Inhibition Of Origin Firing Research Articles

Excessive new origin firing underlies selective glioma stem cell cytotoxicity induced by replication stress response inhibition

Abstract Aims Glioblastoma (GBM) is a treatment refractory cancer of extreme unmet need which exhibits treatment resistance due to a subpopulation of GBM cancer stem cells which have constitutive DNA damage response activation driven by elevated replication stress (RS). RS response inhibition is potently cytotoxic to GSC, however mechanistic understanding will be key to biomarker discovery and successful clinical translation. We investigated response to combined ATR and PARP inhibition (CAiPi) to gain mechanistic insight and inform biomarker development. Method A panel of patient-derived GBM cell lines were cultured as stem enriched (GSCs) or stem depleted (bulk), to characterise response to combined ATR inhibition (VE821 5μM) and PARP inhibition (Olaparib 1μM), by CellTiter-Glo viability assay. Mechanistic investigations included immunofluorescence of 53BP1 nuclear bodies and DNA fibre analysis. Studies into the importance of PARP trapping included another PARPi Veliparib (1μM), and investigations into inhibition of origin firing used the CDK inhibitor Roscovitine. Results Responses to CAiPi in a panel of primary paired GBM GSCs vs differentiated progeny were heterogenous. CAiPi is selectively GSC cytotoxic in a subpopulation of tumours. DNA fibre analysis identified increased new origin firing with PARPi, which was correlated with increased PARP trapping. Inhibition of origin firing by exposure to roscovitine rescued the CAiPi cytotoxic phenotype, suggesting origin firing has an important role in selective GSC cytotoxicity. A population of treatment-sensitive GSCs with increased numbers of 53BP1 nuclear bodies in G1 phase with CAiPi were identified, indicative of under-replication of DNA in S phase. Conclusion Selective GSC cytotoxicity is induced by CAiPi via dysregulation of replication, by both DNA under-replication resulting in DNA lesions, and the novel finding of increased new origin firing in GSC due to PARPi.

Coupling arginine with DNA replication and chemoresistance

How cells adapt DNA replication to meet amino acid demands is an active area of interest across biology. Among a broad range of functions, the extracellular arginine is needed for the availability of newly synthesized histones to promote DNA replication fork progression. We show herein that transient deprivation of extracellular arginine, but not glutamine or leucine, causes a reversible S‐phase arrest. This unexpected dynamic S‐phase arrest feature is independent of mTORC1 signaling inhibition, insufficient metabolites, endogenous de novo arginine biosynthesis, and general amino acid control pathway. Moreover, knockdown of arginyl‐tRNA‐transferase recapitulated the loss of histone H4 lysine 5 and 12 acetylation marks and the reduction of replication fork speed without inhibition of origin firing. Importantly, single round of arginine depletion and addition did not induce ATR/Chk1 activation and DNA double‐stand breaks associated γH2AX. The halt of arginine‐dependent histone H4 synthesis is therefore emerging as a “brake” for DNA replication bypassing checkpoint control. However, repeated arginine fluctuation promote the emergence of nuclear p53‐binding protein 1 bodies in the subsequent G1‐phase, creating vulnerabilities of DNA replications and can be exploited by cancer cells that promote DNA repair and resistance to genotoxic agents. Thus, intratumoral variation in the arginine microenvironment synchronizes DNA replication with therapeutic responses.

Crosslinks and replication: the show must go on

Crosslinks and replication: the show must go on

Checkpoint Regulation of Replication Forks at Sites of DNA Damage

Statement of methods:We have used single‐molecule DNA combing to study how the checkpoint regulates individual origins and forks in response to DNA damage.imageSummary of resultsThe S‐phase DNA damage checkpoint slows replication and allows cells to replicate damaged DNA, but how slowing relates to damage tolerance remains unclear. The standard explanation — that checkpoint delays cell‐cycle progression until the damage can be repaired — does not seem to apply in the case of DNA replication. Instead, replication proceeds in the presence of the damage, but at a slower rate. Replication slowing can be achieved by inhibition of origin firing, slowing of fork progression or a combination of both. In bulk assays, 1uM 4NQO and 0.03% MMS slow S phase to a similar extent. However the combing data shows that 4NQO slows replication by reducing origin firing rate, while MMS slows S phase by stalling forks and reducing the fork rate. While reduction in origin firing rate is checkpoint dependent, reduction in fork rate has checkpoint dependent and independent components. We are currently investigating the mechanistic difference in response to different damaging agents and how the checkpoint regulates fork components to achieve replication slowingAnother key question is how forks replicate quickly through damaged DNA in the absence of the checkpoint. The MMS‐induced lesions we use are believed to stop replicative polymerases. Nonetheless, in the absence of the checkpoint, replication forks pass them without any apparent delay. This bypass seems to be mainly dependent on recombination and translesion polymerase based synthesis. We are working on how forks bypass polymerase‐stalling lesions and what effect this bypass has on genomic stability.Funding Source:NIHGMS

Regulation of DNA replication by the S-phase DNA damage checkpoint

Cells slow replication in response to DNA damage. This slowing was the first DNA damage checkpoint response discovered and its study led to the discovery of the central checkpoint kinase, Ataxia Telangiectasia Mutated (ATM). Nonetheless, the manner by which the S-phase DNA damage checkpoint slows replication is still unclear. The checkpoint could slow bulk replication by inhibiting replication origin firing or slowing replication fork progression, and both mechanisms appear to be used. However, assays in various systems using different DNA damaging agents have produced conflicting results as to the relative importance of the two mechanisms. Furthermore, although progress has been made in elucidating the mechanism of origin regulation in vertebrates, the mechanism by which forks are slowed remains unknown. We review both past and present efforts towards determining how cells slow replication in response to damage and try to resolve apparent conflicts and discrepancies within the field. We propose that inhibition of origin firing is a global checkpoint mechanism that reduces overall DNA synthesis whenever the checkpoint is activated, whereas slowing of fork progression reflects a local checkpoint mechanism that only affects replisomes as they encounter DNA damage and therefore only affects overall replication rates in cases of high lesion density.

ATR and ATM regulate the timing of DNA replication origin firing.

Timing of DNA replication initiation is dependent on S-phase-promoting kinase (SPK) activity at discrete origins and the simultaneous function of many replicons. DNA damage prevents origin firing through the ATM- and ATR-dependent inhibition of Cdk2 and Cdc7 SPKs. Here, we establish that modulation of ATM- and ATR-signalling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM and ATR with caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promoted rapid and synchronous origin firing; conversely, inhibition of Cdc25A slowed DNA replication. Cdk2 was in equilibrium between active and inactive states, and the concentration of replication protein A (RPA)-bound single-stranded DNA (ssDNA) correlated with Chk1 activation and inhibition of origin firing. Furthermore, ATM was transiently activated during ongoing replication. We propose that ATR and ATM regulate SPK activity through a feedback mechanism originating at active replicons. Our observations establish that ATM- and ATR-signalling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis, and are therefore poised to halt replication in the presence of DNA damage.