intra-S Checkpoint Research Articles

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

Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.

The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress.

The DNA-Binding Domain of S. pombe Mrc1 (Claspin) Acts to Enhance Stalling at Replication Barriers.

During S-phase replication forks can stall at specific genetic loci. At some loci, the stalling events depend on the replisome components Schizosaccharomyces pombe Swi1 (Saccharomyces cerevisiae Tof1) and Swi3 (S. cerevisiae Csm3) as well as factors that bind DNA in a site-specific manner. Using a new genetic screen we identified Mrc1 (S. cerevisiae Mrc1/metazoan Claspin) as a replisome component involved in replication stalling. Mrc1 is known to form a sub-complex with Swi1 and Swi3 within the replisome and is required for the intra-S phase checkpoint activation. This discovery is surprising as several studies show that S. cerevisiae Mrc1 is not required for replication barrier activity. In contrast, we show that deletion of S. pombe mrc1 leads to an approximately three-fold reduction in barrier activity at several barriers and that Mrc1’s role in replication fork stalling is independent of its role in checkpoint activation. Instead, S. pombe Mrc1 mediated fork stalling requires the presence of a functional copy of its phylogenetically conserved DNA binding domain. Interestingly, this domain is on the sequence level absent from S. cerevisiae Mrc1. Our study indicates that direct interactions between the eukaryotic replisome and the DNA are important for site-specific replication stalling.

PNAS-4, an Early DNA Damage Response Gene, Induces S Phase Arrest and Apoptosis by Activating Checkpoint Kinases in Lung Cancer Cells

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Our previous study has shown that PNAS-4 induces S phase arrest and apoptosis when overexpressed in A549 lung cancer cells. However, the underlying action mechanism remains far from clear. In this work, we found that PNAS-4 expression in lung tumor tissues is significantly lower than that in adjacent lung tissues; its expression is significantly increased in A549 cells after exposure to cisplatin, methyl methane sulfonate, and mitomycin; and its overexpression induces S phase arrest and apoptosis in A549 (p53 WT), NCI-H460 (p53 WT), H526 (p53 mutation), and Calu-1 (p53(-/-)) lung cancer cells, leading to proliferation inhibition irrespective of their p53 status. The S phase arrest is associated with up-regulation of p21(Waf1/Cip1) and inhibition of the Cdc25A-CDK2-cyclin E/A pathway. Up-regulation of p21(Waf1/Cip1) is p53-independent and correlates with activation of ERK. We further showed that the intra-S phase checkpoint, which occurs via DNA-dependent protein kinase-mediated activation of Chk1 and Chk2, is involved in the S phase arrest and apoptosis. Gene silencing of Chk1/2 rescues, whereas that of ATM or ATR does not affect, S phase arrest and apoptosis. Furthermore, human PNAS-4 induces DNA breaks in comet assays and γ-H2AX staining. Intriguingly, caspase-dependent cleavage of Chk1 has an additional role in enhancing apoptosis. Taken together, our findings suggest a novel mechanism by which elevated PNAS-4 first causes DNA-dependent protein kinase-mediated Chk1/2 activation and then results in inhibition of the Cdc25A-CDK2-cyclin E/A pathway, ultimately causing S phase arrest and apoptosis in lung cancer cells.

Nitrosative stress induces a novel intra-S checkpoint pathway in Schizosaccharomyces pombe involving phosphorylation of Cdc2 by Wee1

Excess production of nitric oxide and reactive nitrogen intermediates causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study the cell cycle regulation under nitrosative stress response. We discovered a novel intra-S-phase checkpoint that is activated in S. pombe under nitrosative stress. The mechanism for this intra-S-phase checkpoint activation is distinctly different than previously reported for genotoxic stress in S. pombe by methyl methane sulfonate. Our flow cytometry data established the fact that Wee1 phosphorylates Cdc2 Tyr15 which leads to replication slowdown in the fission yeast under nitrosative stress. We checked the roles of Rad3, Rad17, Rad26, Swi1, Swi3, Cds1, and Chk1 under nitrosative stress but those were not involved in the activation of the DNA replication checkpoint. Rad24 was found to be involved in intra-S-phase checkpoint activation in S. pombe under nitrosative stress but that was independent of Cdc25.

Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements.

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle.

Replication fork integrity and intra-S phase checkpoint suppress gene amplification.

Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs.

Developmental regulation of the Tetrahymena thermophila origin recognition complex.

The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development.

Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

Mec1-Ddc2 (ATR-ATRIP) controls the DNA damage checkpoint and shows differential cell-cycle regulation in yeast. To find regulators of Mec1-Ddc2, we exploited a mec1 mutant that retains catalytic activity in G2 and recruitment to stalled replication forks, but which is compromised for the intra-S phase checkpoint. Two screens, one for spontaneous survivors and an E-MAP screen for synthetic growth effects, identified loss of PP4 phosphatase, pph3Δ and psy2Δ, as the strongest suppressors of mec1-100 lethality on HU. Restored Rad53 phosphorylation accounts for part, but not all, of the pph3Δ-mediated survival. Phosphoproteomic analysis confirmed that 94% of the mec1-100-compromised targets on HU are PP4 regulated, including a phosphoacceptor site within Mec1 itself, mutation of which confers damage sensitivity. Physical interaction between Pph3 and Mec1, mediated by cofactors Psy2 and Ddc2, is shown biochemically and through FRET in subnuclear repair foci. This establishes a physical and functional Mec1-PP4 unit for regulating the checkpoint response.

Dinaciclib, a CDK Inhibitor, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition

Multiple myeloma (MM) cells are characterized by genomic instability, implicating aberrant DNA damage repair. They exhibit pervasive double strand breaks (DSBs), the most lethal DNA lesions, as indicated by the constitutive abundance of γH2AX foci. DSBs can be repaired through homologous recombination (HR) or nonhomologous end joining (NHEJ). We previously showed elevated HR in MM cells, which were reported to have impaired NHEJ function; they may thus depend on elevated HR for survival. Poly (ADP-ribose) polymerase 1 (PARP1), hyperactivated in MM cells and required for single-strand break (SSB) repair, when inhibited results in accumulation of SSBs that degenerate into lethal DSBs during replication. HR occurs predominantly in the S and G2 cell-cycle phases, and is the preferred route of DSB repair. Should HR be unavailable, as is the case in BRCA-deficient tumor cells, these DSBs persist and/or are repaired by the more error-prone NHEJ, ultimately leading to cell death. Cyclin-dependent kinases (CDKs), upregulated in MM and previously targeted in chemo­therapy, are known to modulate HR. CDK1 and 2 phosphorylate BRCA1, a modification considered essential for BRCA1 to function in checkpoint activation and DSB repair. CDK5 activates the intra-S and G2 checkpoints via ATM phosphorylation and mediates cell sensitivity to PARP inhibition. Previous studies have shown that a proteasome inhibitor, bortezomib, induces a state of “BRCAness” in MM cells and sensitizes the cells to PARP inhibition. We hypothesized that targeting CDKs with dinaciclib (which inhibits CDKs 1, 2, 5 and 9, and is being evaluated for MM treatment), will disrupt HR repair and create a “contextual synthetic lethality” in MM cells when combined with PARP inhibition.We investigated the effects on human MM cell lines of dinaciclib, doxorubicin (a topoisomerase-II inhibitor that induces DSBs), and veliparib (an inhibitor of PARP1/2). Confocal immuno­fluores­cence microscopy was used to quantify γH2AX, BRCA1 and RAD51 foci in order to assess dinaciclib ± veliparib effects on recruitment of HR proteins to DSB sites. PCR arrays and qRT-PCR were used to examine the effects of dinaciclib on mRNA levels of DNA-repair genes, and western blotting to measure levels of phospho-BRCA1 (at Ser1497, a CDK target site) and RAD51 proteins. Dinaciclib effects on HR were measured in a MM cell line (MM.1S) bearing a chromosomally integrated DR-GFP reporter. Cytotoxicity was determined by WST-1 and colony-formation assays. In vivo consequences of dinaciclib ± veliparib were examined in a myeloma xenograft model, CB.17/SCID mice with MM cells injected subcutaneously. Total RNA was extracted from those tumors and analyzed by qRT-PCR to determine in vivoeffects of dinaciclib on HR-repair genes.We observed significant decreases in the percentage of cells with ≥5 BRCA1 foci (47% to 6%, p<0.0001) or ≥5 RAD51 foci (26% to 4%, p<0.0001), in cells treated with veliparib plus dinaciclib, relative to veliparib alone. On the other hand, 19% of veliparib-treated cells showed ≥5 γH2AX foci compared to 52% for cells treated with veliparib plus dinaciclib (p<0.0001). Dinaciclib significantly reduced the transcript levels of genes involved in HR, including BRCA1 and RAD51 both in vitro and invivo, and elicited significant decreases (p≤0.05) in RAD51 protein levels and doxorubicin-induced phosphorylation of BRCA1 on S1497. In a DR-GFP reporter of HR events, 24% of vehicle-treated cells but only 4% of dinaciclib-treated cells were GFP+ (p<0.0001). Dinaciclib + veliparib treatment produced synergistic cytotoxicity (p≤0.05 for synergy) compared to either agent alone. MM cells were implanted subcutaneously, and tumor volume monitored in vivo over a 3-week period. Tumor volume increased 20- fold in mice exposed only to vehicle, but 9-fold with dinaciclib, 19-fold with veliparib, and 1.7-fold with the combination (p≤0.012 compared to vehicle or either agent alone). Log-rank tests for Kaplan-Meier survival curves showed a significant increase in survival (p≤0.004) for the dinaciclib+ veliparib treatment relative to single agent or vehicle treatment.In conclusion, CDK inhibition via dinaciclib inhibits HR in MM cells, and creates a contextual synthetic lethality when combined with PARP inhibition. This combination represents an effective treatment strategy for MM. DisclosuresNo relevant conflicts of interest to declare.

WT1 Protects Leukemic Cells from Cytotoxic Replicative Stress

BACKGROUND:The transcription factor WT1 is a “pan-leukemic marker”. WT1 is overexpressed in a significant number of AML, CML, ALL, and MDS cells. Moreover, the persistence of WT1 expression after chemotherapy indicates residual leukemic stem cells. Hydroxyurea can decrease the white blood cell count in leukemic patients. Hydroxyurea inhibits ribonucleotide reductase and consequently depletes the cellular dNTP pool. This process induces replicative stress and activates the intra-S phase checkpoint. The prolonged arrest of DNA replication leads to double strand DNA breaks and apoptosis. The aim of our study was to investigate whether WT1 has an impact on the survival of AML and CML cells treated with hydroxyurea. Furthermore, we analyzed whether replicative stress affects WT1 expression. We also addressed whether clinically relevant drugs modulating WT1 influence apoptosis, autophagy, and DNA damage signaling in leukemic cells.METHODS:We used human AML and CML cells (MV4-11, RS4-11, NB4, HEL, K562) and primary AML cells with various genetic lesions (FLT3-ITD, MLL-AF4, PML-RARα, JAK2V617F, BCR-ABL; p53 mutant or null) or a complex karyotype. Cells were exposed to 0,5 mM hydroxyurea and analyzed by FACS, Western Blot and mRNA expression analyses. RNAi with morpholinos targeting the WT1 mRNA as well as the tyrosine kinase inhibitor imatinib (0,1-1 µM) and epigenetic modifiers of the histone deacetylase inhibitor family (HDACi: 1,5-5 µM entinostat and 10-100 nM panobinostat) were used to decrease WT1 levels.RESULTS:1.The stability of WT1 is causally linked to the survival of leukemic cells undergoing replicative stress. Hydroxyurea-treated leukemic cells retaining WT1 (K562 and RS4-11 cells) accumulate in S phase and survive. However, cells that lose WT1 (NB4, MV4-11, HEL, and primary AML cells) succumb to apoptosis (57-70% apoptosis after 24 h; p<0,001).2.The specific elimination of WT1 in K562 cells with morpholino antisense nucleotides (10 nM) significantly sensitizes the cells to hydroxyurea-induced apoptosis (26-30% increased apoptosis rates after 24 h; p<0,005).3.The hydroxyurea-induced loss of WT1 in sensitive cells is caspase-dependent but does not require the p53 tumor suppressor. The pan-caspase inhibitor ZVAD-FMK (20-50 µM) and the ectopic overexpression of mitochondrial anti-apoptotic proteins (BCL-XL/BCL2) prevents the hydroxyurea-induced depletion of WT1 and apoptosis in NB4 cells (from 62-69% to 9-14%; p<0,001).4.Moreover, our data reveal that hydroxyurea promotes the occurrence of previously unrecognized WT1 cleavage products. These are different from the known HTRA2-induced WT1 fragments, as they contain the WT1 zinc fingers required for DNA binding. These novel cleavage products also contain the domain for transcriptional activation, but lack the domain for transcriptional repression. We demonstrate that caspases cleave the full-length WT1 protein into such fragments, which accumulate in the nucleus of hydroxyurea-treated NB4 cells.5.Resistance of BCR-ABL-positive K562 CML cells to pro-apoptotic effects of hydroxyurea can be overcome with the tyrosine kinase inhibitor imatinib. This drug inhibits the WT1 promoter and sensitizes the cells to hydroxyurea (22-48% increased apoptosis rate after 24 h; p<0,005).6.The HDACi entinostat and panobinostat suppress the WT1 gene and accelerate the proteasomal degradation of WT1 by an increased expression of the E2 ubiquitin conjugase UBCH8. The depletion of WT1 by HDACi renders K562 cells sensitive towards hydroxyurea-induced killing and causes a synergistic activation of cell death (apoptosis increased by 43-55% after 24 h; p<0,001).7.In K562 cells hydroxyurea/entinostat combinations lead to a caspase-dependent cleavage of the DNA repair enzyme PARP-1 and to the accumulation of double-strand DNA breaks.8.We further show that entinostat significantly shifts the balance from a hydroxyurea-induced autophagy program to apoptosis (from 60% to 16% autophagy; p<0,001) in K562 cells. We reveal that entinostat depletes proteins critical for autophagy and autophagosome-lysosome fusion (Beclin-1 and HDAC6) in these cells.CONCLUSIONS:Our data illustrate that WT1 is a novel biological target of hydroxyurea, independent of a specific genetic lesion and irrespective of a functional p53 pathway. The inhibition of WT1 might be therapeutically exploitable for targeting approaches against leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

DNA damage checkpoint responses in the S phase of synchronized diploid human fibroblasts.

We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m−2 UVC in early, mid- and late-S phase. The endpoints measured were the following: (1) radio-resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC-induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10-fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra-S checkpoint activated by UVC-induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.

H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly.

The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

Abstract 354: The replication stress response defect is associated with tumor-initiating cell formation

Abstract Breast cancer is the most prevalent cancer in women and early diagnosis of breast cancer greatly improves successful treatment and survival rate. Therefore, it is essential to expand current understanding in breast tumorigenesis in order to find specific treatment to target breast cancer in its precancerous or earliest stage. To date, two important observations have been made in terms of precancerous and early breast caner development. First, replication stress response (RSR), also referred to as intra-S checkpoint, serves as a barrier to prevent the occurrence of precancerous lesions and tumorigenesis. When normal cells encounter DNA damages resulting from oncogenic stress, these damages will trigger RSR and provoke cell cycle arrest for repairing damaged DNA or force damaged cells to undergo senescence or apoptosis. However, when RSR is defective, the cells with DNA damage will be able to break the barrier and transform to cancer cells. Second, tumor-initiating cells, which possess stem cell-like properties, are also required for tumorigenesis. Tumor-initiating cells are on the top of the tumor development hierarchy. Therefore, they form in the early stage and then differentiate themselves into nontumorgenic cancer cell to give the heterogeneity of tumor. Even though both processes strongly regulate tumorigenesis, it remains unclear whether defective RSR is associated with tumor-initiating cell formation. To gain insight into breast tumorigenesis from this aspect, RSR-intact cells were given oncogenic stress by exogenous expression of cyclin E, a major oncogene in breast cancer. Once the cells encounter oncogenic-stress-induced DNA damage, several key RSR genes, such as ataxia telangiectasia and Rad3 related (ATR), are then suppressed in order to allow damaged cells to escape from senescence. By measuring the expression changes of transcriptional landscape in RSR-deficient model system, the molecular profile of RSR defective cells are more similar to breast mammary stem cell and breast tumor-initiating cells. Furthermore, the RSR defective cells increase ALDH1, a marker of normal and malignant human mammary stem cell, and increase the ability of mammosphere formation, an indicator of self-renewal property of mammary epithelial stem cells.Importantly, we have also identified EGFR/MEK1 signaling as a key pathway that renders RSR defective cells the ability to escape from senescence or evade apoptosis and acquire tumor-initiating properties. Our findings not only provide insight into the RSR defective function network and the association between RSR defect and tumor-initiating cell formation in early breast cancer development, but also identify the key pathway for breast cancer target therapy and prevention. Citation Format: Curtis Chun-Jen Lin, Hui Dai, Shiaw-Yih Lin. The replication stress response defect is associated with tumor-initiating cell formation. [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 354. doi:10.1158/1538-7445.AM2014-354

Abstract SY33-04: Tumor suppression by the DNA damage response

Abstract The DNA damage response (DDR) is activated by oncogenic stress, but the mechanisms by which this occurs and the particular DDR functions that constitute barriers to tumorigenesis remain unclear. We established a mouse model of sporadic oncogene-driven breast tumorigenesis in a variety of mutant mouse strains with specific DDR deficiencies to reveal a novel role for the Mre11 complex (comprised of Mre11, Nbs1, and Rad50) in the response to oncogene activation. We demonstrate that the Mre11-mediated DDR suppresses tumorigenesis by inducing a G2 arrest and a program of chromatin alteration that, when compromised, results in oncogene-induced mammary hyperplasias that progress to invasive and metastatic breast cancers. In contrast, we provide genetic evidence that the intra-S checkpoint and the DNA damage-induced apoptosis functions of the DDR are dispensable for tumor suppression. These findings provide novel insight into the mechanism of DDR engagement by activated oncogenes, and reveal the particular DDR functions that must be overcome during tumorigenesis. We further demonstrate that impairment of these Mre11-dependent functions promotes metastatic dissemination, particularly upon secondary inactivation of the Ink4a-p19Arf (CDKN2a) locus. These findings provide insight into the mechanism of DDR engagement by activated oncogenes, and reveal a novel genetic interaction between the DDR and Ink4a-p19Arf pathways in suppression of oncogene-driven tumorigenesis and metastasis. Citation Format: John Petrini. Tumor suppression by the DNA damage response. [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 SY33-04. doi:10.1158/1538-7445.AM2014-SY33-04

Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome.

Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4Δ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1Δ cells, and checkpoint-defective mec1Δ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres.

DNA-PKcs is required to maintain stability of Chk1 and Claspin for optimal replication stress response

The ataxia telangiectasia mutated and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis is the major signaling pathway activated in response to replication stress and is essential for the intra-S checkpoint. ATR phosphorylates and activates a number of molecules to coordinate cell cycle progression. Chk1 is the major effector downstream from ATR and plays a critical role in intra-S checkpoint on replication stress. Activation of Chk1 kinase also requires its association with Claspin, an adaptor protein essential for Chk1 protein stability, recruitment and ATR-dependent Chk1 phosphorylation. We have previously reported that, on replication stress, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated by ATR at the stalled replication forks and is required for cellular resistance to replication stresses although the impact of DNA-PKcs onto the ATR signaling pathway remains elusive. Here we report that ATR-dependent Chk1 phosphorylation and Chk1 signaling are compromised in the absence of DNA-PKcs. Our investigation reveals that DNA-PKcs is required to maintain Chk1–Claspin complex stability and transcriptional regulation of Claspin expression. The impaired Chk1 activity results in a defective intra-S checkpoint response in DNA-PKcs–deficient cells. Taken together, these results suggest that DNA-PKcs, in addition to its direct role in DNA damage repair, facilitates ATR-Chk1 signaling pathway in response to replication stress.

Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair

Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G(2)-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin's intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells.

RIF1: A novel regulatory factor for DNA replication and DNA damage response signaling

DNA double strand breaks (DSBs) are highly toxic to the cells and accumulation of DSBs results in several detrimental effects in various cellular processes which can lead to neurological, immunological and developmental disorders. Failure of the repair of DSBs spurs mutagenesis and is a driver of tumorigenesis, thus underscoring the importance of the accurate repair of DSBs. Two major canonical DSB repair pathways are the non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. 53BP1 and BRCA1 are the key mediator proteins which coordinate with other components of the DNA repair machinery in the NHEJ and HR pathways respectively, and their exclusive recruitment to DNA breaks/ends potentially decides the choice of repair by either NHEJ or HR. Recently, Rap1 interacting factor 1 has been identified as an important component of the DNA repair pathway which acts downstream of the ATM/53BP1 to inhibit the 5'-3' end resection of broken DNA ends, in-turn facilitating NHEJ repair and inhibiting homology directed repair. Rif1 is conserved from yeast to humans but its function has evolved from telomere length regulation in yeast to the maintenance of genome integrity in mammalian cells. Recently its role in the maintenance of genomic integrity has been expanded to include the regulation of chromatin structure, replication timing and intra-S phase checkpoint. We present a summary of these important findings highlighting the various aspects of Rif1 functions and discuss the key implications for genomic integrity.