Chk1 Inhibitor Research Articles

Gynecologic Cancer InterGroup CA125 response has a high negative predictive value for CHK1 inhibitor RECIST response in recurrent ovarian cancer

We investigated the association of CA125 response with prognosis and RECIST response/progressive disease (PD) criteria in recurrent high grade serous ovarian cancer (HGSOC) patients treated with a cell cycle checkpoint kinase 1 inhibitor (CHK1i), prexasertib. 81 patients had measurable disease per RECISTv1.1, of which 72 and 70 were measurable by Gynecologic Cancer InterGroup (GCIG) CA125 response and PD criteria, respectively. Univariate and multivariate analyses showed that GCIG CA125 response (n = 32) is associated with improved progression-free survival (PFS) and overall survival (OS) compared to no GCIG CA125 response (n = 40) (median PFS 8.0 vs. 3.5 months [HR: 0.30, 95% CI: 0.18–0.51, p < 0.0001]; median OS 19.8 vs. 10.0 months [HR: 0.38, 95% CI: 0.23–0.64, p < 0.001]) independent of BRCA mutation status, platinum-sensitivity, previous PARP inhibitor therapy, ECOG performance status, and FIGO stage. Notably, GCIG CA125 response had a high negative predictive value (NPV: 93%, 95% CI: 80–98), but poor positive predictive value (PPV: 53%, 95% CI: 35–71) in predicting RECIST response. CA125 PD criteria also showed poor concordance with RECIST PD (PPV 56%, 95% CI: 40–71; NPV 33%, 95% CI: 17–54). Therefore, serum CA125 may be useful as a highly accessible prognostic and predictive biomarker to CHK1i therapy in recurrent HGSOC.

Investigation into the Neuroprotective and Therapeutic Potential of Plant-Derived Chk2 Inhibitors.

Nature provides us with a rich source of compounds with a wide range of applications, including the creation of innovative drugs. Despite advancements in chemically synthesized therapeutics, natural compounds are increasingly significant, especially in cancer treatment, a leading cause of death globally. One promising approach involves the use of natural inhibitors of checkpoint kinase 2 (Chk2), a critical regulator of DNA repair, cell cycle arrest, and apoptosis. Chk2's activation in response to DNA damage can lead to apoptosis or DNA repair, influencing glycolysis and mitochondrial function. In cancer therapy, inhibiting Chk2 can disrupt DNA repair and cell cycle progression, promoting cancer cell death and enhancing the efficacy of radiotherapy and chemotherapy. Additionally, Chk2 inhibitors can safeguard non-cancerous cells during these treatments by inhibiting p53-dependent apoptosis. Beyond oncology, Chk2 inhibition shows potential in treating hepatitis C virus (HCV) infections, as the virus relies on Chk2 for RNA replication in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), in which DNA damage plays a crucial role. Plant-derived Chk2 inhibitors, such as artemetin, rhamnetin, and curcumin, offer a promising future for treating various diseases with potentially milder side effects and broader metabolic impacts compared to conventional therapies. The review aims to underscore the immense potential of natural Chk2 inhibitors in various therapeutic contexts, particularly in oncology and the treatment of other diseases involving DNA damage and repair mechanisms. These natural Chk2 inhibitors hold significant promise for revolutionizing the landscape of cancer treatment and other diseases. Further research into these compounds could lead to the development of innovative therapies that offer hope for the future with fewer side effects and enhanced efficacy.

Synergistic induction of mitotic pyroptosis and tumor remission by inhibiting proteasome and WEE family kinases

Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.

ATR, CHK1 and WEE1 inhibitors cause homologous recombination repair deficiency to induce synthetic lethality with PARP inhibitors

PurposePARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1.Experimental designA panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM).ResultsRucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib.ConclusionsOur data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence “induced synthetic lethality” with PARPi.

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint.

The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.

MDB-101. TARGETING CTPS1 AND NUCLEOTIDE BIOSYNTHESIS PATHWAYS AS A NOVEL THERAPEUTIC APPROACH IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract Although leukemia is the leading pediatric oncology diagnosis, brain tumors are the leading cause of pediatric cancer-related death. Medulloblastoma (MB) is the most common pediatric brain tumor, representing approximately 20% of all diagnoses. MYC-amplified Group 3 medulloblastoma (G3 MB) tumors are the deadliest, with a &amp;lt; 50% 5-year overall survival rate. High risk patients receive large doses of chemo- and radio- therapy (CT/RT) with most survivors experiencing debilitating neurological and cognitive sequelae making the identification of novel targets of the utmost importance. Utilizing whole-genome CRISPR screening, we identified the enzyme CTPS1 as a strong G3 MB-specific vulnerability. Further analysis revealed dependencies on other enzymes in the de novo pyrimidine synthesis pathway, which is corroborated by recent metabolic studies. Targeted knockdown of CTPS1 restricts sphere formation and reduces proliferation of G3 MB tumor lines. Treatment with a specific CTPS1 inhibitor reduced proliferation but did not induce cell death, suggesting a cytostatic mechanism. Previous work has shown that triggering an imbalance in pyrimidine and purine levels leads to replication stress and activation of the ATR/CHK1 pathway. Both genetic and pharmacologic inhibition of CTPS1 leads to CHK phosphorylation, allowing for cell survival despite lack of dNTPs. Previously, the CHK1 inhibitor, prexasertib, demonstrated robust brain accumulation and targeting of G3 MB in pre-clinical models. Dual inhibition of CHK1 and CTPS1 synergizes to significantly reduce cell viability in vitro and tumor growth in vivo. We further demonstrated robust synergy of CHK1 inhibition with either glutamine antagonists or other inhibitors of the de novo pyrimidine synthesis pathway, prolonging in vivo survival in an orthotopic xenograft G3 MB model. The results of this investigation confirm CTPS1 as a novel target in G3 MB, pinpoint a dependency on de novo pyrimidine rather than purine biosynthesis, and identifies a novel rational combination for treatment in MYC-amplified MB patients.

MDB-65. RESULTS FROM THE SJ-ELIOT PHASE 1 CLINICAL TRIAL EVALUATING PREXASERTIB (LY2606368) IN COMBINATION WITH CYCLOPHOSPHAMIDE OR GEMCITABINE FOR CHILDREN AND ADOLESCENTS WITH REFRACTORY OR RECURRENT MEDULLOBLASTOMA

Abstract BACKGROUND SJ-ELIOT (NCT04023669) was a phase 1 trial that explored the combination of the checkpoint kinase inhibitor, prexasertib with the DNA-damaging agents, cyclophosphamide and gemcitabine, in recurrent or refractory medulloblastoma. METHODS Participants ≥ 1 year and &amp;lt; 25 years were stratified to one of two treatment strata: stratum A prexasertib and cyclophosphamide and stratum B prexasertib and gemcitabine. Patients with Group 3/4 medulloblastoma were assigned to either stratum A or B, whereas patients with SHH medulloblastoma were assigned to stratum A. A Rolling-6 design was used. RESULTS Fifteen patients were enrolled on stratum A and six patients on stratum B. The study was closed early due to drug supply issues. In stratum A, three patients were escalated to dose level (DL) 3 and none had dose limiting toxicity (DLT). For stratum B, of the first three patients enrolled on DL1, two had DLTs (grade 3 hypotension) related to drug reactions. Consequently, stratum B was amended to add hydrocortisone prophylaxis. A further three patients were enrolled. One patient had a grade 3 DLT (ALT increase). The remaining two patients electively stopped therapy within the first two courses. One patient on stratum A, with SHH TP53 mutant, MYCN amplified medulloblastoma, achieved a complete sustained response, received 12 cycles, and electively stopped treatment after 408 days. They progressed nine months later. The remaining 14 patients on stratum A progressed. There were no objective responses on stratum B. Median PFS were 1.9 and 2.1 months for stratum A and B respectively. CONCLUSIONS The MTD/RP2D was not established for either arm. In stratum A, DL3 was tolerated, whereas DL1 for Stratum B was not tolerated. Despite robust preclinical data, no efficacy signal was observed. Intriguingly, one patient with a highly aggressive tumor (SHH TP53 mutant, MYCN amplified) appeared to derive a sustained benefit from the combination of prexasertib and cyclophosphamide.

CHK1 inhibitor induced PARylation by targeting PARG causes excessive replication and metabolic stress and overcomes chemoresistance in ovarian cancer

Chemoresistance contributes to the majority of deaths in women with ovarian cancer (OC). Altered DNA repair and metabolic signaling is implicated in mediating therapeutic resistance. DNA damage checkpoint kinase 1 (CHK1) integrates cell cycle and DNA repair in replicating cells, and its inhibition causes replication stress, repair deficiency and cell cycle dysregulation. We observed elevated Poly-ADP-ribosylation (PAR) of proteins (PARylation) and subsequent decrease in cellular NAD+ levels in OC cells treated with the CHK1 inhibitor prexasertib, indicating activation of NAD+ dependent DNA repair enzymes poly-ADP-ribose polymerases (PARP1/2). While multiple PARP inhibitors are in clinical use in treating OC, tumor resistance to these drugs is highly imminent. We reasoned that inhibition of dePARylation by targeting Poly (ADP-ribose) glycohydrolase (PARG) would disrupt metabolic and DNA repair crosstalk to overcome chemoresistance. Although PARG inhibition (PARGi) trapped PARylation of the proteins and activated CHK1, it did not cause any significant OC cell death. However, OC cells deficient in CHK1 were hypersensitive to PARGi, suggesting a role for metabolic and DNA repair crosstalk in protection of OC cells. Correspondingly, OC cells treated with a combination of CHK1 and PARG inhibitors exhibited excessive replication stress-mediated DNA lesions, cell cycle dysregulation, and mitotic catastrophe compared to individual drugs. Interestingly, increased PARylation observed in combination treatment resulted in depletion of NAD+ levels. These decreased NAD+ levels were also paralleled with reduced aldehyde dehydrogenase (ALDH) activity, which requires NAD+ to maintain cancer stem cells. Furthermore, prexasertib and PARGi combinations exhibited synergistic cell death in OC cells, including an isogenic chemoresistant cell line and 3D organoid models of primary patient-derived OC cell lines. Collectively, our data highlight a novel crosstalk between metabolism and DNA repair involving replication stress and NAD+-dependent PARylation, and suggest a novel combination therapy of CHK1 and PARG inhibitors to overcome chemoresistance in OC.

Abstract B006: Data-driven discoveries of molecular mechanism and therapeutic vulnerabilities of CDK12 mutant tumors

Abstract Somatic structural variations (SVs) are common in cancer. Although a small fraction of SVs in breast and ovarian cancers can be attributed to homologous recombination deficiency, the underlying molecular mechanisms for the vast majority of somatic SVs remain unclear. Here, we focus on the roles of transcription and DNA replication collisions in genomic instability in cancer. Such collisions are unavoidable in cells since both transcription and replication use the same DNA as template. We hypothesized that transcription replication collisions (TRCs), if not properly repaired, would lead to collapsed replication forks and result in SVs. To this end, we studied somatic SVs in 6193 high-coverage whole-genome sequenced primary and metastatic tumors from three independent pan-cancer cohorts. A total of 13 conserved SV signatures, representing independent molecular mechanisms, were deconvoluted from these cohorts using non-negative matrix factorization approach. We detected replicated-strand bias, the expected footprint of transcription-replication collision, in large tandem duplications (TDs) across multiple cohorts. This bias was only observed in expressed genes, consistent with TRCs depending on transcription activity. Large TDs were abundant in female-specific (breast, ovarian and uterus), upper gastric-intestinal tract, and prostate cancers. They were associated with CDK12 mutations and worse patient survival. CDK12 is a cyclin-dependent kinase, a key regulator of transcription elongation and termination. Deleting or suppressing CDK12 using CRISPR-Cas9 in prostate cell lines increased RNA:DNA hybrids (R-loops), promoted TRCs, and ultimately led to large TDs. Finally, we found that cells lacking CDK12 were sensitive to WEE1, CHK1 and ATR inhibitors. In summary, our data suggest that large TDs in cancer form due to impaired TRC repair and can be used as a biomarker for prognosis and treatment. Citation Format: Lixing Yang. Data-driven discoveries of molecular mechanism and therapeutic vulnerabilities of CDK12 mutant tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B006.

Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition.

Splicing factor SF3B1 mutations are frequent somatic lesions in myeloid neoplasms that transform hematopoietic stem cells (HSCs) by inducing mis-splicing of target genes. However, the molecular and functional consequences of SF3B1 mutations in human HSCs and progenitors (HSPCs) remain unclear. Here, we identify the mis-splicing program in human HSPCs as a targetable vulnerability by precise gene editing of SF3B1 K700E mutations in primary CD34+ cells. Mutant SF3B1 induced pervasive mis-splicing and reduced expression of genes regulating mitosis and genome maintenance leading to altered differentiation, delayed G2/M progression, and profound sensitivity to CHK1 inhibition (CHK1i). Mis-splicing or reduced expression of mitotic regulators BUBR1 and CDC27 delayed G2/M transit and promoted CHK1i sensitivity. Clinical CHK1i prexasertib selectively targeted SF3B1-mutant immunophenotypic HSCs and abrogated engraftment in vivo. These findings identify mis-splicing of mitotic regulators in SF3B1-mutant HSPCs as a targetable vulnerability engaged by pharmacological CHK1 inhibition. Significance: In this study, we engineer precise SF3B1 mutations in human HSPCs and identify CHK1 inhibition as a selective vulnerability promoted by mis-splicing of mitotic regulators. These findings uncover the mis-splicing program induced by mutant SF3B1 in human HSPCs and show that it can be therapeutically targeted by clinical CHK1 inhibitors.

Methylation of FAM110C is a synthetic lethal marker for ATR/CHK1 inhibitors in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. An epigenetic-based synthetic lethal strategy provides a novel opportunity for PDAC treatment. Finding more DNA damage repair (DDR)-related or cell fate-related molecules with aberrant epigenetic changes is becoming very important. Family with sequence similarity 110C (FAM110C) is a cell fate-related gene and its function in cancer remains unclear. Seven cell lines, 34 cases of intraductal papillary mucinous neoplasm (IPMN), 15 cases of mucinous cystic neoplasm (MCN) and 284 cases of PDAC samples were employed. Methylation-specific PCR, western blot, CRISPR knockout, immunoprecipitation and a xenograft mouse model were used in this study. FAM110C is methylated in 41.18% (14/34) of IPMN, 46.67% (7/15) of MCN and 72.89% (207/284) of PDAC, with a progression trend from IPMN/MCN to pancreatic cancer (P = 0.0001, P = 0.0389). FAM110C methylation is significantly associated with poor overall survival (OS) (P = 0.0065) and is an independent prognostic marker for poor OS (P = 0.0159). FAM110C inhibits PDAC cells growth both in vitro and in vivo, serving as a novel tumor suppressor. FAM110C activates ATM and NHEJ signaling pathways by interacting with HMGB1. Loss of FAM110C expression sensitizes PDAC cells to VE-822 (an ATR inhibitor) and MK-8776 (a CHK1 inhibitor). FAM110C methylation is a potential diagnostic and prognostic marker in PDAC, and its epigenetic silencing sensitizes PDAC cells to ATR/CHK1 inhibitors.

ImmunoSarc II master trial (phase II of sunitinib and nivolumab): Results from the dedifferentiated chondrosarcoma (DDCS) cohort—A GEIS, ISG and UCL study.

11506 Background: Dedifferentiated chondrosarcoma (DDCS) is a rare aggressive CS subtype with a dismal prognosis and no standard of care systemic therapy. For those with advanced, metastatic disease, retrospective chemotherapy studies demonstrate short-term median progression-free survival (PFS) of around 3 to 5.5 months. Benefit from checkpoint and tyrosine kinase inhibitors has been reported, however clinical trial data is scarce, and endpoints are not well defined, thus benchmarking outcomes is challenging. The IMMUNOSARC I master-trial exploring sunitinib plus nivolumab in multiple sarcoma subtypes, detected potential activity in DDCS leading to a specific cohort within the phase II trial IMMUNOSARC II (NCT03277924). Methods: Patients (pts) with ECOG 0-1, centrally confirmed and progressive, measurable DDCS were eligible and treated with sunitinib 37.5 mg/d for 14 days (induction phase), followed by 25 mg/d, in combination with nivolumab 240 mg every 2 weeks until progressive disease (PD) or intolerance. Imaging assessments were performed every 8 weeks. Main endpoint was 6-m PFSR with one-arm survival design (type I error α 0.05, power 0.90, H0 40%, H1 70%) and a Brookmeyer-Crowley like test was assumed with at least 14 of 23 pts required without progression at 6 months. Results: Between December 2019 and October 2023, 24 pts with a median age of 60 years (38-76) were enrolled in 7 centres. Sixteen pts (66%), were male. Pts had received a median of 1 (0-2) prior lines of therapy. In total, 23 pts were evaluable with a median follow-up of 20.1 months (95%CI: 11.6-28.6). Median PFS, according to local site assessment, was 5.6 months (95%CI: 4.5-6.7); the 6m-PFSR was 10 of 23 pts (46%); (95%CI: 23-66) and median overall survival 10.3 months (95%CI: 6.7-13.9). In 19 pts with RECIST measurable response, 5 pts (26.3%) achieved a partial response; 10 (52.6%) stable disease and 4 (21.1%) PD. End-of-treatment reasons were PD in 21 pts (87%), toxicity 1 pt (4.2%) with two pts (8.4%) ongoing. For safety population, the most relevant study drug-related G3-4 toxicities were neutropenia (20.9%), anemia (16.7%), and increased ALT (12.5%). Conclusions: Sunitinib and nivolumab demonstrated encouraging activity in advanced DDCS that did not meet the primary endpoint, but compares favourably with previous analyses of cytotoxic and targeted therapy. The combination is worthy of further exploration in conjunction with reaching a consensus on clinically meaningful endpoints in this challenging population. Translational studies to identify potential predictive biomarkers are ongoing. Clinical trial information: NCT03277924 .

The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i’s) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

Coptisine-mediated downregulation of E2F7 induces G2/M phase arrest in hepatocellular carcinoma cells through inhibition of E2F4/NFYA/NFYB transcription factors

Coptisine (COP) has been shown to exhibit a wide range of anticancer properties, including in hepatocellular carcinoma (HCC). Nevertheless, the precise mechanism of COP in the treatment of HCC remains elusive. This study aims to investigate the potential mechanism of action of COP against HCC. By evaluating the anti-HCC activity of COP in different HCC cells lines and in xenografted nude mice, it was found that COP inhibited HCC in vitro and in vivo. Through RNA-Seq analysis, E2F7 was identified as a potential target of COP against HCC, as well as the cell cycle as a possible pathway. The overexpression of E2F7 and the inhibition of CHK1 demonstrated that COP inhibits the activity of HCC and induces G2/M phase arrest of HCC cells by down-regulating E2F7 and influencing the CHK1/CDC25A pathway. Finally, the promoter fragmentation experiments and chromatin immunoprecipitation revealed that COP down-regulated E2F7 by inhibiting the E2F4/NFYA/NFYB transcription factors. In conclusion, our study demonstrated that COP downregulates E2F7 by affecting key transcription factors, thereby inducing cell cycle arrest and inhibits HCC cell growth. This provides further evidence of the efficacy of COP in the treatment of tumors.

Novel imidazolone derivatives as potential dual inhibitors of checkpoint kinases 1 and 2: Design, synthesis, cytotoxicity evaluation, and mechanistic insights

Applying various drug design strategies including ring variation, substituents variation, and ring fusion, two series of 2-(alkylthio)-5-(arylidene/heteroarylidene)imidazolones and imidazo[1,2-a]thieno[2,3-d]pyrimidines were designed and prepared as dual potential Chk1 and Chk2 inhibitors. The newly synthesized hybrids were screened in NCI 60 cell line panel where the most active derivatives 4b, d-f, and 6a were further estimated for their five dose antiproliferative activity against the most sensitive tumor cells including breast MCF-7 and MDA-MB-468 and non-small cell lung cancer EKVX as well as normal WI-38 cell. Noticeably, increasing the carbon chain attached to thiol moiety at C-2 of imidazolone scaffold elevated the cytotoxic activity. Hence, compounds 4e and 4f, containing S-butyl fragment, exhibited the most antiproliferative activity against the tested cells where 4f showed extremely potent selectivity toward them. As well, compound 6a, containing imidazothienopyrimidine core, exerted significant cytotoxic activity and selectivity toward the examined cells. The mechanistic investigation of the most active cytotoxic analogs was achieved through the evaluation of their inhibitory activity against Chk1 and Chk2. Results revealed that 4f displayed potent dual inhibition of both Chk1 and Chk2 with IC50 equal 0.137 and 0.25 μM, respectively. It also promoted its antiproliferative and Chk suppression activity via EKVX cell cycle arrest at S phase through stimulating the apoptotic approach. The apoptosis induction was also emphasized by elevating the expression of Caspase-3 and Bax, that are accompanied by Bcl-2 diminution. The in silico molecular docking and ADMET profiles of the most active analogs have been carried out to evaluate their potential as significant anticancer drug candidates.

Novel Amidine Derivative K1586 Sensitizes Colorectal Cancer Cells to Ionizing Radiation by Inducing Chk1 Instability.

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.

The luciferase-based in vivo protein–protein interaction assay revealed that CHK1 promotes PP2A and PME-1 interaction

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.

Abstract LB259: Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer

Abstract Epigenetic regulation plays an important role in prostate cancer progression and metastasis. KDM5D is a male-specific histone-modifying enzyme encoded on the Y chromosome that demethylates di- and tri-methylated forms of lysine 4 in histone H3, resulting in transcriptional repression of certain genes. In this study, we investigated the role of KDM5D in resistance to inhibitors targeting the G2/M cell cycle checkpoint kinase CHK1. Loss of KDM5D expression was associated with reduced cell proliferation and resistance to the CHK1 inhibitor SRA737 in castration-resistant (CRPC) and neuroendocrine (NEPC) prostate cancer cell lines. In KDM5D expressing cells, depletion of KDM5D differentially impacted the proliferation of prostate cancer cell lines. The androgen-sensitive LNCaP cell line was particularly sensitive to the knockdown of KMD5D. On the other hand, in CRPC cell line 22Rv1, the knockdown of KDM5D caused resistance to both inhibitors, indicating a dual role of KDM5D in mediating ATR and CHK1 inhibitor sensitivity and cell proliferation in prostate cancer cells. Further analysis demonstrated a self-regulatory cycle between CHK1 and KDM5D in 22Rv1 cells, where depletion of KMD5D reduced CHK1 mRNA, while decreased CHK1 caused upregulation of KDM5D and overexpression of KDM5D resulted in increased CHK1 expression. The relevance of this regulatory mechanism to ATR and CHK1 inhibitor sensitivity is currently under investigation, along with several potential mediators of the differential responses to these inhibitors. Additional ATR and CHK1 inhibitors will also be examined in this context. This study may uncover novel biomarkers for ATR and CHK1 inhibitors that are currently in clinical trials for various cancers. KDM5D may be used to stratify prostate cancer patients receiving these highly potent anticancer agents. Citation Format: Wenxiao Zheng, Yuzhou Chen, Qiming J. Wang. Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB259.

Characterizing functional DNA damage and response caused by the combination of CHK1 and WEE1 inhibitors in ovarian and breast cancer models

BackgroundWe proposed to quantify reduction of functional DNA damage response (DDR) mechanisms caused by the combination of CHK1 and WEE1 inhibitors.MethodsSurvival of cells and tumor growth in-vitro and in-vivo caused by the combination of the CHK1 inhibitor SRA737 and the WEE1 inhibitor adavosertib was studied in OVCAR3 and MDA-MB 436 cells. Functional DNA damage was quantified using in vitro cell free DNA assays.ResultsThe combination of SRA737 and adavosertib caused significant reduction of survival of cells and DNA damage in-vitro and growth inhibition in-vivo. Studies using functional DDR assays found significant changes in the functional capacity of OVCAR3 but not MDA-MB 436 cells to repair DNA damage using multiple mechanisms including intra strand cross link repair, nucleotide excision repair, homologous recombination and non-homologous end joining. This study, for the first time provides a mechanistic insight into differences in the reduction in functional capacity of cells to repair DNA when exposed to CHK1 and WEE1 inhibitors.ConclusionThe combination of the CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib causes growth inhibition in-vitro and in-vivo, but differential functional inhibition of DDR in the models studied.

The CHK1 inhibitor prexasertib in BRCA wild-type platinum-resistant recurrent high-grade serous ovarian carcinoma: a phase 2 trial

The multi-cohort phase 2 trial NCT02203513 was designed to evaluate the clinical activity of the CHK1 inhibitor (CHK1i) prexasertib in patients with breast or ovarian cancer. Here we report the activity of CHK1i in platinum-resistant high-grade serous ovarian carcinoma (HGSOC) with measurable and biopsiable disease (cohort 5), or without biopsiable disease (cohort 6). The primary endpoint was objective response rate (ORR). Secondary outcomes were safety and progression-free survival (PFS). 49 heavily pretreated patients were enrolled (24 in cohort 5, 25 in cohort 6). Among the 39 RECISTv1.1-evaluable patients, ORR was 33.3% in cohort 5 and 28.6% in cohort 6. Primary endpoint was not evaluable due to early stop of the trial. The median PFS was 4 months in cohort 5 and 6 months in cohort 6. Toxicity was manageable. Translational research was an exploratory endpoint. Potential biomarkers were investigated using pre-treatment fresh biopsies and serial blood samples. Transcriptomic analysis revealed high levels of DNA replication-related genes (POLA1, POLE, GINS3) associated with lack of clinical benefit [defined post-hoc as PFS < 6 months]. Subsequent preclinical experiments demonstrated significant cytotoxicity of POLA1 silencing in combination with CHK1i in platinum-resistant HGSOC cell line models. Therefore, POLA1 expression may be predictive for CHK1i resistance, and the concurrent POLA1 inhibition may improve the efficacy of CHK1i monotherapy in this hard-to-treat population, deserving further investigation.