Replication Checkpoint Kinase Research Articles

Abstract 6177: ATRN-119 and ATRN-W1051: Novel and potentially well tolerated ATR and WEE1 inhibitors for targeted cancer treatment

Abstract Previous studies have demonstrated the promise of ATR and WEE1 inhibitors (ATRi and WEE1i) as cancer treatments. Inhibition of these DNA replication checkpoint kinases disrupts normal DNA synthesis and causes replication fork collapse, and these effects are accentuated by numerous cancer-associated mutations. However, a key limitation to this approach in clinical trials has been the occurrence of adverse hematological effects, including anemia, neutropenia, and thrombocytopenia. Herein, we describe two novel inhibitors, ATRN-119 (ATRi) and ATRN-W1051 (WEE1i), that exhibit increased target selectivity that potentially decreases these dose-limiting toxicities. ATRN-119, the first macrocyclic ATRi to enter clinical trials, is highly specific for inhibition of ATR relative to other phosphatidylinositol kinase-related kinases (PIKKs), such as ATM, DNA-PK, and MTOR. This specificity correlates with increased tolerability, which permits continuous dosing in animal models. Importantly, dose scheduling of ATRN-119 that suppresses tumor growth in xenograft mouse models of colon, pancreatic, and prostate cancers causes no appreciable loss of body weight or hematologic toxicity (anemia, neutropenia or thrombocytopenia). Daily dosing is also tolerated in combination with PARP inhibition, which causes significant tumor reduction in a BRCA2-defient PDX model of high-grade serous ovarian cancer. These findings have led a biomarker-driven Phase I clinical trial of ATRN-119 with daily dosing for ovarian and other cancers (Simpkins, PI). To potentially reduce the toxicity of WEE1i, a new potent and selective WEE1 inhibitor, ATRN-W1051, was developed. In vitro, ATRN-W1051 inhibits WEE1 with an IC50 of 2.2 nM and limits the proliferation of ovarian cancer cell lines in the 100 nM to 200 nM range. Importantly, ATRN-W1051 exhibits low off-target inhibition of the PLK family of kinases (PLK1, PLK2 and PLK3), which are substantially inhibited by the leading WEE1i in the clinic (AZD1775 and ZN-c3)1. In addition, ATRN-W1051 also has potentially favorable pharmacokinetic properties that permits 3-8 times lower dosing than AZD1775 or ZN-c3 to achieve similar exposure (AUC0-24) levels2. Notably, daily oral dosing of ATRN-W1051 is well tolerated in mice and suppresses the growth of CCNE1-amplified HGSOC xenografted tumors. These preclinical data suggest that ATRN-W1051 may be a potential therapeutic candidate for CCNE1-overexpressing HGSOC. Together, ATRN-119 and ATRN-W1051 provide promising alternative DNA replication checkpoint inhibitors for the treatment of a variety of cancers. Citation Format: Joseph Vacca, Stephen Rocca, Justin Frye, Steven J. Schnell, Molly Hansbarger, Gregory Korbel, Fiona Simpkins, Eric J. Brown, Oren Gilad. ATRN-119 and ATRN-W1051: Novel and potentially well tolerated ATR and WEE1 inhibitors for targeted cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6177.

Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis

Venetoclax plus cytarabine therapy is approved for elderly acute myeloid leukemia (AML) patients and needs further improvement. We studied the mechanisms of venetoclax plus cytarabine treatment and searched for a third agent to enhance their effects. Cytarabine induces S phase arrest-mediated DNA damage with activation of DNA replication checkpoint kinase 1 (Chk1) through phosphorylation, while venetoclax induces B cell lymphoma 2 (Bcl-2)-interacting mediator of cell death (Bim)-mediated apoptotic DNA damage. Myeloid cell leukemia-1 (Mcl-1) plays negative roles in both events by sequestering Bim and accelerating Chk1 phosphorylation. Venetoclax releases Bim from Bcl-2 with increased Bim binding to Mcl-1. Artesunate, an antimalaria drug, induces Noxa to replace Bim from Mcl-1 and induces synergistic apoptosis with venetoclax accompanied with Mcl-1 reduction. Silencing Mcl-1 or adding venetoclax/artesunate diminishes the cytarabine resistance pathway p-Chk1. The triple combination exhibits S phase arrest with enhanced DNA damage, improves AML colony formation inhibition, and prolongs survival of two mice xenograft models compared to the venetoclax/cytarabine dual combination. Artesunate serves as a bridge for venetoclax and cytarabine combination by Noxa and Bim-mediated apoptosis and Mcl-1 reduction. We provide a new triple combination for AML treatment by targeting the Noxa/Mcl-1/Bim axis to reverse Mcl-1/p-Chk1 resistance of cytarabine therapy.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. Invivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis invitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors.

Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant.

Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress.

Checkpoint-Mediated DNA Polymerase ε Exonuclease Activity Curbing Counteracts Resection-Driven Fork Collapse

DNA polymerase epsilon (Pole) carries out leading strand synthesis with high fidelity owing to its exonuclease activity. Pole polymerase and exonuclease activities are balanced, due to partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. Here we show that stalled Pole drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Pole catalytic subunit Pol2 is phosphorylated on serine 430, which influences partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork functional collapse. Conversely, non-phosphorylatable pol2-S430A expression results in resection-driven collapse of stressed forks. Our findings reveal that checkpoint kinases switch Pole to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Pole roles in genome integrity maintenance.

Abstract PR09: APOBEC3A sensitizes leukemia cells to inhibitors of the replication checkpoint

Abstract The human APOBEC3 family of DNA-cytosine deaminases comprises seven members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 enzymes are best defined by their capacity to restrict viral infection, however several family members are also capable of causing cellular DNA damage that leads to activation of DNA damage responses. Mutational signatures consistent with APOBEC3 activity have been identified in cancer genomes, and elevated expression of APOBEC3 enzymes has been observed in solid tumors. These findings point to a role for APOBEC3 enzymes in cancer-associated mutagenesis, however the impact of APOBEC3 activity within cancer cells has not been examined to date. We hypothesized that A3A is a potential source of mutagenesis in leukemia, since it is endogenously expressed at high levels in hematopoietic cells. To identify hematologic malignancies in which A3A expression is elevated, we examined RNA sequencing from adult and pediatric leukemias in the TCGA and TARGET databases. We found elevated A3A expression in a subset of pediatric and adult acute myeloid leukemia (AML). Additionally, we found that high A3A expression was associated with decreased survival, suggesting that new therapeutic options are important for patients with this type of malignancy. Since we have previously demonstrated that A3A acts on the cellular genome during replication, we hypothesized that abrogation of the replication checkpoint will produce genotoxicity and cell death in leukemias with elevated A3A expression. We therefore investigated the DNA damage response to A3A activity in leukemia cells by generating cell lines with inducible A3A expression. We found increased Ataxia Telangiectasia and Rad3-Related (ATR)-mediated phosphorylation of Chk1 (at serine 317 and serine 345) by Western blotting, and G2 arrest by flow cytometry. These results demonstrate that A3A causes replication checkpoint activation in leukemia cell lines. We investigated the potential for selective targeting of A3A-expressing AML cells by inhibition of replication checkpoint kinases. We found that A3A expression sensitized THP1 cells to ATR kinase inhibition, resulting in decreased viability, apoptosis, and abrogation of cell cycle arrest. We also found that Chk1 inhibition combined with A3A expression results in synthetic lethality in these cells. We determined that synthetic lethality with A3A is specific to the inhibition of ATR-Chk1 pathway and does not occur after inhibition of Ataxia Telangiectasia Mutated (ATM). We propose that A3A acts on ssDNA during replication in leukemia cells. ATR-Chk1 signaling is required for genome protection when A3A is active, and inhibition of this checkpoint results in cytotoxicity. These data reveal a new vulnerability of AML cells with elevated A3A expression and suggest that A3A is a biomarker for treatment with ATR kinase inhibitors. This abstract is also being presented as Poster B18. Citation Format: Abby M. Green, Konstantin Budagyan, Katharina E. Hayer, Matthew D. Weitzman. APOBEC3A sensitizes leukemia cells to inhibitors of the replication checkpoint [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR09.

Abstract IA14: Mechanisms of the ATR-dependent replication stress response

Abstract Replication stress can be caused by DNA damage, difficult to replicate DNA sequences, collisions between replication and transcriptional machineries, and aberrations in the replication timing or other regulatory mechanisms. Cancer cells often have elevated levels of replication stress driven by oncogenes. Replication stress response pathways are important in these contexts to allow cells to complete replication, maintain genome stability, and remain viable. Drugs that increase the replication stress burden or inactivate components of the replication stress response pathway can be useful as cancer therapeutics. For example, inhibitors of the replication checkpoint kinase ATR are currently being tested in clinical trials. To better understand how replication stress response pathways operate, we have taken both genetic and proteomic approaches. These approaches include purifying active and stressed replication fork proteomes using isolation of proteins on nascent DNA (iPOND). iPOND can monitor changes in the replication fork proteome and when combined with mass spectrometry provides an unbiased analysis. These approaches have allowed us characterize the consequences of replication stress and functions of ATR. For example, we found that ATR does not regulate the stability of the replisome itself in response to stress, but it does direct the action of a number of fork remodeling enzymes including SMARCAL1 that act to protect and repair damaged forks. In addition, iPOND identified new replication stress response proteins including ETAA1—a new regulator of the ATR checkpoint kinase. I will present our latest data on the replication stress response emphasizing the results from iPOND proteomic analyses. Citation Format: David Cortez. Mechanisms of the ATR-dependent replication stress response [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA14.

Biochemical Fractionation of Time-Resolved Drosophila Embryos Reveals Similar Transcriptomic Alterations in Replication Checkpoint and Histone mRNA Processing Mutants

In higher eukaryotes, maternally provided gene products drive the initial stages of embryogenesis until the zygotic transcriptional program takes over, a developmental process called the midblastula transition (MBT). In addition to zygotic genome activation, the MBT involves alterations in cell-cycle length and the implementation of DNA damage/replication checkpoints that serve to monitor genome integrity. Previous work has shown that mutations affecting histone mRNA metabolism or DNA replication checkpoint factors severely impact developmental progression through the MBT, prompting us to characterize and contrast the transcriptomic impact of these genetic perturbations. In this study, we define gene expression profiles that mark early embryogenesis in Drosophila through transcriptomic analyses of developmentally staged (early syncytial versus late blastoderm) and biochemically fractionated (nuclear versus cytoplasmic) wild-type (wt) embryos. We then compare the transcriptomic profiles of loss-of-function mutants of the Chk1/Grapes replication checkpoint kinase and the stem loop binding protein (SLBP), a key regulator of replication-dependent histone mRNAs. Our analysis of RNA spatial and temporal distribution during embryogenesis offers new insights into the dynamics of early embryogenesis. In addition, we find that grp and Slbp mutant embryos display profound and highly similar defects in gene expression, most strikingly in zygotic gene expression, compromising the transition from a maternal to a zygotic regulation of development.

Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks.

The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. Here, we successfully observed recruitment of FANCM at stalled forks using a newly developed protocol. We report that the FANCM recruitment depends upon its intrinsic DNA translocase activity, and its DNA-binding partner FAAP24. Moreover, it is dependent on the replication checkpoint kinase, ATR; but is independent of the FA core and FANCD2–FANCI complexes, two essential components of the FA pathway, indicating that the FANCM recruitment occurs downstream of ATR but upstream of the FA pathway. Interestingly, the recruitment of FANCM requires its direct interaction with Bloom syndrome complex composed of BLM helicase, Topoisomerase 3α, RMI1 and RMI2; as well as the helicase activity of BLM. We further show that the FANCM–BLM complex interaction is critical for replication stress-induced FANCM hyperphosphorylation, for normal activation of the FA pathway in response to ICLs, and for efficient traverse of ICLs by the replication machinery. Epistasis studies demonstrate that FANCM and BLM work in the same pathway to promote replication traverse of ICLs. We conclude that FANCM and BLM complex work together at stalled forks to promote both FA repair and replication traverse pathways of ICLs.

Abstract 4384: Selective cross-cohort discovery of transcriptional mechanisms presiding over high-risk neuroblastoma subtype state maintenance

Abstract BACKGROUND: Neuroblastoma (NBL) is the most common extracranial solid tumor in children. High-risk NBLs progress to metastatic disease and have 5-year survival of only ∼40%, despite intensive multimodal therapy. This malignancy is characterized by significant heterogeneity, both clinical and molecular, which is still poorly understood. Rather than focusing on its initiating genetic events, which are highly idiosyncratic, we focused on the core regulatory machinery responsible for implementation and maintenance of tumor state. This approach led to elucidating three molecularly distinct subtypes of high-risk NBLs, as well as the core regulatory machinery responsible for their implementation and stability, including canalization and integration of mutational events and regulation of the genetic programs that represent the hallmarks of this disease. METHODS: We dissected large-scale gene expression profiling data available from TARGET and NRC Consortium by clustering algorithm and established three subtypes of high-risk NBL, followed by identification of master regulators (MR)s of each subtype by Master Regulator Inference algorithm (Lefebvre, C. et al, 2010). We performed extensive experimental validation of MRs by both in-vitro and in-vivo RNAi mediated screening, using cell viability as readout. We then used a variety of experimental assays to elucidate the modular logic controlling disease state and to identify novel NBL subtype specific dependencies. RESULTS: We identified unique MR protein modules for three distinct molecular subtypes of high-risk NBL, which were conserved across independent cohorts. Experimental MR validation identified a TEAD4-MYCN positive feedback loop as the key NBL state maintenance mechanisms in the MYCN amplification associated subtype. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally implement 70% of the subtype gene expression signature. Biologically, MYCN repressed differentiation and TEAD4 activated proliferation, two hallmarks of MYCN-amplified NBL. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk NBL pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. Consistently, TEAD4 inhibition induced loss of NBL cell viability, thus providing novel therapeutic targets. TEAD4 activity was an outstanding predictor of survival, independent of outcome-related variables. CONCLUSION: Our results show that the inference of transcriptional regulators driving distinct molecular subgroups when combined with functional analyses is valuable to uncover the regulatory modules required for sustaining the tumor subtypes. This approach can be used to successfully identify the functional bottlenecks of other cancer subtypes. Citation Format: Presha Rajbhandari, Gonzalo Lopez, Jiyang Yu, Ruth Rodriguez-Barrueco, Mariano Alvarez, Daniel Martinez, Mark Yarmarkovich, Jo Vandesompele, Pieter Mestdagh, Jose M. Silva, Anna Lasorella, Antonio Iavarone, John M. Maris, Andrea Califano. Selective cross-cohort discovery of transcriptional mechanisms presiding over high-risk neuroblastoma subtype state maintenance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4384.

Abstract 3579: APOBEC3 activity sensitizes cells to ATR-Chk1 pathway inhibition

Abstract There are six functional members of the APOBEC3 family of enzymes (A3A-A3C, A3F-A3H), which catalyze the conversion of cytosine to uracil in single-stranded DNA. APOBEC3 enzymes are best described for their ability to deaminate viral and LINE element DNA, thus restricting infection and retrotransposition. However, recent analyses of human tumor sequences reveal mutational signatures consistent with APOBEC3 activity. These signatures indicate the capacity for APOBEC3 enzymes to cause mutations and breaks in cellular DNA and further, their potential to contribute to cellular transformation or clonal evolution of cancer. We have previously shown that APOBEC3 enzymes are capable of causing cellular DNA damage and activation of the DNA damage checkpoint via Ataxia-Telangiectasia Mutated (ATM) kinase signaling. To investigate additional cellular mechanisms that respond to APOBEC3-induced DNA damage, we examined the impact of A3A expression on protein kinases involved in DNA replication checkpoint signaling. Here, we show that A3A expression can also cause cell cycle arrest through activation of the DNA replication checkpoint as indicated by ATM-Related (ATR) kinase phosphorylation of Checkpoint Kinase 1 (Chk1) on S345 and S317. Based on these results, we hypothesized that inhibition of DNA replication checkpoint kinases in tumors with upregulated APOBEC3 enzymes would lead to uncontrolled replication, accumulation of DNA damage, and ultimately cell death. To study the impact of ATR inhibition on cells in which A3A is active, we generated cell lines with inducible A3A expression. We introduced a doxycycline-inducible A3A gene into a human fibroblast cell line and treated the cells with a selective ATR inhibitor (VE822). Using a colony formation assay, we show that ATR inhibition restricts cell growth in the context of A3A expression. We also found that A3A expression led to decreased cell viability following treatment with an ATR inhibitor. We examined the mechanism of cell death and found that ATR inhibition in cells induced to express A3A resulted in increased Annexin V staining, indicative of apoptosis. To confirm that these findings represent effects specific to the DNA replication checkpoint mediated by ATR activation and phosphorylation of Chk1, we employed siRNA knockdown of Chk1. We found that inhibition of Chk1 in the context of A3A expression also resulted in reduced cell growth, decreased viability, and apoptosis compared to uninduced cells. These observations suggest that DNA replication checkpoint signaling is critical for protection of the cellular genome from A3A-induced DNA damage. Upregulation of APOBEC3 activity in tumor cells provides an opportunity for targeted therapy. Thus, the ATR-Chk1 pathway may represent a target for therapeutic intervention in tumors in which APOBEC3 enzymes are active. Citation Format: Konstantin Budagyan, Matthew D. Weitzman, Abby M. Green. APOBEC3 activity sensitizes cells to ATR-Chk1 pathway inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3579.

ATR inhibition preferentially targets homologous recombination-deficient tumor cells.

Homologous recombination (HR) is required for faithful repair of double-strand DNA breaks. Defects in HR repair cause severe genomic instability and challenge cellular viability. Paradoxically, various cancers are HR defective and have apparently acquired characteristics to survive genomic instability. We aimed to identify these characteristics to uncover therapeutic targets for HR-deficient cancers. Cytogenetic analysis of 1143 ovarian cancers showed that the degree of genomic instability was correlated to amplification of replication checkpoint genes ataxia telangiectasia and Rad3-related kinase (ATR) and CHEK1. To test whether genomic instability leads to increased reliance on replication checkpoint signaling, we inactivated Rad51 to model HR-related genomic instability. Rad51 inactivation caused defective HR repair and induced aberrant replication dynamics. Notably, inhibition of Rad51 led to increased ATR/checkpoint kinase-1 (Chk1)-mediated replication stress signaling. Importantly, inhibition of ATR or Chk1 preferentially killed HR-deficient cancer cells. Combined, our data show that defective HR caused by Rad51 inhibition results in differential sensitivity for ATR and Chk1 inhibitors, implicating replication checkpoint kinases as potential drug targets for HR-defective cancers.

The checkpoint-dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress

Guanine nucleotide exchange factors control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence at the N-terminus of Rgf1p, whereas release into the cytoplasm requires two leucine-rich nuclear export sequences at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes nine serine potential phosphorylation Cds1p sites for alanine fails to accumulate in the nucleus in response to replication stress, and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologues of Rgf1p in budding yeast and higher eukaryotes.

CHK1 and WEE1 inhibition combine synergistically to enhance therapeutic efficacy in acute myeloid leukemia ex vivo

Novel combinations targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.

Protection from UV-induced skin carcinogenesis by genetic inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase

Multiple human epidemiologic studies link caffeinated (but not decaffeinated) beverage intake with significant decreases in several types of cancer, including highly prevalent UV-associated skin carcinomas. The mechanism by which caffeine protects against skin cancer is unknown. Ataxia telangiectasia and Rad3-related (ATR) is a replication checkpoint kinase activated by DNA stresses and is one of several targets of caffeine. Suppression of ATR, or its downstream target checkpoint kinase 1 (Chk1), selectively sensitizes DNA-damaged and malignant cells to apoptosis. Agents that target this pathway are currently in clinical trials. Conversely, inhibition of other DNA damage response pathways, such as ataxia telangiectasia mutated (ATM) and BRCA1, promotes cancer. To determine the effect of replication checkpoint inhibition on carcinogenesis, we generated transgenic mice with diminished ATR function in skin and crossed them into a UV-sensitive background, Xpc(-/-). Unlike caffeine, this genetic approach was selective and had no effect on ATM activation. These transgenic mice were viable and showed no histological abnormalities in skin. Primary keratinocytes from these mice had diminished UV-induced Chk1 phosphorylation and twofold augmentation of apoptosis after UV exposure (P = 0.006). With chronic UV treatment, transgenic mice remained tumor-free for significantly longer (P = 0.003) and had 69% fewer tumors at the end of observation of the full cohort (P = 0.019), compared with littermate controls with the same genetic background. This study suggests that inhibition of replication checkpoint function can suppress skin carcinogenesis and supports ATR inhibition as the relevant mechanism for the protective effect of caffeinated beverage intake in human epidemiologic studies.

Autoinhibition and Autoactivation of the DNA Replication Checkpoint Kinase Cds1

Cds1 is the ortholog of Chk2 and the major effector of the DNA replication checkpoint in Schizosaccharomyces pombe. Previous studies have shown that Cds1 is activated by a two-stage mechanism. In the priming stage, the sensor kinase Rad3 and the mediator Mrc1 function to phosphorylate a threonine residue, Thr(11), in the SQ/TQ domain of Cds1. In the autoactivation stage, primed Cds1 molecules dimerize via intermolecular interactions between the phosphorylated Thr(11) in one Cds1 and the forkhead-associated domain of the other. Dimerization activates Cds1, probably by promoting autophosphorylation. To define the mechanisms for the autoactivation of primed Cds1 and the regulation of this process, we carried out genetic and biochemical studies to identify phosphorylatable residues required for checkpoint activation. Our data indicate that dimerization of Cds1 promotes trans-autophosphorylation of a number of residues in the catalytic domain, but phosphorylation of a highly conserved threonine residue (Thr(328)) in the activation loop is the only covalent modification required for kinase activation in vitro and in vivo. Autophosphorylation of Thr(328) and kinase activation in unprimed, monomeric Cds1 are strongly inhibited by the C-terminal 27-amino acid tail of the enzyme. This autoinhibitory effect may play an important role in preventing spontaneous activation of the replication checkpoint during normal cell cycles. The two-stage activation pathway and the autoinhibition mechanism, which are probably shared by other members of the Chk2 family, provide sensitivity, specificity, and noise immunity, properties required for the replication checkpoint.

A tale of two tails: Activation of DNA damage checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1

The DNA damage and replication checkpoint kinase Mec1/ATR is a member of the PI3-kinase related kinases that function in response to various genotoxic stresses. The checkpoint clamp 9-1-1 (Rad9-Rad1-Hus1 in S. pombe and mammals; Ddc1-Rad17-Mec3 in S. cerevisiae) executes two distinct checkpoint functions. In S. cerevisiae, DNA-bound 9-1-1 directly activates Mec1 kinase activity, a function that has not been demonstrated in other organisms. A second, conserved activity of 9-1-1 is that of TopBP1/Cut5/Dpb11 recruitment to stalled replication sites; subsequent activation of Mec1/ATR is carried out by TopBP1/Cut5/Dpb11. Biochemical studies indicate that the mode of Mec1/ATR activation by S. cerevisiae 9-1-1 is analogous to activation by S. cerevisiae Dpb11 or by vertebrate TopBP1: activation is mediated by the intrinsically disordered C-terminal tail of each activator. The relative contributions made by multiple activators of Mec1/ATR are discussed.

Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

Modulation of Cell Cycle–specific Gene Expressions at the Onset of S Phase Arrest Contributes to the Robust DNA Replication Checkpoint Response in Fission Yeas

Fission yeast replication checkpoint kinases Rad3p and Cds1p are essential for maintaining cell viability after transient treatment with hydroxyurea (HU), an agent that blocks DNA replication. Although current studies have focused on the cyclin-dependent protein kinase Cdc2p that is regulated by these checkpoint kinases, other aspects of their functions at the onset of S phase arrest have not been fully understood. In this study, we use genome-wide DNA microarray analyses to show that HU-induced change of expression profiles in synchronized G(2) cells occurs specifically at the onset of S phase arrest. Induction of many core environmental stress response genes and repression of ribosomal genes happen during S phase arrest. Significantly, peak expression level of the MluI-like cell cycle box (MCB)-cluster (G(1)) genes is maintained at the onset of S phase arrest in a Rad3p- and Cds1p-dependent manner. Expression level maintenance of the MCB-cluster is mediated through the accumulation of Rep2p, a putative transcriptional activator of the MBF complex. Conversely, the FKH-cluster (M) genes are repressed during the onset of S phase arrest in a Rad3p-dependent manner. Repression of the FKH-cluster genes is mediated through the decreased levels of one of the putative forkhead transcription factors, Sep1p, but not Fkh2p. Together, our results demonstrate that Rad3p and Cds1p modulate transcriptional response during the onset of S phase arrest.