Chk1 Inhibitor Research Articles

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells

Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

PARP1 and CHK1 coordinate PLK1 enzymatic activity during the DNA damage response to promote homologous recombination-mediated repair.

Polo-like kinase 1 (PLK1) is a master kinase that regulates cell cycle progression. How its enzymatic activity is regulated in response to DNA damage is not fully understood. We show that PLK1 is enriched at double strand breaks (DSBs) within seconds of UV laser irradiation in a PARP-1-dependent manner and then disperses within 10 min in a PARG-dependent manner. Poly(ADP-)ribose (PAR) chains directly bind to PLK1 in vitro and inhibit its enzymatic activity. CHK1-mediated PLK1 phosphorylation at S137 prevents its binding to PAR and recruitment to DSBs but ensures PLK1 phosphorylation at T210 and its enzymatic activity toward RAD51 at S14. This subsequent phosphorylation event at S14 primes RAD51 for CHK1-mediated phosphorylation at T309, which is essential for full RAD51 activation. This CHK1–PLK1–RAD51 axis ultimately promotes homologous recombination (HR)-mediated repair and ensures chromosome stability and cellular radiosensitivity. These findings provide biological insight for combined cancer therapy using inhibitors of PARG and CHK1.

Abstract 1010: A study of combinatorial growth inhibition, cell death and DNA damage repair caused by CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib in TP53 mutated cell lines

Abstract Purpose of study: CHK1 and WEE1 are critical determinants of the G2/M cell cycle checkpoint. The G2/M checkpoint is crucial to enable DNA repair caused by endogenous and exogenous stress. We hypothesised that simultaneous inhibition of CHK1 and WEE1 would cause cell death in TP53 mutated cell lines because of the associated replication stress due to an abrogated G1/S checkpoint. Experimental procedures: The potentiation index of the combination of SRA737 and adavosertib was calculated in six cancer cell lines harbouring TP53 mutations (OVCAR3, COV505, BT20, MDA-MB-436 and CAPAN1, MIAPaCa2) using Cell Titre Blue assays. Cell death and DNA damage was quantified using c-PARP, p-CHK1 Ser345 and gamma H2AX using western blot analysis. In-vivo studies evaluated the efficacy of vehicle control, SRA737, adavosertib and the combination in OVCAR3 and MDA-MB-436 xenograft models. We further explored the mechanisms of DNA damage repair caused by the single agents and the combination using two functional fluorescent multiplex DNA repair assay platforms in OVCAR3 and MDA-MB-436 cells; Exy-SPOT (base excision repair/nucleotide excision repair) and Next-SPOT (double strand break repair). Results: The potentiation index of the addition of SRA737 to adavosertib were 19, 8.6, 8.8, 9.7, 27.2 and 5.7 for COV504, OVCAR3, BT20, MDA-MB-436, CAPAN1 and MIAPaCa2 respectively. The combination of both drugs at single agent GI50 concentrations caused increase in c-PARP, p-CHK1 Ser345 and gamma H2AX in all cell lines studied. We chose to explore the combination in the TP53 mutated OVCAR3 and the TP53 and BRCA1 mutated MDA-MB-436 cell lines. In the OVCAR3 xenograft model, the combination of SRA737 and adavosertib caused tumour regressions and tumour volumes were significantly smaller than controls, p<0001. The combination caused a lesser but significant degree of reduction in the tumour volume in the MDA-MB-436 xenograft model compared to control p=0.003. There was less than a 20% loss of mouse body weight in treatment arms of both xenograft models. Functional DNA damage repair analysis showed the combination of SRA737 and adavosertib caused a significantly greater reduction in oxidative stress related base excision repair (Etheno and 8oxoG lesions) as well as double stranded break repair via alternative end joining in the OVCAR3 cell lines compared to MDA-MB-436 cells, p=0.009, p=0.014 and p=0.005 respectively. Conclusions: The combination of SRA737 and adavosertib caused cell death in TP53 mutated cell lines. There were differences in the DNA damage repair mechanisms in across cell lines and the combination showed significant activity in the xenograft models studied. The combination of SRA737 and adavosertib warrants further evaluation for the treatment of selected TP53 mutated cancers. Citation Format: Adam Stewart, Lisa Pickard, Albert E. Hallsworth, Sylvie Sauvaigo, Giovanna Muggiolu, Florence Raynaud, UDAI BANERJI. A study of combinatorial growth inhibition, cell death and DNA damage repair caused by CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib in TP53 mutated cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1010.

Combined Inhibition of Chk1 and Wee1 As a New Therapeutic Strategy for High-Risk Multiple Myeloma Patients

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite introduction of novels agents that have significantly improved clinical outcomes, most patients relapse and develop drug resistance. Therefore, novel therapeutic strategies to overcome chemotherapy resistance are needed in clinical settings.MM is characterized by genomic instability and high level of replicative stress that arise during the disease's pathogenesis. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways that include the kinases ATR, Chk1 and Wee1. Chk1 is activated by ATR after DNA damage or replicative stress and promote cell cycle arrest through the regulation of S and G2 checkpoints. Wee1 kinase plays a central role in the proper timing of cell division cycle controlling entry into mitosis and DNA replication during S phase. Interestingly, experimental studies have identified that Chk1 inhibitors are strongly synergistic with Wee1 inhibitors in various cancer models. Furthermore, Chk1 and Wee1 inhibitors are currently under clinical investigation.Here, we investigated the therapeutic interest of Chk1 and Wee1 in MM.First we investigated the prognostic value of Chk1 and/or Wee1 gene expression in MM patients. Both Chk1 and Wee1 were significantly overexpressed in human myeloma cell lines (HMCLs, n= 40) compared to bone marrow plasma cells (BMPCs, n=5) (P<001 and P<001 respectively). Chk1 and Wee1 expression were significantly higher in the poor prognosis “proliferation” MM subgroup (P<001 and P<001 respectively).High Chk1 expression and high Wee1 expression could predict for shorter overall survival (OS) in 4 independent cohorts of newly-diagnosed patients (P<0.001 and P=0.007 in the HM cohort (N=206), P<0.001 and P=0.008 in UAMS-TT2 cohort (N=345), P<0.001 and P<0.001 in Hovon cohort (N=282) and P=0.002 and P<0.001 in UAMS-TT3 cohort (N=186)). Same results were obtained for event free survival (EFS). Moreover, Chk1 and Wee1 high expression are associated with a poor prognosis in a cohort of 242 patients treated by Bortezomib monotherapy (Mulligan Cohort, P<0.001 and P=0.001 respectively).Interestingly, combined high expression of Chk1 and Wee1 are associated to significantly shorter survival compared to high expression of Chk1 or Wee1 alone in the 4 independent cohorts investigated. Same results were obtained for event free survival (EFS).According, to these results, we investigated the therapeutic interest of Chk1 and Wee1 inhibitors (AZD7762 and MK-1775) alone and in combination in MM.Chk1 inhibitor (AZD7762) induced a dose dependent cell growth inhibition in 13 HMCLs with a median IC50 of 179nM (range 69-366nM). A correlation between HMCLs sensitivity and P53 mutation was observed (r =0.7, P<0.01).Wee1 inhibitor (MK-1775) induced also a dose dependent inhibition of cell growth in all investigated HMCLs (n=10) with a median of IC50 of 450nM (range 100-800nM).Combination treatment of Wee1 inhibitor IC20 with various doses of Chk1 inhibitor was performed in 4 HMCLs. We identified a high synergy (combination index range 0.4-0.7) associated with a significant and remarkable reduction of Chk1 inhibitor (AZD7762) IC50 (198nM to 20nM for XG6; 166nM to 25nM for AMO1; 386nM to 18nM for XG7 and 163nM to 21nM for OPM2) in all the tested HMCLs. Chk1 and Wee1 inhibitors combination demonstrated a significant increase in apoptosis induction, proliferation inhibition and DNA damage induction compared to Chk1i or Wee1i alone in 3 HMCLs.We investigated the effects of Chk1i, Wee1i and Chk1i/Wee1i combination on primary MM cells of patients (n = 5) co-cultured with their bone marrow microenvironment. After 4 days of treatment, cells were enumerated and the fraction of viable myeloma cells (CD138+) and non-myeloma cells (CD138-) were determined by flow cytometry. Chk1i/Wee1i combination significantly and specifically induced a higher toxicity on myeloma cells of patients compared to Chk1i or Wee1i alone. This toxicity is specific of MM cells since normal bone marrow cells were significantly less affected by the combination treatment (P < 0.01).Taken together, these data suggest that association of Chk1i and Wee1i may represent a promising therapeutic approach in high-risk MM patients characterized by high Chk1 and Wee1 expression. DisclosuresBruyer:Diag2tec: Employment. Martin:Diag2tec: Employment. Cartron:Celgene: Consultancy, Employment; Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:EngMab: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding.

Synthetic Lethal Screening Identifies CHK1 Inhibition As an Exploitable Vulnerability in EZH2 Deficient T-ALL

Loss of function mutations of Enhancer of Zeste (Drosophila) homolog 2 (EZH2), the enzymatic component of Polycomb Repressive Complex 2 (PRC2), are particularly frequent in the early T cell precursor (ETP) subtype of T cell Acute Lymphoblastic Leukaemia (T-ALL), and have been associated with chemotherapy-resistance and a poor prognosis. There is thus a need to identify novel treatment strategies for this subgroup of patients.We hypothesized that inactivation of EZH2 in T-ALL cells induces specific synthetic lethal vulnerabilities that can be exploited using drugs targeting other dependency pathways, potentially resulting in a clinically meaningful therapeutic strategy. Using isogenic Jurkat T-ALL cells, with and without EZH2 -inactivating mutations generated by double-nicking CRISPR Cas9, we performed cell-based drug screening using 220 well-characterized compounds, enriched for drugs targeting epigenetic, cell cycle and DNA repair machinery. Strikingly, we observed that EZH2 -knock out (KO) cells are hypersensitive to checkpoint kinase 1 (CHK1) inhibition by the clinical-grade small molecule MK8776, showing an IC50 reduction of over 2-fold. This effect was similarly observed when testing other CHK1 inhibitors (LY2603618 and CHIR-124), but was absent in the presence of inhibitors of other DNA damage-response kinases such as ATM, ATR and CHK2, demonstrating the specificity of this synthetic lethal interaction. Furthermore, pharmacological inhibition of EZH2 methyltransferase activity by GSK126 in human T-ALL cell lines resulted in sensitization to CHK1 inhibition. Notably, MK8776 resulted in apoptosis of EZH2-deficient cells as determined by Annexin V-PI staining.CHK1 inhibition has previously been shown to result in enhanced DNA damage and accumulation of cells with incompletely replicated DNA. In line with this, EZH2 -KO cells treated with MK8776 exhibited markedly elevated levels of the DNA damage marker yH2AX. Interestingly, the highest proportion of apoptotic cells were found at the G1 phase of the cell cycle when analyzed by TUNEL-PI, suggesting that MK8776 treated EZH2 -KO cells with accumulated DNA damage override the G2 checkpoint, exit mitosis and undergo mitotic catastrophe-related cell death during G1, a feature characteristic of mitotic slippage.To investigate the mechanism further, gene expression of two EZH2 -KO Jurkat clones was compared to their isogenic parental control by RNA-seq. EZH2 -KO cells showed a striking upregulation of genes associated with human ETP-ALL including LYL1, MYCN, KIT and HHEX, a finding that could be recapitulated with GSK126 treatment. Gene set enrichment analysis (GSEA) showed significant changes of expression in MYC target genes. Importantly, MYCN protein was markedly upregulated in both EZH2 -KO and GSK126 treated cells. We speculate that increased MYCN expression induces high levels of replication stress and increased dependency on CHK1 for cell survival. Our findings suggest that specific inhibition of the CHK1 pathway can preferentially target PRC2-deficient leukemic cells, providing a potentially less toxic and more effective treatment for this high-risk subgroup of patients. DisclosuresNo relevant conflicts of interest to declare.

Kinome Expression Profiling in Multiple Myeloma Identifies New Therapeutic Targets

Multiple Myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Active research on MM allowed great improvement in treatment, including proteasomes inhibitors or immunomodulatory agents, that enhanced significantly the patients median survival from 3-4 years in the 90's to 7-8 years nowadays. However there is a vital need for additional therapies since until today, MM is a condition that cannot be cured, and all patients finally relapse. Although numbers of kinases inhibitors are currently in preclinical and clinical development in cancers, kinases have only be poorly studied in MM. Thus only one study (Tiedeman RE Blood 2010) examined the kinome in MM by using a screening of siRNA in Human Myeloma Cell lines (HMCL).Here, in contrast to Tiedeman et al., we evaluated the potential of kinase involvement directly on patient outcome. We first performed kinome in silico analysis in three independent cohorts of patients, and identified 36 kinases significantly and identically involved in MM patient's survival. We built a Kinome Index (KI) from the 36 kinases expression, and showed that KI: 1) is related to OS and EFS prognosis in 3 independent cohorts, 2) is linked to proliferation and poor prognosis subgroups of MM molecular classification, 3) is associated with relapse. Among the 36 targets, 7 had available inhibitors and were not already consistently studied in MM (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK). We then tested the therapeutic interest of these 7 kinase inhibitors using 4 HMCLs representing two IL-6 independent (AMO1, OPM2) and two IL-6 dependent (XG1, XG21) cell lines. All tested inhibitors significantly reduced viability of the cells, and had IC50 from the nanomolar (for MELKi, CHK1i and PLK4i) to the micromolar range (for PBKi, SRPK1i, CDC7-DBF4i and MPS1/TTKi). Annexin, PARP and cell cycle analysis following treatment with selected concentration of the inhibitors in AMO1, showed significant increase in apoptosis, associated for most of them with a perturbation of the cell cycle. Treatment of primary myeloma cells from patients with MELKi, CHK1i and PLK4i induced MM cell death while they didn't impacted significantly the cellular microenvironment. Similarly experiments using murine MM cells, from 5TMM models, confirmed the potential of these three inhibitors.Next, IC20 of the different inhibitors were tested in co-treatment with melphalan, lenaledomide or velcade in XG1, XG21, AMO1 and OPM2 to challenge the capabilities of the selected kinases inhibitors to synergize with major routinely used therapeutics. We didn't observed any synergy for the co-treatments with bortezomib. However, CHK1i, MELKi, PBKi, DBF4i synergized with melphalan to induce MM cell death. Furthermore, CHK1i, MELKi, PLK4i, PBKi, SRPK1i demonstrated also a synergistic effect when combined with lenalidomide, highlighting their potential therapeutic interest in MM. Analysis of apoptosis and cell cycle following co-treatments with melphalan and lenaledomide using AMO1 cell line demonstrated an increase in PARP cleavage and annexin V staining in association with significant cell cycle deregulation. One of the outstanding co-treatment effects was the reversion of the natural AMO1's resistance to lenaledomide with CHK1, MELK and PBK inhibitors used at their IC20. We then tested their therapeutic interest to overcome melphalan drug resistance using wild-type (XG7-WT) and melphalan-resistant (XG7-Mres) cell lines. PLK4 and DBF4 inhibitors demonstrated higher activity on XG7-Mres cells compared to their WT control. In addition CHK1, PBK and DBF4 inhibitors (IC20) were able to re-sensitize XG7-Mres to melphalan at a similar level than XG7-WT. Altogether we identified 36 new potential kinases deregulated in MM in association with a poor prognostic value. Seven of them represent potential new MM therapeutic targets alone or in combination with conventional therapies in high-risk MM patients. DisclosuresBruyer:Diag2tec: Employment. Cartron:Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Celgene: Consultancy, Employment; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:Sanofi: Research Funding; EngMab: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees.

Targeting DNA Repair Mechanisms to Overcome Drug Resistance in Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of adult non-Hodgkin lymphomas. Most DLBCL patients achieve long-term remission after treatment, but a third relapse after conventional Rituximab (R)-based chemotherapy regimens, such as CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) (Siegel et al, 2012).Cancer cells are exposed to chronic replication stress, which impedes the duplication of their genome and induces mitotic catastrophe (Shaheen et al, 2011). Functional DNA repair pathways are therefore important for the survival of cancer cells. This dependence can be exploited therapeutically to hamper repair of the intrinsic DNA damage occurring during replication or to exacerbate DNA damage induced by chemotherapy (Shaheen et al, Blood 2011). Furthermore, high-risk DLBCL patients overexpress genes potentially involved in resistance to CHOP-based regimens, such as genes of the nucleotide excision repair (NER) pathway (Bret et al, Oncotarget 2012, Cell Cycle 2013).We recently developed GEP-based DNA repair scores that allow to identify high-risk DLBCL patients that could benefit from treatment with DNA repair inhibitors (Bret at al, BJH 2015).DLBCL treatments include cyclophosphamide, a nitrogen mustard derivate that induces interstrand crosslinks (ICLs), and doxorubicin, a DNA topoisomerase inhibitor that induces DNA double-strand breaks, DNA adducts and formaldehyde-dependent ICL formation. Inhibiting DNA repair is a promising strategy to improve the efficacy of genotoxic drugs and overcome drug resistance. Our data support the view that inhibitors of DNA damage signaling and DNA repair have potential therapeutic interest in DLBCL.We characterized the drug-response of 16 DLBCL cell lines to 9 DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1/2 inhibitor), AZD6738 (ATR inhibitor), MK8776 (CHK1 inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor), Lomeguatrib (MGMT) and genotoxic agents used in DLBCL treatment (Cyclophosphamide, Gemcitabine, Doxorubicin and Etoposide) (Table 1). All drugs induced significant apoptosis (Annexin V staining and PARP cleavage) and significant inhibition of proliferation (BrdU incorporation) in the different DLBCL cell lines tested (P <0.05). CHK1/2 inhibitor, Wee1 inhibitor, Cyclophosphamide, Gemcitabine and Doxorubicin induced DNA damages monitored by H2AX phosphorylation. Correlating drug response of each compounds with our GEP-based DNA repair scores (Bret et al, BJH 2015), we identified a significant correlation between FANC score and response to ATR inhibitor (AZD6738) and HRR score/BER score and response to Etoposide (P<0.05). High-risk DLBCL patients identified with GEP-based FANC, HRR and BER scores may benefit from treatment by ATR inhibitors or Etoposide respectively.Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 of DNA repair targeted treatments with conventional genotoxic agents in DLBCL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone, a total of 3 synthetic lethal combinations have been identified including cyclophosphamide with CHK1/2 inhibitor (PF477736) (IC50 reduced from 2.63 µM to 1.20 µM), cyclophosphamide and ATR inhibitor (AZD6738) (IC50 reduced from 2.63 µM to 0.74 µM) and doxorubicin with DNAPK inhibitor (NU7441) (IC50 reduced from 57 nM to 14 nM).Furthermore we identified new potent synergistic combinations (combination index < 1) including CHK1/2 inhibitor (PF477736) with etoposide (IC50 reduced from 560 nM to 437 nM), ATR inhibitor (AZD6738) (IC50 reduced from 560 nM to 364 nM) with etoposide and ATM inhibitor (KU55933) with etoposide (IC50 reduced from 560 nM to 269 nM).Despite overall improvements in the treatment of DLBCL, including the use of rituximab, approximately one-third of patients fail to achieve complete remission or experience relapse. This remains a major cause of morbidity and mortality. The DNA repair scores could be useful to identify high-risk DLBCL patients and define the best synthetic lethal approach combining DNA repair inhibitors with conventional chemotherapy. These results open new perspectives to improve the treatment of DLBCL patients and provide new strategies to overcome drug resistance. [Display omitted] DisclosuresCartron:Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Celgene: Consultancy, Employment; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding.

Activation of CDC25A phosphatase is limited by CDK2/cyclin A-mediated feedback inhibition

ABSTRACT Cyclin-dependent kinase (CDK) 1 complexed with cyclin B is a driver of mitosis, while CDK2 drives S phase entry and replicon initiation. CDK2 activity increases as cells progress through S phase, and its cyclin partner switches from cyclin E to cyclin A. Activation of CDK2 requires dephosphorylation of tyrosine-15 by CDC25A. DNA damage activates the checkpoint protein CHK1, which phosphorylates and degrades CDC25A to prevent activation of CDK2 and protect from cell cycle progression before damage is repaired. CHK1 inhibitors were developed to circumvent this arrest and enhance the efficacy of many cancer chemotherapeutic agents. CHK1 inhibition results in the accumulation of CDC25A and activation of CDK2. We demonstrate that inhibition of CDK2 or suppression of cyclin A also results in accumulation of CDC25A suggesting a feedback loop that prevents over activation of this pathway. The feedback inhibition of CDC25A targets phosphorylation of S88-CDC25A, which resides within a CDK consensus sequence. In contrast, it appears that CDK complexes with cyclin B (and possibly cyclin E) stabilize CDC25A in a feed-forward activation loop. While CDK2/cyclin A would normally be active at late S/G2, we propose that this feedback inhibitory loop prevents over activation of CDK2 in early S phase, while still leaving CDK2/cyclin E to catalyze replicon initiation. One importance of this observation is that a subset of cancer cell lines are very sensitive to CHK1 inhibition, which is mediated by CDK2/cyclin A activity in S phase cells. Hence, dysregulation of this feedback loop might facilitate sensitivity of the cells.

Checkpoint Kinase 1 (Chk1) inhibition fails to activate the Stimulator of Interferon Genes (STING) innate immune signalling in a human coculture cancer system

Utilising Checkpoint Kinase 1 (Chk1) inhibitors to increase cytoplasmic DNA may be a potential strategy to increase the sensitivity of tumours to immune checkpoint modulators. The appearance of DNA in the cytoplasm can drive Cyclic GMP-AMP Synthase-2′,3′-Cyclic Guanosine Monophosphate–Adenosine Monophosphate-Stimulator of Interferon Genes (cGAS-cGAMP-STING) inflammatory, anti-tumour T-cell activity via a type I interferon (IFN) and nuclear factor-κB response. In the THP1-Dual reporter cell line, the STING agonist cGAMP activated both reporters, and increased phosphorylation of the innate immune pathway signallers Tank Binding Kinase 1 (TBK1) and Interferon Regulatory Factor (IRF) 3. Inhibition of Chk1 increased TBK1 but not IRF3 phosphorylation and did not induce IRF or NF-κB reporter activation. cGAMP induced a Type I IFN response in THP1 cells whereas inhibition of Chk1 did not. HT29 or HCC1937 cell treatment with a Chk1 inhibitor increased cytoplasmic dsDNA in treated HCC1937 but not HT29 cells and increased IRF reporter activation in cocultured THP1-Dual cells. HT29 cells pre-treated with gemcitabine or camptothecin had elevated cytoplasmic dsDNA and IRF reporter activation in cocultured THP1-Dual cells. Camptothecin or gemcitabine plus a Chk1 inhibitor increased cytoplasmic dsDNA but Chk1 inhibition suppressed IRF reporter activation in cocultured THP1 cells. In THP1-Dual cells treated with cGAMP, Chk1 inhibition suppressed the activation of the IRF reporter compared to cGAMP alone. These results suggest that, in some cellular models, there is little evidence to support the combination of Chk1 inhibitors with immune checkpoint modulators and, in some combination regimes, may even prove deleterious.

Chk1 suppression leads to a reduction in the enhanced radiation-induced invasive capability on breast cancer cells.

Radiation therapy is generally effective for treating breast cancers. However, approximately 30% of patients with breast cancer experience occasional post-treatment local and distant metastasis. Low-dose (0.5 Gy) irradiation is a risk factor that promotes the invasiveness of breast cancers. Although an inhibitor of checkpoint kinase 1 (Chk1) suppresses the growth and motility of breast cancer cell lines, no study has investigated the effects of the combined use of a Chk1 inhibitor and radiation on cancer metastasis. Here, we addressed this question by treating the human breast cancer cell line MDA-MB-231 (in vitro) and mouse mammary tumor cell line 4 T1 (in vitro and in vivo) with γ-irradiation and the Chk1 inhibitor PD407824. Low-dose γ-irradiation promoted invasiveness, which was suppressed by PD407824. Comprehensive gene expression analysis revealed that low-dose γ-irradiation upregulated the mRNA and protein levels of S100A4, the both of which were downregulated by PD407824. We conclude that PD407824 suppresses the expression of S100A4. As the result, γ-irradiation-induced cell invasiveness were inhibited.

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Checkpoint kinases are required for oocyte meiotic progression by the maintenance of normal spindle structure and chromosome condensation

Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.

CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

SummaryLimited therapeutic options are available for advanced colorectal cancer (CRC). Herein, we report that exposure to a neo-synthetic bis(indolyl)thiazole alkaloid analog, nortopsentin 234 (NORA234), leads to an initial reduction of proliferative and clonogenic potential of CRC sphere cells (CR-CSphCs), followed by an adaptive response selecting the CR-CSphC-resistant compartment. Cells spared by the treatment with NORA234 express high levels of CD44v6, associated with a constitutive activation of Wnt pathway. In CR-CSphC-based organoids, NORA234 causes a genotoxic stress paralleled by G2-M cell cycle arrest and activation of CHK1, driving the DNA damage repair of CR-CSphCs, regardless of the mutational background, microsatellite stability, and consensus molecular subtype. Synergistic combination of NORA234 and CHK1 (rabusertib) targeting is synthetic lethal inducing death of both CD44v6-negative and CD44v6-positive CRC stem cell fractions, aside from Wnt pathway activity. These data could provide a rational basis to develop an effective strategy for the treatment of patients with CRC.

Inhibition of DNA Repair in Combination with Temozolomide or Dianhydrogalactiol Overcomes Temozolomide-Resistant Glioma Cells.

Simple SummaryGlioblastoma is the most prevalent and lethal brain tumor. Temozolomide is usually used for the treatment of glioblastoma. The poor prognosis of the tumor is due to drug resistance and tumor heterogeneity. The mechanism of the resistance to temozolomide is various within the same tumor. The aim of the study was to clarify the mechanism of temozolomide resistance and find methods to overcome temozolomide resistance in glioma. Inhibition of DNA repair (homologous recombination or base excision repair) resensitized resistant cells harboring different resistance mechanism to temozolomide. Additionally, a bifunctional DNA-targeting agent, dianhydrogalactiol, showed anti-tumor effect independent of MGMT and mismatch repair status. Further, inhibition of checkpoint or homologous recombination enhanced dianhydrogalactiol-induced cytotoxicity in temozolomide-resistant glioma cells. Although resistance to temozolomide is clinically important issue, selecting suitable treatments for resistance mechanism can improve the prognosis of glioma.Resistance to temozolomide and intratumoral heterogeneity contribute to the poor prognosis of glioma. The mechanisms of temozolomide resistance can vary within a heterogeneous tumor. Temozolomide adds a methyl group to DNA. The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to a futile DNA mismatch repair cycle, formation of double-strand breaks, and eventual cell death when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair (BER). The study aim was to elucidate temozolomide resistance mechanisms and identify methods to overcome temozolomide resistance in glioma. Several temozolomide-resistant clones were analyzed. Increased homologous recombination and mismatch repair system deficiencies contributed to temozolomide resistance. Inhibition of homologous recombination resensitized resistant cells with high homologous recombination efficiency. For the mismatch repair-deficient cells, inhibition of BER by PARP inhibitor potentiated temozolomide-induced cytotoxicity. Dianhydrogalactiol is a bifunctional DNA-targeting agent that forms N7-alkylguanine and inter-strand DNA crosslinks. Dianhydrogalactiol reduced the proliferation of cells independent of MGMT and mismatch repair, inducing DNA double-strand breaks and apoptosis in temozolomide-resistant cells. Further, inhibition of chk1 or homologous recombination enhanced dianhydrogalactiol-induced cytotoxicity in the cells. Selecting treatments most appropriate to the types of resistance mechanisms can potentially improve the prognosis of glioma.

Acceleration or Brakes: Which Is Rational for Cell Cycle-Targeting Neuroblastoma Therapy?

Unrestrained proliferation is a common feature of malignant neoplasms. Targeting the cell cycle is a therapeutic strategy to prevent unlimited cell division. Recently developed rationales for these selective inhibitors can be subdivided into two categories with antithetical functionality. One applies a “brake” to the cell cycle to halt cell proliferation, such as with inhibitors of cell cycle kinases. The other “accelerates” the cell cycle to initiate replication/mitotic catastrophe, such as with inhibitors of cell cycle checkpoint kinases. The fate of cell cycle progression or arrest is tightly regulated by the presence of tolerable or excessive DNA damage, respectively. This suggests that there is compatibility between inhibitors of DNA repair kinases, such as PARP inhibitors, and inhibitors of cell cycle checkpoint kinases. In the present review, we explore alterations to the cell cycle that are concomitant with altered DNA damage repair machinery in unfavorable neuroblastomas, with respect to their unique genomic and molecular features. We highlight the vulnerabilities of these alterations that are attributable to the features of each. Based on the assessment, we offer possible therapeutic approaches for personalized medicine, which are seemingly antithetical, but both are promising strategies for targeting the altered cell cycle in unfavorable neuroblastomas.

Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells

Checkpoint kinases (CHKs) are involved in the DNA damage response in many cancer cells. CHK inhibitors have been used in clinical trials in combination with chemotherapeutics; however, their effect against bladder cancer remains unclear. Here, we investigated the efficacy of combining gemcitabine with MK-8776, a novel CHK1 inhibitor, in four bladder cancer cell lines. The effects of gemcitabine and MK-8776 on cell viability, clonogenicity, cell cycle, and apoptosis were examined alongside in vivo efficacy using murine xenograft tumor models. Combined treatment inhibited the viability and colony formation of bladder cancer cells compared to either single treatment. Although gemcitabine (10 nM) alone increased the cell number in S-phase, it increased the cell number in sub-G1 phase when combined with MK-8776 (0.5 µM). Combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin-V-positive cells, indicating the induction of apoptosis. In vivo, administration of gemcitabine and MK-8776 was well tolerated and suppressed tumor growth. Mechanistically, the combined treatment elevated γH2A.X and suppressed Rad51 expression. Our study demonstrates that MK-8776 and gemcitabine combined induces apoptosis and suppresses proliferation in bladder cancer cells by inhibiting CHKs and DNA repair. Therefore, CHK1 inhibition combined with gemcitabine may be a potential treatment for bladder cancer.

Intracellular PD-L1 regulates DNA damage checkpoints and suppresses Chk1 and PARP inhibitor synthetic lethality

Abstract Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.

Far-infrared irradiation inhibits breast cancer cell proliferation independently of DNA damage through increased nuclear Ca2+/calmodulin binding modulated-activation of checkpoint kinase 2

Far-infrared (FIR) irradiation is reported to inhibit cell proliferation in various types of cancer cells; the underlying mechanism, however, remains unclear. We explored the molecular mechanisms using MDA-MB-231 human breast cancer cells. FIR irradiation significantly inhibited cell proliferation and colony formation compared to hyperthermal stimulus, with no alteration in cell viability. No increase in DNA fragmentation or phosphorylation of DNA damage kinases including ataxia-telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase, and DNA-dependent protein kinase indicated no DNA damage. FIR irradiation increased the phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (p-Chk2-Thr68) but not that of checkpoint kinase 1 at Ser345. Increased nuclear p-Chk2-Thr68 and Ca2+/CaM accumulations were found in FIR-irradiated cells, as observed in confocal microscopic analyses and cell fractionation assays. In silico analysis predicted that Chk2 possesses a Ca2+/calmodulin (CaM) binding motif ahead of its kinase domain. Indeed, Chk2 physically interacted with CaM in the presence of Ca2+, with their binding markedly pronounced in FIR-irradiated cells. Pre-treatment with a Ca2+ chelator significantly reversed FIR irradiation-increased p-Chk2-Thr68 expression. In addition, a CaM antagonist or small interfering RNA-mediated knockdown of the CaM gene expression significantly attenuated FIR irradiation-increased p-Chk2-Thr68 expression. Finally, pre-treatment with a potent Chk2 inhibitor significantly reversed both FIR irradiation-stimulated p-Chk2-Thr68 expression and irradiation-repressed cell proliferation. In conclusion, our results demonstrate that FIR irradiation inhibited breast cancer cell proliferation, independently of DNA damage, by activating the Ca2+/CaM/Chk2 signaling pathway in the nucleus. These results demonstrate a novel Chk2 activation mechanism that functions irrespective of DNA damage.

The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition.

Simple SummaryThe ATR-CHK1 axis of the DNA damage response is crucial for the survival of most colorectal cancer stem cells (CRC-SCs), but a significant fraction of primary CRC-SCs either is resistant to ATR or CHK1 inhibitors or survives the abrogation of the ATR-CHK1 cascade despite an initial response. Here, we demonstrate that the targeting of RAD51 or MRE11 improves the sensitivity of primary CRC-SCs to the CHK1/2 inhibitor prexasertib by sequentially inducing replication stress, the abrogation of cell cycle checkpoints, and the emergence of mitotic defects. This results in the induction of mitotic catastrophe and CRC-SC killing via a caspase-dependent apoptosis.Cancer stem cells (CSCs) drive not only tumor initiation and expansion, but also therapeutic resistance and tumor relapse. Therefore, CSC eradication is required for effective cancer therapy. In preclinical models, CSCs demonstrated high capability to tolerate even extensive genotoxic stress, including replication stress, because they are endowed with a very robust DNA damage response (DDR). This favors the survival of DNA-damaged CSCs instead of their inhibition via apoptosis or senescence. The DDR represents a unique CSC vulnerability, but the abrogation of the DDR through the inhibition of the ATR-CHK1 axis is effective only against some subtypes of CSCs, and resistance often emerges. Here, we analyzed the impact of druggable DDR players in the response of patient-derived colorectal CSCs (CRC-SCs) to CHK1/2 inhibitor prexasertib, identifying RAD51 and MRE11 as sensitizing targets enhancing prexasertib efficacy. We showed that combined inhibition of RAD51 and CHK1 (via B02+prexasertib) or MRE11 and CHK1 (via mirin+prexasertib) kills CSCs by affecting multiple genoprotective processes. In more detail, these two prexasertib-based regimens promote CSC eradication through a sequential mechanism involving the induction of elevated replication stress in a context in which cell cycle checkpoints usually activated during the replication stress response are abrogated. This leads to uncontrolled proliferation and premature entry into mitosis of replication-stressed cells, followed by the induction of mitotic catastrophe. CRC-SCs subjected to RAD51+CHK1 inhibitors or MRE11+CHK1 inhibitors are eventually eliminated, and CRC-SC tumorspheres inhibited or disaggregated, via a caspase-dependent apoptosis. These results support further clinical development of these prexasertib-based regimens in colorectal cancer patients.

Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo

The DNA damage checkpoint is crucial to protect genome integrity.1,2 However, the early embryos of many metazoans sacrifice this safeguard to allow for rapid cleavage divisions that are required for speedy development. At the mid-blastula transition (MBT), embryos switch from rapid cleavage divisions to slower, patterned divisions with the addition of gap phases and acquisition of DNA damage checkpoints. The timing of the MBT is dependent on the nuclear-to-cytoplasmic (N/C ratio)3-7 and the activation of the checkpoint kinase, Chk1.8-17 How Chk1 activity is coupled to the N/C ratio has remained poorly understood. Here, we show that dynamic changes in histone H3 availability in response to the increasing N/C ratio control Chk1 activity and thus time the MBT in the Drosophila embryo. We show that excess H3 in the early cycles interferes with cell-cycle slowing independent of chromatin incorporation. We find that the N-terminal tail of H3 acts as a competitive inhibitor of Chk1 invitro and reduces Chk1 activity invivo. Using a H3-tail mutant that has reduced Chk1 inhibitor activity, we show that the amount of available Chk1 sites in the H3 pool controls the dynamics of cell-cycle progression. Mathematical modeling quantitatively supports a mechanism where titration of H3 during early cleavage cycles regulates Chk1-dependent cell-cycle slowing. This study defines Chk1 regulation by H3 as a key mechanism that coordinates cell-cycle remodeling with developmental progression.