The ATR-Chk1 pathway is essential in cellular responses to DNA damage and replication stress, whereas the role of long noncoding RNAs (lncRNAs) in regulating this pathway remains largely unknown. In this study, we identify an ATR and Chk1 interacting lncRNA (ACIL, also known as LRRC75A-AS1 or SNHG29), which promotes the phosphorylation of Chk1 by ATR upon DNA damages. High ACIL levels are associated with chemoresistance to DNA damaging agents and poor outcome of breast cancer patients. ACIL knockdown sensitizes breast cancer cells to DNA damaging drugs in vitro and in vivo. ACIL protects cancer cells against DNA damages by inducing cell cycle arrest, stabilizing replication forks and inhibiting unscheduled origin firing, thereby guarding against replication catastrophe and contributing to DNA damage repair. These findings demonstrate a lncRNA-dependent mechanism of activating the ATR-Chk1 pathway and highlight the potential of utilizing ACIL as a predictive biomarker for chemotherapy sensitivity, as well as targeting ACIL to reverse chemoresistance in breast cancer.

Multidrug resistance is a substantial obstacle in treating non-small cell lung cancer (NSCLC) with therapies like cisplatin (DDP)-based adjuvant chemotherapy and EGFR-tyrosine kinase inhibitors (TKIs). Aaptamine-7 (AP-7), a benzonaphthyridine alkaloid extracted from Aaptos aaptos sponge, has been shown to exhibit a broad spectrum of anti-tumor activity. However, the anti-cancer activity of AP-7 in combination with DDP and its molecular mechanisms in multidrug-resistant NSCLC are not yet clear. Our research indicates that AP-7 bolsters the growth inhibition activity of DDP on multidrug-resistant NSCLC cells. AP-7 notably disrupts DDP-induced cell cycle arrest and amplifies DDP-induced DNA damage effects in these cells. Furthermore, the combination of AP-7 and DDP downregulates Chk1 activation, interrupts the DNA damage repair-dependent Chk1/CDK1 pathway, and helps to overcome drug resistance and boost apoptosis in multidrug-resistant NSCLC cells and a gefitinib-resistant xenograft mice model. In summary, AP-7 appears to enhance DDP-induced DNA damage by impeding the Chk1 signaling pathway in multidrug-resistant NSCLC, thereby augmenting growth inhibition, both in vitro and in vivo. These results indicate the potential use of AP-7 as a DDP sensitizer in the treatment of multidrug-resistant NSCLC.

Stress is an important initiating factor in promoting Alzheimer\'s disease (AD) pathogenesis. However, the mechanism by which stress induces AD-like cognitive impairment remains to be clarified. Here, we demonstrate that DNA damage is increased in stress hormone Corticotropin-releasing factor (CRF)-treated cells and in brains of mice exposed to chronic restraint stress. Accumulation of DNA damage drives activation of cell cycle checkpoint protein kinase 1 (Chk1), upregulation of cancerous inhibitor of PP2A (CIP2A), tau hyperphosphorylation, and Aβ overproduction, eventually resulting in synaptic impairment and cognitive deficits. Pharmacological intervention targeting Chk1 by specific inhibitor and DNA damage by vitamin C, suppress DNA damage-Chk1-CIP2A signaling pathway in chronic stress animal model, which in turn attenuate AD-like pathologies, synaptic impairments and cognitive deficits. Our study uncovers a novel molecular mechanism of stress-induced AD-like pathologies and provides effective preventive and therapeutic strategies targeting this signaling pathway.

Checkpoint kinase 1 (Chk1) is a key mediator of the DNA damage response that regulates cell cycle progression, DNA damage repair, and DNA replication. Small-molecule Chk1 inhibitors sensitize cancer cells to genotoxic agents and have shown preclinical activity as single agents in cancers characterized by high levels of replication stress. However, the underlying genetic determinants of Chk1-inhibitor sensitivity remain unclear. Although treatment options for advanced colorectal cancer are limited, radiotherapy is effective. Here, we report that exposure to a novel amidine derivative, K1586, leads to an initial reduction in the proliferative potential of colorectal cancer cells. Cell cycle analysis revealed that the length of the G2/M phase increased with K1586 exposure as a result of Chk1 instability. Exposure to K1586 enhanced the degradation of Chk1 in a time- and dose-dependent manner, increasing replication stress and sensitizing colorectal cancer cells to radiation. Taken together, the results suggest that a novel amidine derivative may have potential as a radiotherapy-sensitization agent that targets Chk1.

Despite the recent advancement in diagnosis and therapy, pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is still the most lethal cancer with a low five-year survival rate. There is an urgent need to develop new therapies to address this issue. In this study, we developed a treatment strategy by modifying tumor suppressor miRNAs, miR-15a and miR-194, with the chemotherapeutic gemcitabine (Gem) to create Gem-modified mimics, Gem-miR-15a and Gem-miR-194, respectively. In a panel of PDAC cell lines, we found that Gem-miR-15a and Gem-miR-194 induce cell-cycle arrest and apoptosis, and these mimics are potent inhibitors with IC50 values up to several hundred fold less than their native counterparts or Gem alone. Furthermore, we found that Gem-miR-15a and Gem-miR-194 retained miRNA function by downregulating the expression of several key targets including WEE1, CHK1, BMI1, and YAP1 for Gem-miR-15a, and FOXA1 for Gem-miR-194. We also found that our Gem-modified miRNA mimics exhibit an enhanced efficacy compared to Gem in patient-derived PDAC organoids. Furthermore, we observed that Gem-miR-15a significantly inhibits PDAC tumor growth in vivo without observing any noticeable signs of toxicity. Overall, our results demonstrate the therapeutic potential of Gem-modified miRNAs as a treatment strategy for PDAC.

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Replication stress (RS) is a characteristic state of cancer cells as they tend to exchange precision of replication for fast proliferation and increased genomic instability. To overcome the consequences of improper replication control, malignant cells frequently inactivate parts of their DNA damage response (DDR) pathways (the ATM-CHK2-p53 pathway), while relying on other pathways which help to maintain replication fork stability (ATR-CHK1). This creates a dependency on the remaining DDR pathways, vulnerability to further destabilization of replication and synthetic lethality of DDR inhibitors with common oncogenic alterations such as mutations of TP53, RB1, ATM, amplifications of MYC, CCNE1 and others. The response to RS is normally limited by coordination of cell cycle, transcription and replication. Inhibition of WEE1 and PKMYT1 kinases, which prevent unscheduled mitosis entry, leads to fragility of under-replicated sites. Recent evidence also shows that inhibition of Cyclin-dependent kinases (CDKs), such as CDK4/6, CDK2, CDK8/19 and CDK12/13 can contribute to RS through disruption of DNA repair and replication control. Here, we review the main causes of RS in cancers as well as main therapeutic targets-ATR, CHK1, PARP and their inhibitors.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors\' combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

WEE1 and CHEK1 (CHK1) kinases are critical regulators of the G2/M cell cycle checkpoint and DNA damage response pathways. The WEE1 inhibitor AZD1775 and the CHK1 inhibitor SRA737 are in clinical trials for various cancers, but have not been examined in prostate cancer, particularly castration-resistant (CRPC) and neuroendocrine prostate cancers (NEPC). Our data demonstrated elevated WEE1 and CHK1 expressions in CRPC/NEPC cell lines and patient samples. AZD1775 resulted in rapid and potent cell killing with comparable IC50s across different prostate cancer cell lines, while SRA737 displayed time-dependent progressive cell killing with 10- to 20-fold differences in IC50s. Notably, their combination synergistically reduced the viability of all CRPC cell lines and tumor spheroids in a concentration- and time-dependent manner. Importantly, in a transgenic mouse model of NEPC, both agents alone or in combination suppressed tumor growth, improved overall survival, and reduced the incidence of distant metastases, with SRA737 exhibiting remarkable single agent anticancer activity. Mechanistically, SRA737 synergized with AZD1775 by blocking AZD1775-induced feedback activation of CHK1 in prostate cancer cells, resulting in increased mitotic entry and accumulation of DNA damage. In summary, this preclinical study shows that CHK1 inhibitor SRA737 alone and its combination with AZD1775 offer potential effective treatments for CRPC and NEPC.