Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma

Loss-of-function mutations in the RB1 tumour suppressor are key drivers in cancer, including osteosarcoma. RB1 loss-of-function compromises genome-maintenance and hence could yield vulnerability to therapeutics targeting such processes. Here we demonstrate selective hypersensitivity to clinically-approved inhibitors of Poly-ADP-Polymerase1,2 inhibitors (PARPi) in RB1-defective cancer cells, including an extended panel of osteosarcoma-derived lines. PARPi treatment results in extensive cell death in RB1-defective backgrounds and prolongs survival of mice carrying human RB1-defective osteosarcoma grafts. PARPi sensitivity is not associated with canonical homologous recombination defect (HRd) signatures that predict PARPi sensitivity in cancers with BRCA1,2 loss, but is accompanied by rapid activation of DNA replication checkpoint signalling, and active DNA replication is a prerequisite for sensitivity. Importantly, sensitivity in backgrounds with natural or engineered RB1 loss surpasses that seen in BRCA-mutated backgrounds where PARPi have established clinical benefit. Our work provides evidence that PARPi sensitivity extends beyond cancers identifiable by HRd and advocates PARP1,2 inhibition as a personalised strategy for RB1-mutated osteosarcoma and other cancers.

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

Approaching Protein Barriers: Emerging Mechanisms of Replication Pausing in Eukaryotes

During nuclear DNA replication multiprotein replisome machines have to jointly traverse and duplicate the total length of each chromosome during each cell cycle. At certain genomic locations replisomes encounter tight DNA-protein complexes and slow down. This fork pausing is an active process involving recognition of a protein barrier by the approaching replisome via an evolutionarily conserved Fork Pausing/Protection Complex (FPC). Action of the FPC protects forks from collapse at both programmed and accidental protein barriers, thus promoting genome integrity. In addition, FPC stimulates the DNA replication checkpoint and regulates topological transitions near the replication fork. Eukaryotic cells have been proposed to employ physiological programmed fork pausing for various purposes, such as maintaining copy number at repetitive loci, precluding replication-transcription encounters, regulating kinetochore assembly, or controlling gene conversion events during mating-type switching. Here we review the growing number of approaches used to study replication pausing in vivo and in vitro as well as the characterization of additional factors recently reported to modulate fork pausing in different systems. Specifically, we focus on the positive role of topoisomerases in fork pausing. We describe a model where replisome progression is inherently cautious, which ensures general preservation of fork stability and genome integrity but can also carry out specialized functions at certain loci. Furthermore, we highlight classical and novel outstanding questions in the field and propose venues for addressing them. Given how little is known about replisome pausing at protein barriers in human cells more studies are required to address how conserved these mechanisms are.

Measuring the effect of environmental stress on cell survival during replication stress

DNA replication checkpoint ensures cell fitness under replication stress by restraining fork progression and arresting cell cycle. Without checkpoint proteins, cells die in a replication inhibitor hydroxyurea (HU). However, cellular environment may affect their survival in HU. Therefore, the main goal of this study was to examine the effect of environmental stress and to study how it promotes survival in replication checkpoint mutants of fission yeast (rad3∆, mrc1∆, cds1∆). Our viability assays showed a significant increase in these mutants survival in heat-shock + HU compared to HU alone. Cell-cycle staging suggests that cells are altered after heat shock, affecting their response to HU. We measured the consequences to this enhanced survival and found that surviving population exhibits altered DNA mis-segregation and mutation rate. Collectively, our work points to a general cellular response to various environmental stressors that affects survival under replication stress, and may be applicable to human disease.

Measuring the effect of environmental stress on cell survival during replication stress

DNA replication checkpoint ensures cell fitness under replication stress by restraining fork progression and arresting cell cycle. Without checkpoint proteins, cells die in a replication inhibitor hydroxyurea (HU). However, cellular environment may affect their survival in HU. Therefore, the main goal of this study was to examine the effect of environmental stress and to study how it promotes survival in replication checkpoint mutants of fission yeast (rad3∆, mrc1∆, cds1∆). Our viability assays showed a significant increase in these mutants survival in heat-shock + HU compared to HU alone. Cell-cycle staging suggests that cells are altered after heat shock, affecting their response to HU. We measured the consequences to this enhanced survival and found that surviving population exhibits altered DNA mis-segregation and mutation rate. Collectively, our work points to a general cellular response to various environmental stressors that affects survival under replication stress, and may be applicable to human disease.

USP11 suppresses CHK1 activation by deubiquitinating CLASPIN

CLASPIN is an essential mediator of ATR-dependent CHK1 activation in the DNA replication checkpoint. K6-linked polyubiquitination of CLASPIN promotes its chromatin loading and subsequent CHK1 activation. Here, we found that ubiquitin-specific protease 11 (USP11) deubiquitinates the K6-linkage polyubiquitinated form of CLASPIN. Under steady-state conditions, USP11 interacts with CLASPIN, reducing CLASPIN K6-linked ubiquitination levels. In response to replication stress, USP11 is phosphorylated by ATR and subsequently disassociated from CLASPIN, promoting CLASPIN chromatin loading, CHK1 activation and ultimately genome stability. Taken together, our findings uncover a novel function of USP11 in negatively regulating CHK1 activation by suppressing CLASPIN chromatin loading.

Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma

Loss-of-function mutations in the RB1 tumour suppressor are key drivers in cancer, including osteosarcoma. RB1 loss-of-function compromises genome-maintenance and hence could yield vulnerability to therapeutics targeting such processes. Here we demonstrate selective hypersensitivity to clinically-approved inhibitors of Poly-ADP-Polymerase1,2 inhibitors (PARPi) in RB1-mutated cancer cells including an extended panel of osteosarcoma-derived lines. PARPi treatment results in extensive cell death in RB1-mutated backgrounds and prolongs survival of mice carrying human RB1-mutated osteosarcoma grafts. PARPi sensitivity is not associated with canonical homologous recombination defect (HRd) signatures, which predict PARPi sensitivity in cancers with BRCA1,2 loss, but is accompanied by rapid activation of DNA replication checkpoint signalling, and active DNA replication is a prerequisite for sensitivity. Importantly, sensitivity in backgrounds with natural or engineered RB1 loss surpasses that seen in BRCA-mutated backgrounds where PARPi have established clinical benefit. Our work provides evidence that PARPi sensitivity extends beyond cancers identifiable by HRd and advocates PARP1,2 inhibition as a novel, personalised strategy for RB1-mutated osteosarcoma and other cancers.

Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress

The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.