Checkpoint Initiation Research Articles

Pioglitazone as a Treatment Option for Ataxia-Telangiectasia

Abstract Introduction/Objective The ataxia-telangiectasia mutated (ATM) gene product regulates cell cycle checkpoints, the cellular redox state, and DNA break repair. ATM loss causes the rare disorder ataxia-telangiectasia (A-T), characterized by progressive ataxia, telangiectasias, immune dysfunction, and an elevated cancer risk. The clinical course of A-T is relentless, causing death by the early twenties. Methods/Case Report A-T cells show extreme sensitivity to oxidants and ionizing radiation, impaired glutathione (GSH) synthesis, genomic instability, poor cell cycle checkpoint initiation, and increased reactive oxygen species. A-T cells also show unusual iron responses, with iron chelators increasing A-T cell growth, oxidant exposure resistance, and genomic stability, while labile iron exerts toxic effects not seen in ATM wild-type cells. Since GSH is dysregulated in A-T cells and much of the cellular iron is bound up in iron-sulfur clusters, we examined the cellular sulfur pool in A-T cells with and without an ATM expression vector, and in ATM wild-types cells. Free hydrogen sulfide and protein- bound sulfide levels were unchanged with ATM expression. However, iron-sulfur clusters (the acid-labile sulfur faction) were low in the A-T cells and dramatically increased with ATM expression. ATM expression also increased the expression of NFS1, NFU1, and several mitochondrial complex I iron-sulfur proteins. As NFS1 and NFU1 play central roles in the synthesis of iron-sulfur clusters, and ATM expression increases these clusters, our data reveals a role for ATM in iron metabolism. Interestingly, the type II diabetes drug pioglitazone increased the acid-labile sulfur and protein bound sulfur factions in both cell types. It also increased the A-T cell reduced GSH, cell viability following oxidative stress, and decreased oxidative stress-induced double-stranded DNA break formation. Results (if a Case Study enter NA) Our finding that pioglitazone increased both cellular acid-labile and protein-bound sulfur fractions, indicates that these sulfur pools may be a therapeutic target for some type II diabetes drugs. Our data also indicates that pioglitazone may have value in treating A-T Conclusion We propose that pioglitazone could be a promising A-T treatment drug. More clinical research and trials are needed to determine the potential advantages and side effects of using pioglitazone in A-T.

Non-canonical role of the ATR pathway in axon regeneration as a mechanosensitive brake.

Non-canonical role of the ATR pathway in axon regeneration as a mechanosensitive brake.

Abstract PO-036: Safety outcomes of resuming anticancer treatment in patients with lung cancer affected by COVID-19 illness

Abstract Background: We aimed to describe how patients with lung cancer affected by COVID-19 recovered and were subsequently treated. Methods: From March 4th to May 12th, 2020, we have previously identified 23 COVID-19 lung cancer patients confirmed by SARS-CoV-2 RT-PCR in our institution (HGUGM; Madrid, Spain). After symptom recovery patients were tested weekly with nasopharyngeal swabs for SARS-CoV2. We did not start any anticancer treatment until at least 1 PCR confirmed a negative SARS-CoV-2 result. We reviewed treatment records, hospitalization, and outcomes of patients who recovered from COVID-19. Results: With a median follow-up of 9.7 weeks after COVID-19 diagnosis (range 3.7-13 weeks), 15 patients are clinically recovered (65%) and 8 patients died (35%) from COVID-19. In COVID-19 survivors, time to SARS-CoV-2 PCR negativization from symptoms onset was 31 days (range 13-54 days). Three patients remain SARS-CoV-2 RT-PCR positive to date. Of the 12 patients who negativized for SARS-CoV-2, 8 pts have started systemic anticancer treatment as follows: immune checkpoint inhibitors (n=3), chemotherapy (n=3), and initiation of definitive thoracic chemoradiation (n=2). All 3 pts on immunotherapy resumed the same treatment that they were receiving before COVID-19 (pembrolizumab, atezolizumab), as 2 out of 3 pts did with chemotherapy (pemetrexed; carboplatin + paclitaxel). With a median time on treatment of 40 days (range 22-48 days), only 1 patient has developed treatment-related adverse events so far (grade 1 thrombocytopenia after cycle 1, and grade 2 neutropenia after cycle 2 of carboplatin plus etoposide in a patient with relapsed SCLC previously treated with chemoradiation). The 4 patients with negative SARS-CoV-2 PCR who did not start any further treatment had either already completed planned treatment just before COVID-19 or were under surveillance without evidence of disease. Readmission was required in 4/15 patients: 1 pt due to tumor progression and pulmonary embolism, 1 pt due to brain edema from known brain metastasis, 1 pt due to Salmonella gastroenteritis, and 1 pt with hematuria due to prostate cancer. Of note, 1 patient who previously tested negative for SARS-CoV-2 -and who already resumed chemotherapy tested positive again upon admission without COVID-19-related symptoms. Conclusions: Lung cancer patients who survived COVID-19 can be considered for cancer treatment without preliminary early safety concerns. Long-term efficacy and safety evaluation by treatment modality is needed. Citation Format: Antonio Calles, Mar Galera, Inmaculada Aparicio, Manuel Alva, Marianela Bringas, Natalia Gutierrez, Javier Soto, Victoria Tirado, Rosa Alvarez. Safety outcomes of resuming anticancer treatment in patients with lung cancer affected by COVID-19 illness [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-036.

RecQ DNA Helicase Rqh1 Promotes Rad3ATR Kinase Signaling in the DNA Replication Checkpoint Pathway of Fission Yeast.

Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome, looking for mutants defective in response to the replication stress induced by hydroxyurea. In addition to the previously reported mutant with a C-to-Y change at position 307 encoded by tel2 (tel2-C307Y mutant) (Y.-J. Xu, S. Khan, A. C. Didier, M. Wozniak, et al., Mol Cell Biol 39:e00175-19, 2019, https://doi.org/10.1128/MCB.00175-19), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations, except for a start codon mutation, are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect, and heterologous expression of human RECQ1, BLM, and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved, and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.

Sarcoidosis related to checkpoint and BRAF/MEK inhibitors in melanoma.

Therapy for advanced melanoma has deeply changed in the last decade with the introduction of checkpoint and BRAF/MEK inhibitors. Granulomatous reactions have been reported related to these drugs. We performed a systematic review of all the cases described in the medical literature by the search (("Melanoma"[Mesh]) AND ("Sarcoidosis"[Mesh] OR "Granuloma"[Mesh])). Ninety-one patients under immunotherapy were included in the analyses. The time from the initiation of the immunotherapy until the onset of sarcoidosis or sarcoid-like reaction (SLR) was 7.1months (SD 9). Peripheral lymph nodes as the mode of onset were seen more frequently in patients under CTLA-4 inhibitors (p=.016) whereas in patients under BRAF/MEK inhibitors used to be in the form of specific skin lesions (p=.006). Chest X-ray stage I-II was the rule in the CTLA-4 and PD-1 groups. On the contrary, stage 0 accounted for 80% of the patients in the BRAF/MEK group examined for pulmonary involvement. Specific skin involvement was the most common manifestation apart from pulmonary involvement. It was more frequent in patients under BRAF/MEK inhibitors and especially in the form of papules. Splenic involvement was found also more frequently in patients under CTLA-4 inhibitors. Specific treatment for sarcoidosis/SLR was prescribed in 50 patients (58.8%), without differences among groups. Almost all patients presented a good prognosis independently of the decision made regarding their previous immunotherapy. CONCLUSION: Physicians should bear in mind the possibility of sarcoidosis/SLR after the initiation of checkpoint or BRAF/MEK inhibitors in patients diagnosed with advanced melanoma, especially in the form of skin involvement and mediastinal and peripheral lymph nodes. It is important to achieve an accurate diagnosis to rule out the possibility of cancer involvement. What to do with these drugs is yet to be clarified. It seems reasonable to prioritize cancer treatment so it is not mandatory to stop these drugs.

SLFN11 inhibits checkpoint maintenance and homologous recombination repair.

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

Natural Loss of Mps1 Kinase in Nematodes Uncovers a Role for Polo-like Kinase 1 in Spindle Checkpoint Initiation

The spindle checkpoint safeguards against chromosome loss during cell division by preventing anaphase onset until all chromosomes are attached to spindle microtubules. Checkpoint signal is generated at kinetochores, the primary attachment site on chromosomes for spindle microtubules. Mps1 kinase initiates checkpoint signaling by phosphorylating the kinetochore-localized scaffold protein Knl1 to create phospho-docking sites for Bub1/Bub3. Mps1 is widely conserved but is surprisingly absent in many nematode species. Here, we show that PLK-1, which targets a substrate motif similar to that of Mps1, functionally substitutes for Mps1 in C. elegans by phosphorylating KNL-1 to direct BUB-1/BUB-3 kinetochore recruitment. This finding led us to re-examine checkpoint initiation in human cells, where we found that Plk1 co-inhibition significantly reduced Knl1 phosphorylation and Bub1 kinetochore recruitment relative to Mps1 inhibition alone. Thus, the finding that PLK-1 functionally substitutes for Mps1 in checkpoint initiation in C. elegans uncovered a role for Plk1 in species that have Mps1.

Yet another job for Dna2: Checkpoint activation.

Mec1 (ATR in humans) is the principal kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. Checkpoint initiation requires stimulation of Mec1 kinase activity by specific activators. The complexity of checkpoint initiation in yeast increases with the complexity of chromosomal states during the different phases of the cell cycle. In G1 phase, the checkpoint clamp 9-1-1 is both necessary and sufficient for full activation of Mec1 kinase whereas in G2/M, robust checkpoint function requires both 9-1-1 and the replisome assembly protein Dpb11 (human TopBP1). A third activator, Dna2, is employed specifically during S phase to stimulate Mec1 kinase and to initiate the replication checkpoint. Dna2 is an essential nuclease-helicase that is required for proper Okazaki fragment maturation, for double-strand break repair, and for protecting stalled replication forks. Remarkably, all three Mec1 activators use an unstructured region of the protein, containing two critically important aromatic residues, in order to activate Mec1. A role for these checkpoint activators in channeling aberrant replication structures into checkpoint complexes is discussed.

G1 checkpoint establishment in vivo during embryonic liver development.

BackgroundThe DNA damage-mediated cell cycle checkpoint is an essential mechanism in the DNA damage response (DDR). During embryonic development, the characteristics of cell cycle and DNA damage checkpoint evolve from an extremely short G1 cell phase and lacking G1 checkpoint to lengthening G1 phase and the establishment of the G1 checkpoint. However, the regulatory mechanisms governing these transitions are not well understood. In this study, pregnant mice were exposed to ionizing radiation (IR) to induce DNA damage at different embryonic stages; the kinetics and mechanisms of the establishment of DNA damage-mediated G1 checkpoint in embryonic liver were investigated.ResultsWe found that the G2 cell cycle arrest was the first response to DNA damage in early developmental stages. Starting at E13.5/E15.5, IR mediated inhibition of the G1 to S phase transition became evident. Concomitantly, IR induced the robust expression of p21 and suppressed Cdk2/cyclin E activity, which might involve in the initiation of G1 checkpoint. The established G1 cell cycle checkpoint, in combination with an enhanced DNA repair capacity at E15.5, displayed biologically protective effects of repairing DNA double-strand breaks (DSBs) and reducing apoptosis in the short term as well as reducing chromosome deletion and breakage in the long term.ConclusionOur study is the first to demonstrate the establishment of the DNA damage-mediated G1 cell cycle checkpoint in liver cells during embryogenesis and its in vivo biological effects during embryonic liver development.

Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery

Initiation of the DNA replication checkpoint in yeast is mainly mediated by Mec1 protein kinase, the ortholog of human ATR, while its homolog Tel1, the ortholog of human ATM, has a minor replication checkpoint function. Checkpoint initiation requires stimulation of Mec1 kinase activity by specific activators. Saccharomyces cerevisiae Dna2, a nuclease-helicase that is essential for Okazaki fragment maturation, is employed specifically during S phase to stimulate Mec1 kinase and initiate the replication checkpoint. Mutations (W128A and Y130A) in the unstructured N terminus of Dna2 abrogate its checkpoint function in vitro and in vivo. Dna2 shows partial redundancy for the replication checkpoint with checkpoint initiators 9-1-1 (S. cerevisiae Ddc1-Mec3-Rad17 and human Rad9-Rad1-Hus1) and Dpb11, the ortholog of human TopBP1. A triple mutant that eliminates the checkpoint functions of all three initiators abrogates the Mec1-dependent checkpoint.

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well-understood DNA damage response via establishment of G2 checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re-entry. The aim of this study was to investigate the role of Chk1 in the recovery from G2 checkpoint arrest in HCT116 (human colorectal cancer) wt, p53–/– and p21–/– cell lines following H2O2 treatment. Firstly, DNA damage caused G2 checkpoint activation via Chk1. Secondly, overriding G2 checkpoint led to (i) mitotic slippage, cell cycle re-entry in G1 and subsequent G1 arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1-involved G2 arrest with respect to p53/p21WAF1: absence of either protein led to late G2 arrest instead of the classic G2 arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G2 arrest correlated with downstream senescence, but late G2 arrest led to mitotic catastrophe, although both cell cycle re-entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long-term DNA damage responses causing cell cycle re-entry. We propose that recovery from oxidative DNA damage-induced G2 arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21WAF1. The general relevance of Chk1 as an important determinant of recovery from G2 checkpoint arrest was verified in HT29 colorectal cancer cells.

An optimistic checkpointing and message logging approach for consistent global checkpoint collection in distributed systems

Checkpointing and rollback recovery are widely used techniques for achieving fault-tolerance in distributed systems. In this paper, we present a novel checkpointing algorithm which has the following desirable features: A process can independently initiate consistent global checkpointing by saving its current state, called a tentative checkpoint. Other processes come to know about a consistent global checkpoint initiation through information piggy-backed with the application messages or limited control messages if necessary. When a process comes to know about a new consistent global checkpoint initiation, it takes a tentative checkpoint after processing the message (not before processing the message as in existing communication-induced checkpointing algorithms). After a process takes a tentative checkpoint, it starts logging the messages sent and received in memory. When a process comes to know that every other process has taken a tentative checkpoint corresponding to current consistent global checkpoint initiation, it flushes the tentative checkpoint and the message log to the stable storage. The tentative checkpoints together with the message logs stored in the stable storage form a consistent global checkpoint. Two or more processes can concurrently initiate consistent global checkpointing by taking a new tentative checkpoint; in that case, the tentative checkpoints taken by all these processes will be part of the same consistent global checkpoint. The sequence numbers assigned to checkpoints by a process increase monotonically. Checkpoints with the same sequence number form a consistent global checkpoint. We also present the performance evaluation of our algorithm.

Structural Biochemistry of a Bacterial Checkpoint Protein Reveals Diadenylate Cyclase Activity Regulated by DNA Recombination Intermediates

To reveal mechanisms of DNA damage checkpoint initiation, we structurally and biochemically analyzed DisA, a protein that controls a Bacillus subtilis sporulation checkpoint in response to DNA double-strand breaks. We find that DisA forms a large octamer that consists of an array of an uncharacterized type of nucleotide-binding domain along with two DNA-binding regions related to the Holliday junction recognition protein RuvA. Remarkably, the nucleotide-binding domains possess diadenylate cyclase activity. The resulting cyclic diadenosine phosphate, c-di-AMP, is reminiscent but distinct from c-di-GMP, an emerging prokaryotic regulator of complex cellular processes. Diadenylate cyclase activity is unaffected by linear DNA or DNA ends but strongly suppressed by branched nucleic acids such as Holliday junctions. Our data indicate that DisA signals DNA structures that interfere with chromosome segregation via c-di-AMP. Identification of the diadenylate cyclase domain in other eubacterial and archaeal proteins implies a more general role for c-di-AMP in prokaryotes.

A synchronous checkpointing protocol for mobile distributed systems: probabilistic approach

Coordinated checkpointing is a method that minimises number of processes to checkpoint for an initiation. It may require blocking of processes, extra synchronisation messages or useless checkpoints. We propose a minimum process coordinated checkpointing algorithm where the number of useless checkpoints and blocking are reduced using a probabilistic approach that computes an interacting set of processes on checkpoint initiation. A process checkpoints if the probability that it will get a checkpoint request in current initiation is high. A few processes may be blocked but they can continue their normal computation and may send messages. We also modified methodology to maintain exact dependencies.

DNA damage checkpoint maintenance through sustained Chk1 activity.

The G2 DNA damage checkpoint prevents mitotic entry in the presence of DNA damage. This requires the activation of the phosphoinositide-3-kinase-related protein kinases ATR and ATM in human cells and the ATR homologue Rad3 in the fission yeast Schizosaccharomyces pombe. Rad3 activates the effector protein kinase Chk1 by phosphorylation. However, in fission yeast, inactivation of Rad3 following checkpoint activation has no impact on checkpoint duration. This demonstrates that Rad3 is not required for checkpoint maintenance and that the processes of checkpoint initiation and maintenance are distinct. Chk1 is required for checkpoint initiation but its role in checkpoint maintenance has not been investigated. We show here that Chk1 kinase activity is rapidly induced following irradiation and is maintained for the duration of a checkpoint arrest. On entry to mitosis, there is a transient decrease in Chk1 activity and phosphorylation, but Chk1 activity remains higher than that observed in unirradiated cells. We have generated temperature-sensitive alleles of chk1, which phenocopy chk1 deletion at the non-permissive temperature. Using these alleles, we have shown that inactivation of Chk1 during a checkpoint arrest leads to premature checkpoint termination, resulting in catastrophic mitoses that are a hallmark of checkpoint failure. Therefore, unlike Rad3, Chk1 is an important determinant of both checkpoint initiation and maintenance.

Concurrent checkpoint initiation and recovery algorithms on asynchronous ring networks

Checkpointing with rollback recovery is a well-known method for achieving fault-tolerance in distributed systems. In this work, we introduce algorithms for checkpointing and rollback recovery on asynchronous unidirectional and bi-directional ring networks. The proposed checkpointing algorithms can handle multiple concurrent initiations by different processes. While taking checkpoints, processes do not have to take into consideration any application message dependency. The synchronization is achieved by passing control messages among the processes. Application messages are acknowledged. Each process maintains a list of unacknowledged messages. Here we use a logical checkpoint, which is a standard checkpoint (i.e., snapshot of the process) plus a list of messages that have been sent by this process but are unacknowledged at the time of taking the checkpoint. The worst case message complexity of the proposed checkpointing algorithm is O( kn) when k initiators initiate concurrently. The time complexity is O( n). For the recovery algorithm, time and message complexities are both O( n).

Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase.

Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.