MLN4924/belinostat co-administration synergistically induced apoptosis in AML cells with various genetic backgrounds (e.g., wt- or mutant- p53 or FLT3-ITD), while p53 knock-down or enforced FLT3-ITD expression increased sensitivity to this regimen. MLN4924 blocked belinostat-induced NF-κB activation and belinostat co-administration up-regulated Bim, while Bim shRNA knockdown abrogated lethality. Following MLN4924/belinostat co-exposure, an RT2 Array revealed a distinct expression profile of DNA damage response (DDR) network genes. Significantly, belinostat diminished MLN4924-induced ATR/Chk1/Wee1 activation and Cdt1 up-regulation, abrogating the intra-S checkpoint. Notably, shRNA knockdown of Chk1 or Wee1 significantly sensitized cells to MLN4924 in the absence of belinostat. Moreover, belinostat also blocked MLN4924-mediated up-regulation of multiple proteins involved in HR (e.g., CtIP, BRCA1 S1524 and S1423 phosphorylation, FANCD2-L), while co-administration increased H3K56 and H4K16 acetylation, indicating defective NHEJ repair. As a consequence, co-treatment induced robust DSBs, chromosome pulverization, and apoptosis. This regimen sharply induced apoptosis in primary MDS/AML blasts (n = 47) bearing various poor-prognostic mutations, defined by NGS using the Cancer Hotspot Panel v2, as well as CD34+/CD38-/CD123+ populations enriched for leukemia-initiating cells (n = 25), but was minimally toxic to normal CD34+ cells. Finally, MLN4924/belinostat co-treatment profoundly diminished tumor burden and prolonged survival in both s.c. and i.v. AML xenograft models, with minimal toxicity, via the PD events observed in vitro. Collectively, these findings indicate that MLN4924 and HDACIs interact reciprocally to disable the DDR and promote DNA damage and cell death in AML cells, including those with unfavorable genetic features and primitive progenitors, while sparing their normal counterparts. This strategy warrants further consideration in AML. Citation Format: Liang Zhou, Shuang Chen, Yu Zhang, Yun Leng, Lihong Li, Hui Lin, Maciej Kmieciak, Kathryn A. Rizzo, Catherine I. Dumur, Andrea Ferreira-Gonzalez, Yun Dai, Steven Grant. The NAE inhibitor pevonedistat (MLN4924) interacts with the HDAC inhibitor belinostat via disruption of the intra-S checkpoint and both HR and NHEJ DNA repair in AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2015-LB-258

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

Effects of phosphorus deficiency and adenosine 5′-triphosphate (ATP) on growth and cell cycle of the dinoflagellate Prorocentrum donghaiense

Phosphorus (P) is one of the major nutrients required for growth of phytoplankton. While dissolved inorganic phosphorus (DIP) is the preferred form of P, phytoplankton can utilize dissolved organic phosphorus (DOP) when DIP is limited. How DIP deficiency affects the progression of the cell division cycle and how well DOP can restore normal cell cycle in phytoplankton is unclear. Here we investigated the effects of DIP limitation and supply of adenosine 5′-triphosphate (ATP) as sole P-source on the growth and cell cycle in Prorocentrum donghaiense, one of the most common harmful algae bloom dinoflagellates in East China Sea. Part of the culture under DIP-replete condition progressed through a complete cell cycle (G1→S→G2M) in 24h with a typical diel rhythm of each phase, exhibiting a daily growth rate of 0.40d−1 and average cell size 11.19±0.06μm in equivalent spherical diameter. Under phosphorus deficiency, population growth of P. donghaiense averaged at 0.09d−1 and the cell cycle was blocked in G1 phase, while cell size averaged at 11.65±0.05μm. When DIP was replaced by ATP at equivalent molar concentration, the cultures displayed similar growth rate, cell size, and cell cycle oscillation as in the DIP-replete treatment. The results suggest that phosphorus deficiency blocks the cell cycle due to the inability to complete DNA duplication or check-point protein phosphorylation while still allowing photosynthesis and metabolisms, and that DOP such as ATP can fully complement DIP in P. donghaiense.

Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) and LMP2A in Nasopharyngeal Carcinoma Cells.

Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins 1 (LMP1) and LMP2A are consistently expressed in NPC and can cooperate in bitransgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were coexpressed in the EBV-negative NPC cell line HK1 and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A coexpression reduced γH2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5 μM) of the topoisomerase II inhibitor etoposide. Regulation of γH2AX occurred before the onset of caspase activation without modulation of other DNA damage signaling mediators, including ATM, Chk1, or Chk2, and additionally was suppressed by inducers of DNA single-strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A coexpressing cells recovered from cytotoxic doses of etoposide; however, LMP1 expression was sufficient for this effect. LMP1 and LMP2A coexpression did not enhance cell growth, with a moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells. NPC is characterized by clonal EBV infection and accounts for >78,000 annual cancer cases with increased incidence in regions where EBV is endemic, such as southeast Asia. The latent proteins LMP1 and LMP2A coexpressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A coexpression suppressed activation of the DNA damage response (DDR) protein γH2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A-coexpressing cells and indicating distinct LMP1-dependent effects in the restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the cooperative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

Identification of a candidate rad1 subunit for the kinetoplastid 9-1-1 (rad9-hus1-rad1) complex.

The trimeric 9-1-1 (Rad9-Hus1-Rad1) complex plays an important role in the eukaryotic DNA damage response by recruiting DNA repair factors and checkpoint mediators to damaged sites. Extensively characterised in mammals and yeast, evidence is now emerging that 9-1-1 function is conserved beyond the relatively narrow evolutionary range of the Opisthokonts. Kinetoplastid Rad9 and Hus1 proteins have been identified and shown to be involved in the DNA damage response but Rad1 has remained elusive. In this study, PSI-BLAST iterative database searching, phylogenetic and structural modeling techniques are used to identify and characterise candidate Rad1 proteins in kinetoplastid organisms.

A Divergent Role of the SIRT1-TopBP1 Axis in Regulating Metabolic Checkpoint and DNA Damage Checkpoint

DNA replication is executed only when cells have sufficient metabolic resources and undamaged DNA. Nutrient limitation and DNA damage cause a metabolic checkpoint and DNA damage checkpoint, respectively. Although SIRT1 activity is regulated by metabolic stress and DNA damage, its function in these stress-mediated checkpoints remains elusive. Here we report that the SIRT1-TopBP1 axis functions as a switch for both checkpoints. With glucose deprivation, SIRT1 is activated and deacetylates TopBP1, resulting in TopBP1-Treslin disassociation and DNA replication inhibition. Conversely, SIRT1 activity is inhibited under genotoxic stress, resulting in increased TopBP1 acetylation that is important for the TopBP1-Rad9 interaction and activation of the ATR-Chk1 pathway. Mechanistically, weshowed that acetylation of TopBP1 changes the conformation of TopBP1, thereby facilitating its interaction with distinct partners in DNA replication and checkpoint activation. Taken together, our studies identify the SIRT1-TopBP1 axis as a key signaling mode in the regulation of the metabolic checkpoint and the DNA damage checkpoint.

Abstract 3439: Recurrent CDC25C mutations drive malignant transformation in FPD/AML

Abstract Germline mutations in RUNX1 cause familial platelet disorder with predisposit ion to acute myelogenous leukemia (FPD/AML) which is characterized by platelet defects and hematological malignancies. The molecular pathogenesis is poorly understood because of the rarity of this disease, which has impeded the clinical improvement to refine cancer therapy and to identify patients at risk for the onset of malignancies in FPD/AML. Thus, we conducted a nationwide survey to identify patients with familial thrombocytopenia or hematological malignancies. After screening germline RUNX1 mutations in 73 index patients, 13 patients in 7 pedigrees were diagnosed as having FPD/AML, of which 7 had developed hematological malignancies. The mutational spectrum of FPD/AML was unveiled by whole-exome sequencing and targeted deep sequencing, and we identified CDC25C mutation as a recurrent target of FPD/AML, which was found in 7 of 13 (53 %) patients, including 4 FPD/AML patients who had not developed malignancies, although the variant allele frequencies were much lower than those found in patients with malignancies. Furthermore, GATA2 mutations were subclonal in 3 of 7 individuals with CDC25C abnormalities. Deep sequencing enabled an accurate estimation of allele frequencies for individual mutations and prediction of hierarchical architecture, which disclosed that CDC25C mutations form a founding pre-leukemic clone, followed by stepwise acquisition of subclonal mutations that contributed to leukemia progression. Single cell sequencing validated the estimation of the clonal evolution model. In all of the mutated forms of CDC25C, their binding capacity with c-TAK1 was reduced, resulting in decreased phosphorylation of CDC25C at Ser216. As a consequence, mutated CDC25C failed to bind 14-3-3 protein efficiently and stayed in the nucleus at G1/S phase. In accordance with these observations, CDC25C mutants enhanced mitosis entry, which was exaggerated by low dose radiation-induced DNA damage. These results suggest that CDC25C mutation results in disruption of the DNA checkpoint machinery, indicating that CDC25C is a novel target in human cancer. It is known that FPD/AML-associated RUNX1 mutations evokes DNA damage and activates the DNA checkpoint mechanism. We found, however, that introduction of CDC25C mutation results in the marked enhancement of mitotic entry in spite of co-expression of RUNX1 mutation in Ba/F3 cells. Thus, premature mitosis by loss of DNA checkpoint mechanisms in the presence of mutated CDC25C may contribute to malignant transformation of RUNX1-mutated cells. In conclusion, malignant transformation of FPD/AML is formed by stepwise acquisition of mutations and clonal selection, which is initiated by a CDC25C mutation in the pre-leukemic phase and further driven by mutations in GATA2 or other genes. These findings will facilitate the diagnosis and monitoring of patients with FPD/AML who are at an increased risk of developing hematological malignancy. Citation Format: Akihide Yoshimi, Takashi Toya, Masahito Kawazu, Toshihide Ueno, Ayato Tsukamoto, Hiromitsu Iizuka, Masahiro Nakagawa, Yasuhito Nannya, Shunya Arai, Motoshi Ichikawa, Hironori Harada, Kensuke Usuki, Yasuhide Hayashi, Etsuro Ito, Keita Kirito, Hideaki Nakajima, Hiroyuki Mano, Mineo Kurokawa. Recurrent CDC25C mutations drive malignant transformation in FPD/AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3439. doi:10.1158/1538-7445.AM2014-3439

Chk1 inhibition as a novel therapeutic strategy for treating triple-negative breast and ovarian cancers.

BackgroundChk1 inhibitors are currently in clinical trials as putative potentiators of cytotoxic chemotherapy drugs. Chk1 inhibitors may exhibit single agent anti-tumor activity in cancers with underlying DNA repair, DNA damage response or DNA replication defects.MethodsHere we describe the cellular effects of the pharmacological inhibition of the checkpoint kinase Chk1 by the novel inhibitor V158411 in triple-negative breast cancer and ovarian cancer. Cytotoxicity, the effect on DNA damage response and cell cycle along with the ability to potentiate gemcitabine and cisplatin cytotoxicity in cultured cells was investigated. Western blotting of proteins involved in DNA repair, checkpoint activation, cell cycle and apoptosis was used to identify potential predictive biomarkers of Chk1 inhibitor sensitivity.ResultsThe Chk1 inhibitors V158411, PF-477736 and AZD7762 potently inhibited the proliferation of triple-negative breast cancer cells as well as ovarian cancer cells, and these cell lines were sensitive compared to ER positive breast and other solid cancer cells lines. Inhibition of Chk1 in these sensitive cell lines induced DNA damage and caspase-3/7 dependent apoptosis. Western blot profiling identified pChk1 (S296) as a predictive biomarker of Chk1 inhibitor sensitivity in ovarian and triple-negative breast cancer and pH2AX (S139) in luminal breast cancer.ConclusionsThis finding suggests that Chk1 inhibitors either as single agents or in combination chemotherapy represents a viable therapeutic option for the treatment of triple-negative breast cancer. pChk1 (S296) tumor expression levels could serve as a useful biomarker to stratify patients who might benefit from Chk1 inhibitor therapy.

Collaborator of ARF (CARF) Regulates Proliferative Fate of Human Cells by Dose-dependent Regulation of DNA Damage Signaling

Collaborator of ARF (CARF) has been shown to directly bind to and regulate p53, a central protein that controls tumor suppression via cellular senescence and apoptosis. However, the cellular functions of CARF and the mechanisms governing its effect on senescence, apoptosis, or proliferation are still unknown. Our previous studies have shown that (i) CARF is up-regulated during replicative and stress-induced senescence, and its exogenous overexpression caused senescence-like growth arrest of cells, and (ii) suppression of CARF induces aneuploidy, DNA damage, and mitotic catastrophe, resulting in apoptosis via the ATR/CHK1 pathway. In the present study, we dissected the cellular role of CARF by investigating the molecular pathways triggered by its overexpression in vitro and in vivo. We found that the dosage of CARF is a critical factor in determining the proliferation potential of cancer cells. Most surprisingly, although a moderate level of CARF overexpression induced senescence, a very high level of CARF resulted in increased cell proliferation. We demonstrate that the level of CARF is crucial for DNA damage and checkpoint response of cells through ATM/CHK1/CHK2, p53, and ERK pathways that in turn determine the proliferative fate of cancer cells toward growth arrest or proproliferative and malignant phenotypes. To the best of our knowledge, this is the first report that demonstrates the capability of a fundamental protein, CARF, in controlling cell proliferation in two opposite directions and hence may play a key role in tumor biology and cancer therapeutics.

RNF 4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.

Genotoxic Anti-Cancer Agents and Their Relationship to DNA Damage, Mitosis, and Checkpoint Adaptation in Proliferating Cancer Cells

When a human cell detects damaged DNA, it initiates the DNA damage response (DDR) that permits it to repair the damage and avoid transmitting it to daughter cells. Despite this response, changes to the genome occur and some cells, such as proliferating cancer cells, are prone to genome instability. The cellular processes that lead to genomic changes after a genotoxic event are not well understood. Our research focuses on the relationship between genotoxic cancer drugs and checkpoint adaptation, which is the process of mitosis with damaged DNA. We examine the types of DNA damage induced by widely used cancer drugs and describe their effects upon proliferating cancer cells. There is evidence that cell death caused by genotoxic cancer drugs in some cases includes exiting a DNA damage cell cycle arrest and entry into mitosis. Furthermore, some cells are able to survive this process at a time when the genome is most susceptible to change or rearrangement. Checkpoint adaptation is poorly characterised in human cells; we predict that increasing our understanding of this pathway may help to understand genomic instability in cancer cells and provide insight into methods to improve the efficacy of current cancer therapies.

TRIP/NOPO E3 ubiquitin ligase promotes ubiquitylation of DNA polymerase η

We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.

Chemoprevention by Resveratrol in Head-and-Neck Cancer: Molecular Biology and Therapeutics

The nutraceutical resveratrol (trans-3,4',5 Trihydroxystilbene) is a potent but non-toxic chemopreventive agent. Resveratrol arrested HNSCC cells, but not primary cells, in the S phase by damaging DNA, activating the checkpoint and producing apoptosis (Tyagi et al., 2005; 2011). We propose that resveratrol slows DNA replication by inhibition of DNA polymerase resulting in DNA replication fork breakage, damage and apoptosis in cancer cells that lack DNA repair and checkpoint pathways. Our pre-clinical studies are focused on HNSCC because of the possibility of using oral administration of resveratrol to prevent recurrence and secondary malignancies in HNSCC patients after radiation and/or chemotherapy.

Bufalin induces cell death in human lung cancer cells through disruption of DNA damage response pathways.

Bufalin is a key component of a Chinese medicine (Chan Su) and has been proved effective in killing various cancer cells. Its role in inducing DNA damage and the inhibition of the DNA damage response (DDR) has been reported, but none have studied such action in lung cancer in detail. In this study, we demonstrated bufalin-induced DNA damage and condensation in NCI-H460 cells through a comet assay and DAPI staining, respectively. Western blotting indicated that bufalin suppressed the protein levels associated with DNA damage and repair, such as a DNA dependent serine/threonine protein kinase (DNA-PK), DNA repair proteins breast cancer 1, early onset (BRCA1), 14-3-3 σ (an important checkpoint keeper of DDR), mediator of DNA damage checkpoint 1 (MDC1), O6-methylguanine-DNA methyltransferase (MGMT) and p53 (tumor suppressor protein). Bufalin could activate phosphorylated p53 in NCI-H460 cells. DNA damage in NCI-H460 cells after treatment with bufalin up-regulated its ATM and ATR genes, which encode proteins functioning as sensors in DDR, and also up-regulated the gene expression (mRNA) of BRCA1 and DNA-PK. But bufalin suppressed the gene expression (mRNA) of p53 and 14-3-3 σ, however, bufalin did not significantly affect the mRNA of MGMT. In conclusion, bufalin induced DNA damage in NCI-H460 cells and also inhibited its DNA repair and checkpoint function.