Cell Cycle Checkpoint Activation Research Articles

In Fanconi anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress

Fanconi Anemia (FA) is an inherited disorder of DNA-repair due to mutation in one of 20+ interrelated genes that repair intra-strand DNA crosslinks and rescue collapsed or stalled replication forks. The most common hematologic abnormality in FA is anemia, but progression to bone marrow failure (BMF), clonal hematopoiesis, or acute myeloid leukemia (AML) may also occur. In prior studies, we found that Fanconi DNA-repair is required for successful emergency granulopoiesis; the process for rapid neutrophil production during the innate immune response. Specifically, Fancc-/- mice did not develop neutrophilia in response to emergency granulopoiesis stimuli, but instead exhibited apoptosis of bone marrow hematopoietic stem cells (HSCs) and differentiating neutrophils. Repeated emergency granulopoiesis challenges induced BMF in most Fancc-/- mice, with AML in survivors. In contrast, we found equivalent neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice and wild type (WT) mice, without BMF. Since termination of emergency granulopoiesis is triggered by accumulation of bone marrow neutrophils, we hypothesize neutrophilia protects Fancc-/-Tp53+/- bone marrow from the stress of a sustained inflammation that is experienced by Fancc-/- mice. In the current work, we found that blocking neutrophil accumulation during emergency granulopoiesis led to BMF in Fancc-/-Tp53+/- mice, consistent with this hypothesis. Blocking neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice (but not WT) impaired cell cycle checkpoint activity, also found in Fancc-/- mice. Mechanisms for loss of cell cycle checkpoints during infections challenges may define molecular markers of FA progression, or suggest therapeutic targets for bone marrow protection in this disorder.

Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Abstract PO2-24-01: Kif11 Inhibition Preferentially Kills TP53-mutant Breast Cancer Cells

Abstract Background Triple-Negative Breast Cancer (TNBC) makes up 15-20% of breast cancer diagnoses and is more common in younger women, those with BRCA mutations, and Black patients. Furthermore, TNBCs are defined by their lack of expression of the targetable receptors: estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR), resulting in few targeted therapy options for TNBC patients. One potential therapeutic target in TNBC is mutant p53, since 85-90% of TNBCs harbor TP53 mutations. However, the mutant p53 protein is challenging to target directly. Therefore, we have sought to identify survival pathways critical to TP53 mutant cells. Through a combined in vitro and in silico drug screen, we identified that the Kif11 inhibitor SB-743921 differentially kills TP53 mutant breast cancer cells compared to TP53 wild-type cells. Hypothesis Based on this screening data, we hypothesized that Kif11 inhibition causes TP53 mutant cells to undergo mitotic catastrophe resulting in cell death due to failure of activation of p53-mediated cell cycle checkpoints. Methods Clinical data was obtained from cBioPortal from the TCGA and METABRIC cohorts. Expression data analysis and survival analyses were performed using GraphPad Prism. Cell growth assays were conducted by seeding cells in 96 well plates, treating under designated conditions, then staining with Hoechst 33342 for cell counting on the ImageXpress Pico. Cell death was determined by co-staining with Hoechst 33342 and DRAQ7 followed by imaging and analysis on the ImageXpress Pico and by Annexin V and Propidium Iodide Staining followed by flow cytometry analysis. Cell cycle analysis was conducted following synchronization with Lovastatin and Mevalonate release followed by collection of samples, fixation, PI staining, and flow cytometry analysis. Immunofluorescence imaging was conducted by staining with anti-alpha-tubulin-AlexaFluor488 and DAPI and imaging at 20x and 63x on the ImageXPress PICO. In vitro expression experiments were carried out through qPCR and western blotting. < Results In human breast cancers, KIF11 is more highly expressed in TP53 mutant, as compared to wild-type breast cancers, and in TNBCs, as compared to other breast cancer subtypes. Patients with tumors that had higher expression of Kif11 had poorer overall survival outcomes. Inhibition of Kif11 with the small molecule inhibitor SB-743921 induces greater death of TP53 mutant as compared to wild-type breast cancer cells. Kif11 inhibition leads to a cell cycle block in both TP53 mutant and wild-type cells, but TP53 mutant cells then die following this cell cycle block. Kif11 inhibition causes mitotic dysfunction, including monopolar spindle formation, in mutant and wild-type cells, but greater incidence of spindle abnormalities and multinucleated cells in TP53 mutants. Introduction of a TP53 mutation into TP53 wild-type cells also induces cell death following Kif11 inhibition. Conclusions The Kif11 mitotic kinesin is more highly expressed in TP53 mutant and triple-negative breast cancers than in TP53 wild-type breast cancers, and high expression is associated with poorer survival outcomes. In TP53 mutant cells, Kif11 inhibition results in mitotic dysfunction and cell death due to mitotic catastrophe. Acknowledgments Research reported in this publication was supported by the John Charles Cain Endowment and the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers TL1TR003169 and UL1TR003167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would also like to thank the MDACC NORTH Campus Flow Cytometry and Cellular Imaging Core Facility for their assistance. Citation Format: Amanda Lanier, William Tahaney, Jing Qian, Cassandra Moyer, Yanxia Ma, Banu Arun, Abhijit Mazumdar, Powel Brown. Kif11 Inhibition Preferentially Kills TP53-mutant Breast Cancer Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-01.

Abstract 522: TP53mutations promote death of breast cancer cells following Kif11 inhibition

Abstract Background: Triple-Negative Breast Cancer (TNBC) comprises 15-20% of breast cancer diagnoses. These aggressive breast cancers are characterized by their lack of expression of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR), resulting in few targeted therapy options for TNBC patients. While 85-90% of TNBCs contain mutations in TP53, mutant p53 is challenging to target directly. Instead, we aim to identify other survival pathways critical for the growth and survival of TP53 mutant cells. Through a combined in vitro and in silico drug screen, we identified the Kif11 inhibitor SB-743921 as more effective in TP53 mutant compared to wild-type breast cancer cells. Hypothesis: We hypothesized that Kif11 inhibition causes TP53 mutant cells to undergo cell death due to failure of activation of p53-mediated cell cycle checkpoints. Methods: Clinical data from the TCGA and METABRIC cohorts was obtained from cBioPortal. Expression and survival analyses were performed using GraphPad Prism. Cell growth assays were conducted by seeding cells, treating under designated conditions, then staining with Hoechst 33342 for cell counting on the ImageXpress Pico. Cell death was determined by staining with Annexin V and PI or DAPI Staining followed by flow cytometry analysis. Cell cycle analysis was conducted following synchronization with Lovastatin and Mevalonate release followed by imaging analysis of cell lines expressing the Fast FUCCI reporter or PI staining and flow cytometry analysis. Immunofluorescence imaging was conducted by staining with anti-alpha-tubulin-AlexaFluor488 and DAPI and imaging on the Nikon TI2 or ImageXPress PICO. In vitro expression experiments were carried out through qPCR and western blotting. Results: KIF11 is most highly expressed in TP53 mutant and triple-negative tumors and high expression of KIF11 is associated with poorer overall survival. In breast cancer cell lines, Kif11 inhibition leads to a cell cycle block in both TP53 mutant and wild-type cells, but TP53 mutant cells then die following this cell cycle block. Furthermore, Kif11 inhibition causes mitotic dysfunction, including monopolar spindle formation and multinucleated cells in TP53 mutants. Introduction of a TP53 mutation into TP53 wild-type cells also induces cell death following Kif11 inhibition. Conclusions: KIF11 is highly expressed in aggressive breast cancers and is associated with poorer prognosis. Using the small molecule inhibitor SB-74321 that has been tested in phase I/II clinical trials with favorable tolerability, we found that Kif11 inhibition results in mitotic dysfunction and death of TP53 mutant breast cancer cells due to mitotic catastrophe. These results suggest that Kif11 is a promising target for the treatment of TP53 mutant TNBCs. Citation Format: Amanda L. Lanier, William Tahaney, Jing Qian, Cassandra Moyer, Yanxia Ma, Banu Arun, Abhijit Mazumdar, Powel H. Brown. TP53mutations promote death of breast cancer cells following Kif11 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 522.

MBNL1 Regulates Programmed Postnatal Switching Between Regenerative and Differentiated Cardiac States.

Discovering determinants of cardiomyocyte maturity is critical for deeply understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration, but elucidation of endogenous developmental regulators of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. MBNL1 (muscleblind-like 1) regulates fibroblast, thymocyte, and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. MBNL1 gain- and loss-of-function mouse models were studied at several developmental time points and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro assays to determine the mechanisms through which MBNL1 exerts its effects. MBNL1 is coexpressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deficiency was insufficient to promote regeneration in the adult heart because of cell cycle checkpoint activation. Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Targeting loss of cardiomyocyte maturity and downregulation of cell cycle inhibitors through MBNL1 deletion was not sufficient to promote adult regeneration.

The ATR inhibition by Elimusertib enhances the radiosensitivity of MDA-MB-231 triple negative breast cancer in vitro

Purpose DNA damage response (DDR) is the principal mechanism regulating genomic stability and cell cycle checkpoint activation by coordinating DNA repair and apoptotic pathways. Ataxia telangiectasia and Rad3-related protein (ATR) play a significant role in the DDR due to its capability to detect a wide spectrum of DNA damage. Therefore, targeting DDR, specifically ATR, is a promising therapeutic strategy in cancer treatment. Furthermore, the inhibition of ATR sensitizes cancer cells to radiotherapy (RT). Herein, we, for the first time, investigated the synergistic effects of Elimusertib (BAY-1895344) as a highly potent selective ATR inhibitor with RT combination in triple-negative breast cancer (TNBC), in vitro. Methods MDA-MB-231 TNBC cells were firstly treated with different concentrations of Elimusertib for 24 h and then exposed to 4 and 8 Gy of X-ray irradiation. After post-irradiation for 72 h, WST-1, Annexin V, cell cycle, acridine orange/propidium iodide, mitochondria staining and western blot analysis were conducted. Results Our findings showed that 4 Gy irradiation and lower doses (especially 2 and 4 nM) of Elimusertib combination exerted a considerable anticancer activity at 72 h post-irradiation through apoptotic cell death, marked nuclear and mitochondrial damages and the suppression of ATR-Chk1 based DDR mechanism. Conclusion ATR inhibition by Elimusertib in combination with RT may be a promising new treatment strategy in the treatment of TNBC. However, further experiments should be performed to elucidate the underlying molecular mechanisms of the therapeutic efficacy of this combination treatment and its association with DNS repair mechanisms in TNBC, in vitro and in vivo.

Suppression of NBS1 Upregulates CyclinB to Induce Olaparib Sensitivity in Ovarian Cancer.

Background: Deficiencies in DNA damage repair responses promote chemotherapy sensitivity of tumor cells. The Nibrin homolog encoding gene Nijmegen Breakage Syndrome 1 (NBS1) is a crucial component of the MRE11-RAD50-NBN complex (MRN complex) and is involved in the response to DNA double-strand breaks (DSBs) repair that has emerged as an attractive strategy to overcome tumor drug resistance, but the functional relationship between NBS1 regulated DNA damage repair and cell cycle checkpoints has not been fully elucidated. Methods: In this study, lentivirus-mediated RNAi was used to construct NBS1-downregulated cells. Flow cytometry, qPCR, and immunohistochemistry were used to explore the regulatory relationship between NBS1 and CyclinB in vivo and in vitro. Results: Our findings suggest that NBS1 deficiency leads to defective homologous recombination repair. Inhibition of NBS1 expression activates CHK1 and CyclinB signaling pathways leading to cell cycle arrest and sensitizes ovarian cancer cells to Olaparib treatment in vitro and in vivo. NBS1-deficient ovarian cancer cells tend to maintain sensitivity to chemotherapeutic drugs through activation of cell cycle checkpoints. Conclusions: NBS1 may be a potential therapeutic target for epithelial ovarian cancer as it plays a role in the regulation of the DNA damage response and cell cycle checkpoints. Suppression of NBS1 upregulates CyclinB to induce Olaparib sensitivity in ovarian cancer.

Inactivation of DNA Polymerase Theta (PolΘ) Is Synthetic Lethal in DNMT3A Mutated Myeloid Malignancies - Potential Clinical Applications

Somatic mutations in DNMT3A are associated with unfavorable outcome in patients with AML, MPN and CML. DNMT3A mutations promote resistance to anthracyclines (including daunorubicin, the component of standard “7+3” induction therapy), interferon alpha, and ABL1 kinase inhibitor imatinib. Thus, malignant clones carrying DNMT3A mutations may be difficult to eliminate using standard treatments. AML, MPN and CML cells harbor oncogenic tyrosine kinase (OTK) such as FLT3(ITD), JAK2(V617F) and BCR-ABL1, respectively. We reported before that elevated levels of formaldehyde generated by altered serine/one-carbon cycle metabolism contributed to accumulation of highly lethal DNA double-strand breaks (DSBs) in OTK-positive cells. To protect malignant cells from DSB-induced apoptosis, OTKs regulate DNA damage response (DDR) mechanisms involving DSBs sensing (ATM and ATR kinases) and repairing (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, Polθ-dependent microhomology-mediated end-joining = TMEJ) as well as these activating cell cycle checkpoints (CHK1 and CHK2 kinases). Unfortunately, DNMT3A mutations caused resistance of OTK-positive cells to numerous DDR inhibitors (DDRis). DDRis sensitivity screen and synthetic lethal CRISPR/Cas9 screen revealed that OTK-positive cells with DNMT3A mutations are uniquely sensitive to the inhibition of DNA polymerase theta (Polθ encoded by POLQ gene). This effect was dependent on generation of formaldehyde by serine/one-carbon cycle metabolism. Abrogation of DNMT3A function by CRISPR/Cas9 targeting, Cre-loxP gene deletion, and heterozygous R882H mutation resulted in hypersensitivity of OTK-positive murine AML-like cells and human primary AML cells to Polθ inhibitors (Polθis) due to accumulation of toxic DSBs and activation of cGAS/STING pro-apoptotic pathway. Moreover, simultaneous loss of functional DNMT3A combined with inactivation of Polθ (by CRISPR/Cas9 targeting, insertion of neo-resistance gene, and D2230A+Y2231A polymerase inactive mutant) caused accumulation of DSBs, and reduced OTK-driven clonogenic potential and leukemogenic activity in mice. Polθ is abundantly overexpressed in OTK-positive DNMT3A-deficient cells due to enhanced POLQ mRNA stability and elevated translation of Polθ protein but not due to altered POLQ methylation and Polθ protein stability. Polθ is a key element not only in TMEJ of DSBs with limited end-resection, but also in replication fork restart and in single-strand DNA (ssDNA) gap filling. OTK-positive DNMT3A-deficient cells displayed hyperactivity of Polθ-mediated TMEJ and replication fork restart, but not ssDNA gap filling. These effects were accompanied by increased loading of Polθ on DNA damage detected by Polθ foci formation and chromatin extraction. Moreover, DNMT3A deficiency modulates chromatin architecture at DSBs to limit DNA end-resection thus favoring TMEJ over HR. Furthermore, we tested the effectiveness of Polθis combined with FDA approved drugs (quizartinib, etoposide, cytarabine, azacytidine) against FLT3(ITD)-positive DNMT3A-deficient cells (primary patient cells and cell lines) in vitro and in vivo. The combination of Polθis + quizartinib and Polθis + etoposide completely eradicated clonogenic activity of these cells while Polθis + cytarabine and Polθis + azacytidine exerted modest and weak effects, respectively, when compared to individual compound treatments. These drug combinations were only modestly toxic to normal bone marrow cells. Treatment with Polθi or etoposide reduced the percentage of GFP+ FLT3(ITD)-positive DNMT3A-deficient leukemia cells in peripheral blood of the mice by ~2-fold and prolonged survival time by ~1.5-fold. Remarkably, the combination of Polθi and etoposide eradicated leukemia cells below detectable levels in 6/12 mice with no visible toxicity. Median survival time of the mice will be recorded. Altogether, we discovered that Polθ protects OTK-positive DNMT3A-deficient myeloid malignant cells from the toxic effects of DSBs and identified Polθ as a novel therapeutic target.

Cell cycle responses to Topoisomerase II inhibition: Molecular mechanisms and clinical implications.

DNA Topoisomerase IIA (Topo IIA) is an enzyme that alters the topological state of DNA and is essential for the separation of replicated sister chromatids and the integrity of cell division. Topo IIA dysfunction activates cell cycle checkpoints, resulting in arrest in either the G2-phase or metaphase of mitosis, ultimately triggering the abscission checkpoint if non-disjunction persists. These events, which directly or indirectly monitor the activity of Topo IIA, have become of major interest as many cancers have deficiencies in Topoisomerase checkpoints, leading to genome instability. Recent studies into how cells sense Topo IIA dysfunction and respond by regulating cell cycle progression demonstrate that the Topo IIA G2 checkpoint is distinct from the G2-DNA damage checkpoint. Likewise, in mitosis, the metaphase Topo IIA checkpoint is separate from the spindle assembly checkpoint. Here, we integrate mechanistic knowledge of Topo IIA checkpoints with the current understanding of how cells regulate progression through the cell cycle to accomplish faithful genome transmission and discuss the opportunities this offers for therapy.

Targeted inhibition of the methyltransferase SETD8 synergizes with the Wee1 inhibitor adavosertib in restraining glioblastoma growth

Despite intense research efforts, glioblastoma remains an incurable brain tumor with a dismal median survival time of 15 months. Thus, identifying new therapeutic targets is an urgent need. Here, we show that the lysine methyltransferase SETD8 is overexpressed in 50% of high-grade gliomas. The small molecule SETD8 inhibitor UNC0379, as well as siRNA-mediated inhibition of SETD8, blocked glioblastoma cell proliferation, by inducing DNA damage and activating cell cycle checkpoints. Specifically, in p53-proficient glioblastoma cells, SETD8 inhibition and DNA damage induced p21 accumulation and G1/S arrest whereas, in p53-deficient glioblastoma cells, DNA damage induced by SETD8 inhibition resulted in G2/M arrest mediated by Chk1 activation. Checkpoint abrogation, by the Wee1 kinase inhibitor adavosertib, induced glioblastoma cell lines and primary cells, DNA-damaged by UNC0379, to progress to mitosis where they died by mitotic catastrophe. Finally, UNC0379 and adavosertib synergized in restraining glioblastoma growth in a murine xenograft model, providing a strong rationale to further explore this novel pharmacological approach for adjuvant glioblastoma treatment.

The SGLT2 inhibitor canagliflozin suppresses growth and enhances prostate cancer response to radiotherapy

Radiotherapy is a non-invasive standard treatment for prostate cancer (PC). However, PC develops radio-resistance, highlighting a need for agents to improve radiotherapy response. Canagliflozin, an inhibitor of sodium-glucose co-transporter-2, is approved for use in diabetes and heart failure, but is also shown to inhibit PC growth. However, whether canagliflozin can improve radiotherapy response in PC remains unknown. Here, we show that well-tolerated doses of canagliflozin suppress proliferation and survival of androgen-sensitive and insensitive human PC cells and tumors and sensitize them to radiotherapy. Canagliflozin blocks mitochondrial respiration, promotes AMPK activity, inhibits the MAPK and mTOR-p70S6k/4EBP1 pathways, activates cell cycle checkpoints, and inhibits proliferation in part through HIF-1α suppression. Canagliflozin mediates transcriptional reprogramming of several metabolic and survival pathways known to be regulated by ETS and E2F family transcription factors. Genes downregulated by canagliflozin are associated with poor PC prognosis. This study lays the groundwork for clinical investigation of canagliflozin in PC prevention and treatment in combination with radiotherapy.

Circadian control of cisplatin-DNA adduct repair and apoptosis in culture cells.

Cisplatin, a widely prescribed chemotherapeutic agent for treating solid tumors, induces DNA adducts and activates cellular defense mechanisms, including DNA repair, cell cycle checkpoint control, and apoptosis. Considering the circadian rhythmicity displayed by most chemotherapeutic agents and their varying therapeutic efficacy based on treatment timing, our study aimed to investigate whether the circadian clock system influences the DNA damage responses triggered by cisplatin in synchronized cells. We examined the DNA damage responses in circadian-synchronized wild-type mouse embryonic fibroblasts (WT-MEF; clock-proficient cells), cryptochrome1 and 2 double knock-out MEF (CRYDKO; clock-deficient cells), and mouse hepatocarcinoma Hepa1c1c7 cells. Varying the treatment time resulted in a significant difference in the rate of platinum-DNA adduct removal specifically in circadian-synchronized WT-MEF, while CRYDKO did not exhibit such variation. Moreover, diurnal variation in other DNA damage responses, such as cell cycle checkpoint activity indicated by p53 phosphorylation status and apoptosis measured by DNA break frequency, was observed only in circadian-synchronized WT-MEF, not in CRYDKO or mouse hepatocarcinoma Hepa1c1c7 cells. These findings highlight that the DNA damage responses triggered by cisplatin are indeed governed by circadian control exclusively in clock-proficient cells. This outcome bears potential implications for enhancing or devising chronotherapy approaches for cancer patients.

Broadening the scope of WEE1 inhibitors: identifying novel drug candidates via computational approaches and drug repurposing

The protein kinase Wee1 plays a vital role in the G2/M cell cycle checkpoint activation, triggered by double-stranded DNA disruptions. It fulfills this task by phosphorylating and consequently deactivating the cyclin B linked to Cdk1/Cdc2 at the Tyr15 residue, leading to a G2 cell cycle halt and subsequent delay of mitosis post DNA damage. Despite advancements, only the Wee1 inhibitor MK1775 has made it to Phase II clinical trials, presenting a challenge in innovative chemical structure development for small molecule discovery. To navigate this challenge, we employed an e-pharmacophore model of the MK1775-WEE1 complex (PDB ID: 5V5Y), using in silico screening of FDA-approved drugs. We chose six drugs for analog creation, guided by docking scores, key residue interactions, and ligand occupancy. Utilizing the ‘DrugSpaceX’ database, we generated 2,776 analogues via expert-defined transformations. Our findings identified DE90612 as the top-ranked analogue, followed by DE363106, DE489678, DE395383, DE90548, DE689343, DE395019, and DE538066. These analogues introduced unique structures not found in other databases. A t-SNE structurally diversified distribution map unveiled promising transformations linked to Temozolomide for WEE1 inhibitor development. Simulations of the WEE1-DE90612 complex (a Temozolomide analogue) for 200 nanoseconds demonstrated stability, with DE90612 forging robust bonds with active site residues and sustaining vital contacts at ASN376 and CYS379. These results underscore DE90612's potential inhibitory properties at the WEE1 binding site, warranting additional in vitro and in vivo exploration for its anticancer activity. Our approach outlines a promising pathway for creating diverse WEE1 inhibitors with suitable biological properties for potential oncology therapeutics. Communicated by Ramaswamy H. Sarma

Pre-rRNA Facilitates TopBP1-Mediated DNA Double-Strand Break Response.

In response to genotoxic stress-induced DNA damage, TopBP1 mediates ATR activation for signaling transduction and DNA damage repair. However, the detailed molecular mechanism remains elusive. Here, using unbiased protein affinity purification and RNA sequencing, it is found that TopBP1 is associated with pre-ribosomal RNA (pre-rRNA). Pre-rRNA co-localized with TopBP1 at DNA double-strand breaks (DSBs). Similar to pre-rRNA, ribosomal proteins also colocalize with TopBP1 at DSBs. The recruitment of TopBP1 to DSBs is suppressed when cells are transiently treated with RNA polymerase I inhibitor (Pol I-i) to suppress pre-rRNA biogenesis but not protein translation. Moreover, the BRCT4-5 of TopBP1 recognizes pre-rRNA and forms liquid-liquid phase separation (LLPS) with pre-rRNA, which may be the molecular basis of DSB-induced foci of TopBP1. Finally, Pol I-i treatment impairs TopBP1-associated cell cycle checkpoint activation and homologous recombination repair. Collectively, this study reveals that pre-rRNA plays a key role in the TopBP1-dependent DNA damage response.

P-287 Morphokinetic analysis of early embryos subjected to double stranded DNA damage provides evidence for cell cycle checkpoint activity

Abstract Study question Is there evidence of cell cycle checkpoint activation in preimplantation embryos with double stranded breaks (DSBs) in their DNA, induced using CRISPR? Summary answer Cells with persistent double strand DNA damage exhibit longer timeframes to reach key morphological milestones than those with successful repair, consistent with checkpoint activation. What is known already In most cell types, DNA damage leads to activation of cell cycle checkpoints that halt the cycle while DNA repair is attempted. It has been hypothesised that early embryos lack (or have relaxed) checkpoints, and that this may be necessary to allow the embryo to divide rapidly and synchronously during the first few mitoses. It is thought that maternal (oocyte derived) inhibitors may be responsible for suppressing checkpoints until activation of the embryonic genome. Inadequate checkpoint control may be responsible for the genetic instability and sensitivity to DNA damage seen during early preimplantation stages, which is of clinical importance. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods Precise induction of DNA damage involved injection of CRISPR-Cas9 ribonuclear complex (RNP), along with the sperm at the time of fertilization (ICSI). All embryos were disaggregated on day-3 and their cells subjected to whole genome amplification. Amplified products underwent low-pass next generation sequencing to detect segmental aneuploidy related to failure of DNA repair, while the site targeted using CRISPR-Cas9 was PCR amplified and sequenced to confirm whether it had successfully undergone repair. Main results and the role of chance Embryos that successfully repaired the induced DNA damage showed timings of pronuclei appearance/disappearance and cell divisions that were indistinguishable from control embryos. Rates of embryo arrest prior to completion of the second mitotic division were also similar between the two groups (25% versus 24%). In contrast, embryos with unresolved DNA damage, as evidenced by the detection of chromosomal fragments involving a breakpoint at the site targeted using CRISPR, were delayed in reaching the same developmental milestones (p < 0.0001 for time to first cleavage division) and displayed a much higher incidence of arrest (63% before the second mitotic division; p = 0.0002). The delay in mitotic progression in blastomeres with DNA damage represents strong evidence that a checkpoint is active in the cells of cleavage stage embryos, sensing double strand DNA breaks and slowing the cell cycle. However, the fact that 36% of embryos with unresolved DNA damage continued to progress, albeit at a slower rate, is consistent with the notion that checkpoint control is less stringent in early human embryos. This relaxation of control is likely to contribute to the genetic instability seen at the cleavage stage, including a risk of unresolved DNA damage and a high frequency of chromosomal malsegregation (causing mosaicism). Limitations, reasons for caution While altered cell cycle timings are consistent with checkpoint activation, other factors could conceivably influence the duration of key morphokinetic events. Complimentary experiments to test the functionality of specific checkpoints and/or the activity of their individual components will be required for definitive proof that embryonic checkpoints are active but weakened. Wider implications of the findings Segmental aneuploidy, micronucleation and mosaicism are frequently seen in human preimplantation embryos and are associated with reduced likelihood of ongoing pregnancy. Such abnormalities are symptomatic of genetic instability associated with excessively permissive checkpoints. These results highlight the need for culture systems that protect embryos from cellular stressors, especially DNA damage. Trial registration number N.A.

UbcH5c-dependent activation of DNA-dependent protein kinase in response to replication-mediated DNA double-strand breaks

Camptothecin (CPT) exhibits strong cytotoxicity by inducing DNA double-strand breaks (DSBs) through DNA replication. Unlike radiation-induced DSBs, which have two DNA ends, CPT-induced DSBs are considered to have only one DNA end. However, the differences in cellular responses to one-ended and two-ended DSBs are not well understood. Our previous study showed that proteasome inhibitor treatment suppressed CPT-induced activation of DNA-PK, a factor required for non-homologous end-joining in DSB repair, suggesting that the ubiquitin-proteasome pathway is involved in DNA-PK activation in response to one-ended DSBs. To identify the ubiquitination factors required for DNA-PK activation, we screened an siRNA library against E2 ubiquitin-conjugating enzymes and identified UbcH5c. Knockdown of UbcH5c suppressed DNA-PK activation caused by CPT, but not by the radio-mimetic drug neocarzinostatin. UbcH5c-dependent DNA-PK activation occurred independent of DNA end resection. Furthermore, loss of UbcH5c reduced DNA-PK-dependent chromosomal aberrations and suppressed the activation of cell cycle checkpoint in response to CPT. These results suggest that UbcH5c regulates DNA-PK activation in response to one-ended DSBs caused by replication fork collapse. To our knowledge, this is the first report of a DSB repair-related factor that is differentially involved in the response to one- and two-ended DSBs.

Selective inhibition of ATM-dependent double-strand break repair and checkpoint control synergistically enhances the efficacy of ATR inhibitors.

Ataxia Telangiectasia and Rad3-Related Protein (ATR) kinase regulates a key cell regulatory node for maintaining genomic integrity by preventing replication fork collapse. ATR inhibition has been shown to increase replication stress resulting in DNA double strand breaks (DSBs) and cancer cell death, and several inhibitors are under clinical investigation for cancer therapy. However, activation of cell cycle checkpoints controlled by Ataxia Telangiectasia Mutated (ATM) kinase could minimize the lethal consequences of ATR inhibition and protect cancer cells. Here, we investigate ATR-ATM functional relationship and potential therapeutic implications. In cancer cells with functional ATM and p53 signaling, selective suppression of ATR catalytic activity by M6620 induced G1 phase arrest to prevent S-phase entry with unrepaired DSBs. The selective ATM inhibitors, M3541 and M4076, suppressed both ATM-dependent cell cycle checkpoints and DSB repair, lowered the p53 protective barrier and extended the life of ATR inhibitor induced DSBs. Combination treatment amplified the fraction of cells with structural chromosomal defects and enhanced cancer cell death. ATM inhibitor synergistically potentiated the ATR inhibitor efficacy in cancer cells in vitro and increased ATR inhibitor efficacy in vivo at doses that did not show overt toxicities. Further, a combination study in 26 patient-derived xenograft models of triple negative breast cancer with the newer generation ATR inhibitor M4344 and ATM inhibitor M4076 demonstrated substantial improvement in efficacy and survival compared to single-agent M4344, suggesting a novel and potentially broad combination approach to cancer therapy.

A Selective Nano Cell Cycle Checkpoint Inhibitor Overcomes Leukemia Chemoresistance.

Cell cycle checkpoint activation promotes DNA damage repair, which is highly associated with the chemoresistance of various cancers including acute myeloid leukemia (AML). Selective cell cycle checkpoint inhibitors are strongly demanded to overcome chemoresistance, but remain unexplored. A selective nano cell cycle checkpoint inhibitor (NCCI: citric acid capped ultra-small iron oxide nanoparticles) that can catalytically inhibit the cell cycle checkpoint of AML to boost the chemotherapeutic efficacy of genotoxic agents is now reported. NCCI can selectively accumulate in AML cells and convert H2 O2 to • OH to cleave heat shock protein 90, leading to the degradation of ataxia telangiectasia and Rad3-related proteinand checkpoint kinase 1, and the subsequent dysfunction of the G2/M checkpoint. Consequently, NCCI revitalizes the anti-AML efficacy of cytarabine that is previously ineffective both in vitro and in vivo. This study offers new insights into designing selective cell cycle checkpoint inhibitors for biomedical applications.

Plumbagin Exhibits Genotoxicity and Induces G2/M Cell Cycle Arrest via ROS-Mediated Oxidative Stress and Activation of ATM-p53 Signaling Pathway in Hepatocellular Cells

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB), a naturally occurring naphthoquinone mainly isolated from the plant Plumbago zeylanica L., has been proven to possess anticancer activities towards multiple types of cancer. Although there has been an increasing amount of research regarding its anticancer effects, the association between oxidative stress, genotoxicity and the cell cycle arrest induced by PLB still remains unclear. Therefore, it is important to investigate their potential connections and the involvement of DNA damage and the ataxia telangiectasia mutated protein (ATM)-p53 signaling pathway in PLB's anticancer mechanism. The present study showed that PLB exposure significantly reduced HCC cell viability and colony formation. In addition, PLB-induced G2/M cell cycle arrest, oxidative stress, and DNA damage was detected, which could be almost blocked by NAC pretreatment. PLB could trigger a DNA damage response by activating cell cycle checkpoints such as ATM, checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2) and p53. Meanwhile, the key modulator of the G2/M transition factor, Cell Division Cycle 25C (cdc25C), was significantly downregulated in an ROS-dependent manner. Furthermore, pretreatment with ATM and p53 inhibitors (KU55933 and Pifithrin-α) could reduce the occurrence of G2/M cell cycle arrest by inhibiting the activation of the ATM-p53 pathway. Taken together, these results indicate that ROS-mediated oxidative stress plays a key role in PLB-induced G2/M cell cycle arrest mediated by the ATM-p53 pathway.

DNA Repair and Therapeutic Strategies in Cancer Stem Cells.

First-line cancer treatments successfully eradicate the differentiated tumour mass but are comparatively ineffective against cancer stem cells (CSCs), a self-renewing subpopulation thought to be responsible for tumour initiation, metastasis, heterogeneity, and recurrence. CSCs are thus presented as the principal target for elimination during cancer treatment. However, CSCs are challenging to drug target because of numerous intrinsic and extrinsic mechanisms of drug resistance. One such mechanism that remains relatively understudied is the DNA damage response (DDR). CSCs are presumed to possess properties that enable enhanced DNA repair efficiency relative to their highly proliferative bulk progeny, facilitating improved repair of double-strand breaks induced by radiotherapy and most chemotherapeutics. This can occur through multiple mechanisms, including increased expression and splicing fidelity of DNA repair genes, robust activation of cell cycle checkpoints, and elevated homologous recombination-mediated DNA repair. Herein, we summarise the current knowledge concerning improved genome integrity in non-transformed stem cells and CSCs, discuss therapeutic opportunities within the DDR for re-sensitising CSCs to genotoxic stressors, and consider the challenges posed regarding unbiased identification of novel DDR-directed strategies in CSCs. A better understanding of the DDR mediating chemo/radioresistance mechanisms in CSCs could lead to novel therapeutic approaches, thereby enhancing treatment efficacy in cancer patients.