Dependent DNA Damage Response Research Articles

The immediate-early protein 1 of human herpesvirus 6B interacts with NBS1 and inhibits ATM signaling

Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host’s telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.

Abstract A033: Exploring synthetic lethality of targeting miR346-unfolded protein response dependent DNA damage response mechanisms in treatment-resistant prostate cancer

Abstract Background: The Unfolded Protein Response (UPR) is a key homeostatic mechanism that is activated during endoplasmic reticulum (ER) stress caused by both intrinsic (genomic instability, metabolic demand) and extrinsic (chemo/radiotherapy, nutrient deprivation, hypoxia) factors. IRE1 is the main UPR transducer, signalling the transcription factor XBP1, and IRE1 Regulated Dependent Decay (RIDD) to resolve ER stress by controlling the expression of mRNA and miRNAs. IRE1 also regulates aspects of the DNA damage response (DDR) and IRE1-binding drugs have clinical effects as monotherapies or as adjuvants to chemotherapeutics. One miRNA involved in both the DDR and IRE1 biology is miR-346, which we have shown to cause widespread DNA damage in prostate cancer (PCa), sensitising PCa to DNA damaging drugs and associating with improved PCa outcomes. Rationale: In RNAseq data datasets we have shown that miR-346 causes a widespread transcriptional downregulation of DDR markers whilst we observed the opposite in IRE1-CRISPR-KO PCa cells. We have also developed novel IRE1 modulators that sensitise cancers to chemotherapy in vivo. These findings offer a strong rationale to explore co-targeting of miR-346 and IRE1 in the presence of DNA damaging agents to treat aggressive forms of PCa. Consequently, we want to elucidate the exact biology underpinning the miR346-IRE1/XBP1/RIDD-DDR axis to prognosticate treatment groups with gene signatures as proxies of PARPi/chemotherapeutic susceptibility. Impact: If miR-346 is a downstream effector of IRE1 activity, then an IRE1 prognostic signature can act as proxy for stratification and selection of patients for miR-346 treatment as sensitiser to DNA damaging agents whilst if miR-346 and IRE1 work in parallel, we could achieve clinically significant results by exploiting synthetic lethality. By stratifying large cohorts through IRE1/miR-346/DDR signatures (personalised precision medicine) we can increase the pool of patients for whom DNA damaging agents are effective by inducing a BRCAness phenotype, improving treatment responses and quality of life. Citation Format: Dimitrios Doultsinos, Ian Mills, Claire Fletcher. Exploring synthetic lethality of targeting miR346-unfolded protein response dependent DNA damage response mechanisms in treatment-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A033.

Abstract 1 Distinctive and Differentiation-State Dependent DNA Damage Response of Hematopoietic Stem Cells to Genotoxic Noxae

IntroductionHematopoietic stem cells (HSCs) sustain lifelong blood production and can be used in allogenic stem cell transplantation for treatment of hematological malignancies, yet the mechanisms of their DNA damage response (DDR) remain largely unexplored. Genotoxic damage can be induced by chemotherapeutical agents (eg, etoposide, a topoisomerase II inhibitor) or by methylating agents (eg, nitrosamines). Related double strand breaks (DSBs) at the level of immature cells are thus presumed to trigger leukemia.ObjectiveHuman quiescent and cycling HSCs were reported to exhibit distinct responses to genotoxic stress upon in vitro exposure to sublethal doses of etoposide or N-nitroso-N-methylurea (MNU). In this context, we aim to identify and compare the mechanisms involved in the determination of HSC stem and progenitor cell fate upon DNA damage to gain more insights into cytotoxic anticancer drug- and contaminant-related hematological toxicity.MethodsQuiescent human HSCs (CD34+) were isolated from umbilical cord blood, sorted and directly exposed to etoposide or MNU. Besides, CD34+ were expanded in-vitro in medium containing broad acting cytokines for 5 days prior genotoxic exposure. Sublethal doses were assessed by Alamar Blue assay. Propidium iodide staining and colony-forming unit assay (CFU) were used for cell cycle analysis and tracking of cell functionality.ResultsIn vitro inhibitory concentrations were identified for human HSCs treated with etoposide (IC50 = 5 µM) and MNU (IC50 = 1 mM). Treatment of quiescent HSCs with lower genotoxin concentrations induces a cell cycle arrest in the G0/G1-phase, whereas higher concentrations activate apoptosis-related genes and completely abrogate further differentiation into colony-forming progenitors. In contrast, cycling HSCs exhibit an impaired proliferation, upregulation of autophagy-related genes, reduced proportions of viable CD34+ cells, and similar effects regarding differentiation capacity into colony-forming progenitors. However, cells exposed to lower genotoxin doses exhibit a higher regeneration capacity 3 days upon treatment.DiscussionThese results confirm that the DDR following DSB-induction differs between quiescent and cycling HSCs. Quiescent HSCs exhibit a stronger DDR, probably related to their longer lifespan. To avoid malignant transformation, it is necessary to preclude damage accumulation through cell cycle withdrawal and apoptosis. In contrast, more committed cells rather inline toward damage repair and restoration through activation of, for example, non-homologous end joining or mismatch repair genes.

P476: LOSS OF CHD4 IN ACUTE MYELOID LEUKAEMIA GIVES RISE TO INCREASED DNA DAMAGE REPAIR DEFECTS AND ALTERED CELL CYCLE PROGRESSION

Background: Acute Myeloid Leukaemias (AMLs) are an array of blood cell disorders arising from alterations within myeloid precursors. Cancer genome studies highlighted alterations within the nucleosome remodelling and deacetylation (NuRD) complex occur in over 20% of all cancers. The NuRD complex controls nucleosome assembly and chromatin accessibility; CHD4 is a core subunit of the NuRD which is involved in a multitude of functions including epigenetic regulation, cell cycle progression and DNA replication and repair. CHD4 is linked to PARP dependent DNA damage response (DDR) pathway by rapid but transient accumulation at sites of damage, preventing transcription which enables homologous recombination (HR). Aims: We aim to explore the function of CHD4-NuRD in AML using a CHD4 knockout (CHD4-/-) isogenic cell line models created using CRISPR/Cas9 to assess the mechanism of function of CHD4 within AML disease progression and evaluate if there is targetable therapeutic potential. Methods: CRISPR-Cas9 was used to generate isogenic CHD4-/- models in two representative AML cell lines. CHD4-/- models underwent functional studies; proliferation, cell cycle and protein expression assays were used to phenotype models before inducing DNA damage at a clinically relevant dose and reassessing cell response. A high throughput drug screen was performed to identify DNA damage compounds that showed significant sensitivity in CHD-/- models and validation was assessed using single compound treatments and co-culture assays. Results: Analysis of publicly available TCGA AML transcriptomic data set (N=161), found that AML patients (N=82) who express below median CHD4 levels have a poorer prognostic outcome suggesting a link between stalled transcription enabling HR-based DDR and patient survival (P=0.047). Analysis of the intermediate cytogenetic risk group showed patients with a low CHD4 expression had a worse outcome; which was comparable to the adverse risk group patients (P=0.032), indicating that loss of CHD4 may contribute to a more aggressive phenotype AML in this group. Phenotype analysis of CHD4-/- models showed DNA damage markers 53BP1 and PARP expression increases significantly, highlighting the role of CHD4 within the DDR. This increased DNA damage response was accompanied by reduced cellular proliferation in CHD4-/- cells. Accordingly, cell cycle analysis showed a decrease in cells entering the G2/M phase alongside an increase in cells in G1 and S phase suggesting a potential cell cycle checkpoint deficiency causing stunted proliferation in CHD4-/- cells. Of note, we did not observe a significant compensatory increase in CHD3-NuRD components expression in the absence of CHD4 suggesting there is no compensation for the loss of CHD4. We measured behaviour of CHD4-/- cells to stress-inducing conditions; in response to irradiation and chemotherapy drugs commonly used in AML. CHD4-/- cells display much stronger reduction in cellular proliferation post-2Gy irradiation compared to control while cell cycle analysis showed further cellular arrest in G2/M. Furthermore, a drug screen (160 DNA damaging compounds) showed an increased sensitivity of CHD4-/- cells to compounds targeting DNA/RNA synthesis pathways, including Tubercidin and Cytarabine, indicating that loss of CHD4 results in hyper-increase in damage. Summary/Conclusion: Our data has identified a role of CHD4 in AML progression mediated through loss of cell cycle progression control and an elevated response to DNA damage. Therefore, targeting cells with lower CHD4 expression with compounds targeting DNA/RNA synthesis may have an impact on patient outcome.

Processing of DNA Clamp Loader Subunit DnaX Is Important in the Absence of Caulobacter Cell Division Inhibitors

The DNA clamp loader is critical to the processivity of DNA replication and to the coordination of synthesis on the leading and lagging strands. In bacteria, the major subunit of the clamp loader, DnaX, has two forms: the essential full‐length DnaX tau and a shorter DnaX gamma. These forms are conserved across bacterial species and three distinct mechanisms have been found to create them such as ribosomal frameshift or transcriptional slippage. In Caulobacter crescentus, the full‐length DnaX tau is partially proteolyzed by ClpXP into two shorter DnaX gamma forms. Despite being discovered over 30 years ago, the purpose of maintaining multiple DnaX forms is unknown.To examine the purpose of encoding two forms, we created a nonproteolyzable allele of DnaX (tau‐only) which had no observable growth or morphological defects but showed a mild sensitivity to DNA damage. Adding an additional plasmid encoded copy of dnaX(tau‐only) to the dnaX(tau‐only) strain causes growth defects, filamentation, and increased RecA induction. These defects are not observed when adding a plasmid copy of dnaX(tau‐only) to the wild type strain, suggesting that an excess of DnaX tau is toxic, and likely causes replication stress.To better define the pathways that are affected in the absence of DnaX gamma, we performed transposon sequencing analysis to identifying genes that cause synthetic interactions with the dnaX(tau‐only) allele. We found that the insertions in two cell division inhibitors, sidA and didA, were underrepresented in the dnaX(tau‐only) strain compared to wild type. Consistent with this finding, we observed that adding a plasmid copy of dnaX‐tau‐only in a ΔsidA or ΔdidA strain causes an extreme growth defect and filamentation. Interestingly, although sidA is expressed as part of the RecA/LexA‐dependent DNA damage response in Caulobacter, we do not see a similar filamentation when we add an exogenous copy of the dnaX‐tau‐only allele to a ΔrecA strain. This indicates that the uninduced level of sidA expressing is sufficient to suppress the growth and filamentation phenotypes. The observation that cell division inhibitors are critical in the absence of proper DnaX processing suggests that the coordination of DNA replication and cell division is not intact in the dnaX‐tau‐only allele.

2118-P: Regulation of Ataxia-Telangiectasia and Rad3-Related (ATR)–Dependent DNA Damage Response by Nitric Oxide in ß-Cells

Nitric oxide, generated in β-cells in response to cytokines, induces DNA damage that activates ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage response (DDR). While the DDR does not contribute to DNA repair in β-cells, it does regulate β-cell apoptosis when the nitric oxide production falls below a threshold level. Indeed, nitric oxide at high micromolar levels, while inducing DNA damage, also inhibits DDR and protects β-cells from apoptosis. This ability of nitric oxide, a potent inhibitor of mitochondrial oxidation, to attenuate DDR activation correlates with a 10-fold reduction in ATP and the actions are selective to β-cells. Unlike most other cell types, β-cells cannot compensate for mitochondrial inhibition by increasing glycolysis, leading to a loss in ATP levels which correlates with DDR inhibition. We now show that nitric oxide also inhibits the activation of a second DDR transducer kinase, ATR. ATR is activated in response to single-strand DNA breaks and replication stress. We showed that low levels of nitric oxide activate ATR-dependent DDR signaling by inducing replication stress. However, much like with ATM, at higher concentrations nitric oxide and other inhibitors of mitochondrial respiration that reduce ATP levels by more than 10-fold, also inhibit ATR selectively in β-cells. Nitric oxide and these mitochondrial poisons do not inhibit ATR signaling in non-β-cells as they maintain ATP levels by increasing glycolysis under mitochondrial inhibition. Importantly, when non-β-cells are forced to utilize oxidative phosphorylation for ATP generation, nitric oxide and other mitochondrial inhibitors attenuate ATR signaling in a manner similar to β-cells. Together, these studies suggest that inhibition of the DDR by nitric oxide in β-cells is dependent on its ability to inhibit mitochondrial oxidative metabolism and deplete ATP, and suggest that the inhibition of DDR signaling may be a protective response to prevent β-cell apoptosis. Disclosure C. Yeo: None. B. Oleson: None. J.A. Corbett: None. J.K. Schnuck: None. Funding National Institutes of Health (DK052194, AI-44458)

Mediator of DNA Damage Checkpoint Protein 1 Facilitates V(D)J Recombination in Cells Lacking DNA Repair Factor XLF.

DNA double-strand breaks (DSBs) trigger the Ataxia telangiectasia mutated (ATM)-dependent DNA damage response (DDR), which consists of histone H2AX, MDC1, RNF168, 53BP1, PTIP, RIF1, Rev7, and Shieldin. Early stages of B and T lymphocyte development are dependent on recombination activating gene (RAG)-induced DSBs that form the basis for further V(D)J recombination. Non-homologous end joining (NHEJ) pathway factors recognize, process, and ligate DSBs. Based on numerous loss-of-function studies, DDR factors were thought to be dispensable for the V(D)J recombination. In particular, mice lacking Mediator of DNA Damage Checkpoint Protein 1 (MDC1) possessed nearly wild-type levels of mature B and T lymphocytes in the spleen, thymus, and bone marrow. NHEJ factor XRCC4-like factor (XLF)/Cernunnos is functionally redundant with ATM, histone H2AX, and p53-binding protein 1 (53BP1) during the lymphocyte development in mice. Here, we genetically inactivated MDC1, XLF, or both MDC1 and XLF in murine vAbl pro-B cell lines and, using chromosomally integrated substrates, demonstrated that MDC1 stimulates the V(D)J recombination in cells lacking XLF. Moreover, combined inactivation of MDC1 and XLF in mice resulted in synthetic lethality. Together, these findings suggest that MDC1 and XLF are functionally redundant during the mouse development, in general, and the V(D)J recombination, in particular.

Mutational analysis of theFAM175A gene in patients with premature ovarian insufficiency.

The family with sequence similarity 175 member A gene (FAM175A; also known as ABRAXAS1, CCDC98 and ABRA1), a member of the DNA repair family, contributes to the BRCA1 (BRCA1 DNA repair associated)-dependent DNA damage response and is associated with age at natural menopause. However, it remains poorly understood whether sequence variants in FAM175A are causative for premature ovarian insufficiency (POI). The aim of this study was to investigate whether mutations in the gene FAM175A were present in patients with POI. A total of 400 women with idiopathic POI and 498 control women with regular menstruation (306 age-matched women and 192 women over 40 years old) were recruited. After Sanger sequencing of FAM175A, functional experiments were carried out to explore the deleterious effects of the identified variation. DNA damage was subsequently induced by mitomycin C (MMC), and DNA repair capacity and G2-M checkpoint activation were evaluated by examining the phosphorylation level of H2AX (H2A histone family, member X) and the percentage of mitotic cells, respectively. One rare single-nucleotide polymorphism, rs755187051 in gene FAM175A, c.C727G (p.L243V), was identified in two patients but absent in the 498 controls. The functional experiments demonstrated that overexpression of variant p.L243V in HeLa cells resulted in a similar sensitivity to MMC-induced damage compared with cells transfected with wild-type FAM175A. Moreover, after treatment with MMC, there were no differences in DNA repair capacity and G2-M checkpoint activation between the mutant and wild-type genes. Our results suggest that the p.L243V variant of FAM175A may not be causative for POI. The contribution of FAM175A to POI needs further exploration.

Clampdown of inflammation in aging and anticancer therapies by limiting upregulation and activation of GPCR, CXCR4

One of the major pathological outcomes of DNA damage during aging or anticancer therapy is enhanced inflammation. However, the underlying signaling mechanism that drives this is not well understood. Here, we show that in response to DNA damage, ubiquitously expressed GPCR, CXCR4 is upregulated through the ATM kinase-HIF1α dependent DNA damage response (DDR) signaling, and enhances inflammatory response when activated by its ligand, chemokine CXCL12. A pharmacologically active compound screen revealed that this increased inflammation is dependent on reduction in cAMP levels achieved through activation of Gαi through CXCR4 receptor and PDE4A. Through in vivo analysis in mice where DNA damage was induced by irradiation, we validated that CXCR4 is induced systemically after DNA damage and inhibition of its activity or its induction blocked inflammation as well as tissue injury. We thus report a unique DNA damage-linked inflammatory cascade, which is mediated by expression level changes in a GPCR and can be targeted to counteract inflammation during anticancer therapies as well as aging.

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.

USP13 regulates the RAP80-BRCA1 complex dependent DNA damage response

BRCA1 regulates multiple cellular pathways that maintain genomic stability including cell cycle checkpoints, DNA repair, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. Receptor-associated protein 80 (RAP80) helps recruit BRCA1 to double-strand breaks (DSBs) through the scaffold protein CCDC98 (Abraxas) and facilitates DNA damage response (DDR). However, the regulation of RAP80-BRCA1 complex is still unclear. Here we report that a deubiquitinase, USP13, regulates DDR by targeting RAP80. Mechanistically, USP13 is phosphorylated by ATM following DNA damage which, in turn, facilitates its DSB localization. USP13, in turn, deubiquitinates RAP80 and promotes RAP80 recruitment and proper DDR. Depleting or inhibiting USP13 sensitizes ovarian cancer cells to cisplatin and PARP inhibitor (olaparib) while overexpression of USP13 renders ovarian cancer cells resistant to chemotherapy. Overall, we identify USP13 as a regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination axis dynamically regulates RAP80-BRCA1 complex foci formation and function.

Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

Emerging evidence is revealing that exosomes contribute to many aspects of physiology and disease through intercellular communication. However, the biological roles of exosome secretion in exosome-secreting cells have remained largely unexplored. Here we show that exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. The inhibition of exosome secretion results in the accumulation of nuclear DNA in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. This event provokes the innate immune response, leading to reactive oxygen species (ROS)-dependent DNA damage response and thus induce senescence-like cell-cycle arrest or apoptosis in normal human cells. These results, in conjunction with observations that exosomes contain various lengths of chromosomal DNA fragments, indicate that exosome secretion maintains cellular homeostasis by removing harmful cytoplasmic DNA from cells. Together, these findings enhance our understanding of exosome biology, and provide valuable new insights into the control of cellular homeostasis.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.

PPM1D Truncating Mutations Confer Chemotherapy Resistance in Hematopoietic Stem Cells, Which Is Reversible By PPM1D Inhibition

One of the adverse consequences of chemotherapy exposure is the development of therapy-related myeloid neoplasms (t-MNs). However, the cause and origin of most t-MNs are unknown, and the prognosis remains dismal. Novel work has shown that in addition to TP53, PPM1D is selectively mutated in 15% of therapy related MDS (Lindsley et al., ASH Abstract). Truncating mutations of PPM1D are also found to commonly occur in clonal hematopoiesis (Jaiswal et al., NEJM 2014; Genovese et al., NEJM 2014; Xie et al., Nat Med 2014), as well as in the blood of cancer patients, particularly after exposure to chemotherapy (Ruark et al., Nature 2013; Swisher et al., JAMA Oncol 2016). We hypothesized that PPM1D truncating mutations confer chemotherapy resistance, causing selective outgrowth of PPM1D mutant hematopoietic stem cells in states of genotoxic stress. In addition, since PPM1D mutations lead to a gain of function, we examined the potential of targeting PPM1D-mutant cells pharmacologically.The protein phosphatase PPM1Dis a direct regulator of TP53 activity and the DNA damage response pathway (Fiscella et al., PNAS 1997). Consequently, gain-of-function PPM1D mutations lead to decreased TP53 activity. To examine whether PPM1D mutations drive chemotherapy resistance through an abrogation of the TP53 dependent DNA damage response, we engineered PPM1D-mutant subclones using the CRISPR-Cas9 system in the TP53 wild-type AML cell line MOLM-13. PPM1D exon 6 truncation led to increased expression of PPM1D and resistance to DNA damaging agents, including cytarabine, cyclophosphamide and cisplatin. In addition, PPM1D-mutant cells exhibited a selective advantage over wild-type cells in the presence of chemotherapy, expanding 100-fold over a 24-day period (Fig. 1). While treatment with chemotherapy induced phosphorylation of Chk1 and p53, cell cycle arrest and apoptosis in wild-type cells, this response was abrogated in PPM1D-mutant cells due to gain of function of PPM1D. Using phosphoproteomic analysis, we further demonstrate decreased phosphorylation of known and novel targets in PPM1D-mutant compared to wild-type cells.We next investigated the effect of PPM1D mutation on normal marrow progenitors in response to chemotherapy treatment in vivo. We performed a competition experiment in which mouse bone marrow c-Kit+ cells expressing Cas9 were transduced with guide RNAs targeting exon 6 of PPM1D or a control guide, and then transplanted into mice in a 1:5 ratio. We observed a selective outgrowth of PPM1D-mutant myeloid cells in the peripheral blood of mice after exposure to chemotherapy. In addition, we found expansion of PPM1D-mutant cells in the lineage negative, c-Kit+ Sca-1+ population, which is enriched for hematopoietic stem cells and multipotent progenitors, indicating that PPM1D-mutant stem and progenitor cells have a competitive advantage over wild-type cells after exposure to genotoxic stress.The generation of a selective, allosteric inhibitor of PPM1D (Gilmartin et al., Nat Chem Biol 2014) allowed us to examine whether pharmacologic inhibition of PPM1D decreases the chemotherapy resistance or survival of PPM1D-mutant cells. We found that PPM1D-mutant cells have a significantly increased sensitivity to PPM1D inhibition when compared to wild-type controls. In addition, PPM1D inhibitor treatment was able to re-sensitize mutant cells to chemotherapy and abrogate the selective outgrowth of PPM1D-mutant cells during chemotherapy exposure. Lastly, we demonstrate that the proteome-wide phosphorylation profile characteristic of PPM1D-mutant cells can be reversed through treatment with the PPM1D inhibitor.In sum, these results demonstrate that PPM1D mutations confer a competitive advantage to hematopoietic stem cells undergoing genotoxic stress by abrogating the DNA damage response, and are likely to be the initiating mutation in a large proportion of t-MNs. Due to the gain-of-function nature of this mutation, PPM1D-mutant cells are differentially sensitive to treatment with a PPM1D inhibitor. PPM1D inhibition may therefore provide an opportunity for the prevention and targeted treatment of hematologic malignancies that harbor PPM1D mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of ATR-dependent feedback activation of Chk1 sensitises cancer cells to Chk1 inhibitor monotherapy

The Chk1 and ATR kinases are critical mediators of the DNA damage response pathway and help protect cancer cells from endogenous and oncogene induced replication stress. Inhibitors of both kinases are currently being evaluated in clinical trials. Chk1 inhibition with V158411 increases DNA damage and activates the ATR, ATM and DNA-PKcs dependent DNA damage response pathways. Inhibiting ATR, ATM and/or DNA-PKcs has the potential to increase the therapeutic activity of Chk1 inhibitors. ATR inhibition but not ATM or DNA-PKcs inhibition potentiated the cytotoxicity of V158411 in p53 mutant and wild type human cancer cell lines. This increased cytotoxicity correlated with increased nuclear DNA damage and replication stress in a dose and time dependent manner. γH2AX induction following Chk1 inhibition protected cells from caspase-dependent apoptosis. Inhibition of ATR increased Chk1 inhibitor induced cell death independently of caspase activation. The effect of ATR, ATM and/or DNA-PK inhibition on Chk1 inhibitor induced replication stress was dependent on the concentration of Chk1 inhibitor. ATR inhibition potentiated Chk1 inhibitor induced replication stress and cytotoxicity via the abrogation of ATR-dependent feedback activation of Chk1 induced by Chk1 inhibitor generated replication stress. This study suggests that combining an ATR inhibitor to lower the threshold by which a Chk1 inhibitor induces replication stress, DNA damage and tumour cell death in a wide range of cancer types may be a useful clinical approach.

DNA Repair and Cytokines: TGF-β, IL-6, and Thrombopoietin as Different Biomarkers of Radioresistance.

Double strand breaks (DSBs) induced by radiotherapy are highly cytotoxic lesions, leading to chromosomal aberrations and cell death. Ataxia-telangiectasia-mutated (ATM)-dependent DNA-damage response, non-homologous end joining, and homologous recombination pathways coordinately contribute to repairing DSBs in higher eukaryotes. It is known that the expression of DSB repair genes is increased in tumors, which is one of the main reasons for radioresistance. The inhibition of DSB repair pathways may be useful to increase tumor cell radiosensitivity and may target stem cell-like cancer cells, known to be the most radioresistant tumor components. Commonly overexpressed in neoplastic cells, cytokines confer radioresistance by promoting proliferation, survival, invasion, and angiogenesis. Unfortunately, tumor irradiation increases the expression of various cytokines displaying these effects, including transforming growth factor-beta and interleukin-6. Recently, the capabilities of these cytokines to support DNA repair pathways and the ATM-dependent DNA response have been demonstrated. Thrombopoietin, essential for megakaryopoiesis and very important for hematopoietic stem cell (HSC) homeostasis, has also been found to promote DNA repair in a highly selective manner. These findings reveal a novel mechanism underlying cytokine-related radioresistance, which may be clinically relevant. Therapies targeting specific cytokines may be used to improve radiosensitivity. Specific inhibitors may be chosen in consideration of different tumor microenvironments. Thrombopoietin may be useful in fending off irradiation-induced loss of HSCs.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA.

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.

Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana*[S

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).

CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Inactivating UBE2M Impacts the DNA Damage Response and Genome Integrity Involving Multiple Cullin Ligases

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.