KDM6B sensitizes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key cell death effector in PARthanatos. Knockout of KDM6B or loss of KDM6B demethylase activity conferred cancer cells resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

Immune checkpoint blockade in the treatment of malignant tumor: current statue and future strategies

After being stagnant for decades, there has finally been a paradigm shift in the treatment of cancer with the emergence and application of immune checkpoint inhibitors (ICIs). The most extensively utilized ICIs are targeting the pathways involving programmed death-1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4). PD-1, as an crucial immune inhibitory molecule, by and large reasons the immune checkpoint response of T cells, making tumor cells get away from immune surveillance. Programmed cell death ligand-1 (PD-L1) is exceptionally expressed in most cancers cells and approves non-stop activation of the PD-1 pathway in the tumor microenvironment. PD-1/PD-L1 inhibitors can block the combination of PD-1 and PD-L1, inhibit hostile to regulatory signals, and restore the activity of T cells, thereby bettering immune response. The current researchers assume that the efficacy of these drugs is related to PD-L1 expression in tumor tissue, tumor mutation burden (TMB), and other emerging biomarkers. Although malignant tumors can benefit from the immunotherapy of PD-1/PD-L1 inhibitors, formulating a customized medication model and discovering biomarkers that can predict efficacy are the new trend in the new era of malignant tumor immunotherapy. This review summarizes the mechanism of action of PD-1/PD-L1 inhibitors, their clinical outcomes on various malignant tumors, their efficacy biomarkers, as well as predictive markers of irAEs.

Protein Phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the Spindle Position Checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5 and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.

Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.

The Heritability of Replication Problems

The major challenge of DNA replication is to provide daughter cells with intact and fully duplicated genetic material. However, various endogenous or environmental factors can slow down or stall DNA replication forks; these replication problems are known to fuel genomic instability and associated pathology, including cancer progression. Whereas the mechanisms emphasizing the source and the cellular responses of replicative problems have attracted much consideration over the past decade, the propagation through mitosis of genome modification and its heritability in daughter cells when the stress is not strong enough to provoke a checkpoint response in G2/M was much less documented. Some recent studies addressing whether low replication stress could impact the DNA replication program of the next generation of cells made the remarkable discovery that DNA damage can indeed be transmitted to daughter cells and can be processed in the subsequent S-phase, and that the replication timing program at a subset of chromosomal domains can also be impacted in the next generation of cells. Such a progression of replication problems into mitosis and daughter cells may appear counter-intuitive, but it could offer considerable advantages by alerting the next generation of cells of potentially risky loci and offering the possibility of an adaptive mechanism to anticipate a reiteration of problems, notably for cancer cells in the context of resistance to therapy.

Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic NES or NLS sequences or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.

Checkpoint Control of DNA Repair in Yeast

Budding yeast has been a model organism for understanding how DNA damage is repaired and how cells minimize genetic instability caused by arresting or delaying the cell cycle at well-defined checkpoints. However, many DNA damage insults are tolerated by mechanisms that can both be error-prone and error-free. The mechanisms that tolerate DNA damage and promote cell division are less well-understood. This review summarizes current information known about the checkpoint response to agents that elicit both the G2/M checkpoint and the intra-S phase checkpoint and how cells adapt to unrepaired DNA damage. Tolerance to particular bulky DNA adducts and radiomimetic agents are discussed, as well as possible mechanisms that may control phosphatases that deactivate phosphorylated proteins.

Multiple metabolic signals including AMPK and PKA regulate glucose-stimulated double strand break resection in yeast

DNA double strand breaks (DSBs) are cytotoxic lesions repaired by non-homologous end joining (NHEJ) and homologous recombination (HR), with 5' strand resection being the committed step in transition from NHEJ to HR. We previously discovered that gal1 yeast, which cannot metabolize galactose, were unable to perform efficient 5' resection even though DSBs were formed. Adding glucose or restoring GAL1 restored resection, suggesting that carbon source metabolism signals to DSB repair. Here we demonstrate that any fermentable carbon source, including raffinose, can stimulate resection and that the stimulatory effect of glucose was associated with decreased, not increased, cellular ATP. The effect was cell cycle dependent and did not occur in G1, while glucose augmented the G2/M checkpoint arrest even in cells deficient in resection. AMP-activated protein kinase pathway mutants showed only low basal resection despite glucose addition but had normal checkpoint arrest, indicating a primary role for Snf1 specifically in glucose-stimulated resection. The metabolic inputs to resection were multifactorial, however, with loss of the transcriptional repressor Mig1 leading to increased basal resection, three distinct patterns of deficiency with loss of the protein kinase A catalytic subunits, Tpk1, Tpk2 andTpk3, and a resection delay in yeast lacking the lysine demethylase Rph1 that helped separate early and late phase responses to glucose. These results reveal multiple interrelated metabolic signals that optimize DSB resection efficiency while independently amplifying the G2/M checkpoint response.