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.

A Phase I Expansion Cohort Study Evaluating the Safety and Efficacy of the CHK1 Inhibitor LY2880070 with Low-dose Gemcitabine in Patients with Metastatic Pancreatic Adenocarcinoma.

PURPOSE: Combining gemcitabine with CHK1 inhibition has shown promise in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we report the findings from a phase I expansion cohort study (NCT02632448) investigating low-dose gemcitabine combined with the CHK1 inhibitor LY2880070 in patients with previously treated advanced PDAC. PATIENTS AND METHODS: Patients with metastatic PDAC were treated with gemcitabine intravenously at 100 mg/m2 on days 1, 8, and 15, and LY2880070 50 mg orally twice daily on days 2-6, 9-13, and 16-20 of each 21-day cycle. Pretreatment tumor biopsies were obtained from each patient for correlative studies and generation of organoid cultures for drug sensitivity testing and biomarker analyses. RESULTS: Eleven patients with PDAC were enrolled in the expansion cohort between August 27, 2020 and July 30, 2021. Four patients (36%) experienced drug-related grade 3 adverse events. No objective radiologic responses were observed, and all patients discontinued the trial by 3.2 months. In contrast to the lack of efficacy observed in patients, organoid cultures derived from biopsies procured from two patients demonstrated strong sensitivity to the gemcitabine/LY2880070 combination and showed treatment-induced upregulation of replication stress and DNA damage biomarkers, including pKAP1, pRPA32, and γH2AX, as well as induction of replication fork instability. CONCLUSIONS: No evidence of clinical activity was observed for combined low-dose gemcitabine and LY2880070 in this treatment-refractory PDAC cohort. However, the gemcitabine/LY2880070 combination showed in vitro efficacy, suggesting that drug sensitivity for this combination in organoid cultures may not predict clinical benefit in patients.

Derivation of Naïve Human Embryonic Stem Cells Using a CHK1 Inhibitor.

Embryonic development is a continuum in vivo. Transcriptional analysis can separate established human embryonic stem cells (hESC) into at least four distinct developmental pluripotent stages, two naïve and two primed, early and late relative to the intact epiblast. In this study we primarily show that exposure of frozen human blastocysts to an inhibitor of checkpoint kinase 1 (CHK1) upon thaw greatly enhances establishment of karyotypically normal late naïve hESC cultures. These late naïve cells are plastic and can be toggled back to early naïve and forward to early primed pluripotent stages. The early primed cells are transcriptionally equivalent to the post inner cell mass intermediate (PICMI) stage seen one day following transfer of human blastocysts into in vitro culture and are stable at an earlier stage than conventional primed hESC.

Aberrant Activation of Cell-Cycle-Related Kinases and the Potential Therapeutic Impact of PLK1 or CHEK1 Inhibition in Uterine Leiomyosarcoma.

PURPOSE: Uterine leiomyosarcoma is among the most aggressive gynecological malignancies. No effective treatment strategies have been established. This study aimed to identify novel therapeutic targets for uterine leiomyosarcoma based on transcriptome analysis and assess the preclinical efficacy of novel drug candidates. EXPERIMENTAL DESIGN: Transcriptome analysis was performed using fresh-frozen samples of six uterine leiomyosarcomas and three myomas. The Ingenuity Pathway Analysis (IPA) was used to identify potential therapeutic target genes for uterine leiomyosarcoma. Afterward, our results were validated using three independent datasets, including 40 uterine leiomyosarcomas. Then, the inhibitory effects of several selective inhibitors for the candidate genes were examined using SK-UT-1, SK-LMS-1, and SKN cell lines. RESULTS: We identified 512 considerably dysregulated genes in uterine leiomyosarcoma compared with myoma. The IPA revealed that the function of several genes, including CHEK1 and PLK1, were predicted to be activated in uterine leiomyosarcoma. Through an in vitro drug screening, PLK1 or CHEK1 inhibitors (BI-2536 or prexasertib) were found to exert a superior anticancer effect against cell lines at low nanomolar concentrations and induce cell-cycle arrest. In SK-UT-1 tumor-bearing mice, BI-2536 monotherapy remarkably suppressed tumorigenicity. Moreover, the prexasertib and cisplatin combination therapy inhibited tumor proliferation and prolonged the time to tumor progression. CONCLUSIONS: We identified upregulated expressions of PLK1 and CHEK1; their kinase activity was activated in uterine leiomyosarcoma. BI-2536 and prexasertib demonstrated a significant anticancer effect. Therefore, cell-cycle-related kinases may present a promising therapeutic strategy for the treatment of uterine leiomyosarcoma.

p53-NEIL1 co-abnormalities induce genomic instability and promote synthetic lethality with Chk1 inhibition in multiple myeloma having concomitant 17p13(del) and 1q21(gain).

Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.

CHK1 inhibition exacerbates replication stress induced by IGF blockade.

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

DNA damage triggers an interplay between wtp53 and c-Myc affecting lymphoma cell proliferation and Kaposi sarcoma herpesvirus replication.

The induction of DNA damage together with the interference with DNA repair represents a promising strategy in cancer treatment. Here we show that the PARP-1/2/3 inhibitor AZD2461 in combination with the CHK1 inhibitor UCN-01 altered the DNA damage response and reduced cell proliferation in PEL cells, an aggressive B cell lymphoma highly resistant to chemotherapies. AZD2461/UCN-01 combination activated p53/p21 and downregulated c-Myc in these cells, leading to a reduced expression level of RAD51, molecule involved in DNA repair. The effect of AZD2461/UCN-01 on c-Myc and p53/p21 was inter-dependent and, besides impairing cell proliferation, contributed to the activation of the replicative cycle of KSHV, carried in a latent state in PEL cells. Finally, we found that the pharmacological or genetic inhibition of p21 counteracted the viral lytic cycle activation and further reduced PEL cell proliferation, suggesting that it could induce a double beneficial effect in this setting. This study unveils that, therapeutic approaches, based on the induction of DNA damage and the reduction of DNA repair, could be used to successfully treat this malignant lymphoma.

Downregulation of c-Myc expression confers sensitivity to CHK1 inhibitors in hematologic malignancies.

Checkpoint kinase 1 inhibitors (CHK1i) have shown impressive single-agent efficacy in treatment of certain tumors, as monotherapy or potentiators of chemotherapy in clinical trials, but the sensitive tumor types and downstream effectors to dictate the therapeutic responses to CHK1i remains unclear. In this study we first analyzed GDSC (Genomics of Drug Sensitivity in Cancer) and DepMap database and disclosed that hematologic malignancies (HMs) were relatively sensitive to CHK1i or CHK1 knockdown. This notion was confirmed by examining PY34, a new and potent in-house selective CHK1i, which exhibited potent anti-HM effect in vitro and in vivo, as single agent. We demonstrated that the downregulation of c-Myc and its signaling pathway was the common transcriptomic profiling response of sensitive HM cell lines to PY34, whereas overexpressing c-Myc could partially rescue the anticancer effect of PY34. Strikingly, we revealed the significant correlations between downregulation of c-Myc and cell sensitivity to PY34 in 17 HM cell lines and 39 patient-derived cell (PDC) samples. Thus, our results demonstrate that HMs are more sensitive to CHK1i than solid tumors, and c-Myc downregulation could represent the CHK1i efficacy in HMs.

FGFR2 loss sensitizes MYCN-amplified neuroblastoma CHP134 cells to CHK1 inhibitor-induced apoptosis.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i-sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer-related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i-mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor-mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN-amplified neuroblastomas.

CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.

We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.

A novel oral inhibitor for one-carbon metabolism and checkpoint kinase 1 inhibitor as a rational combination treatment for breast cancer.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

Discovery and Development of a Potent, Selective, and Orally Bioavailable CHK1 Inhibitor Candidate: 5-((4-((3-Amino-3-methylbutyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)picolinonitrile.

Checkpoint kinase 1 (CHK1) plays an important role in the DNA damage response pathway, being a potential anti-cancer drug target. In this study, we used a strategy for trifluoromethyl substitution to obtain orally bioavailable CHK1 inhibitors to overcome the limitations of lead compound 1, which can only be administered intravenously. After detailed investigation, we identified compound 6c as an oral CHK1 inhibitor, which demonstrated a considerably higher plasma exposure in mice. Compound 6c also showed good kinase selectivity. Moreover, it exhibited a significant antiproliferative effect in MV-4-11 cells singly and a synergistic effect in combination with gemcitabine in HT-29, A549, and RPMI-8226 cells. Additionally, compound 6c could inhibit tumor growth in the MV-4-11 xenograft mouse model. The combination of 6c and gemcitabine exhibited synergistic effect in the HT-29 xenograft mouse model. Thus, compound 6c was found to be a selective and oral potential anticancer CHK1 inhibitor.

Genetic vulnerabilities upon inhibition of DNA damage response.

Because of essential roles of DNA damage response (DDR) in the maintenance of genomic integrity, cellular homeostasis, and tumor suppression, targeting DDR has become a promising therapeutic strategy for cancer treatment. However, the benefits of cancer therapy targeting DDR are limited mainly due to the lack of predictive biomarkers. To address this challenge, we performed CRISPR screens to search for genetic vulnerabilities that affect cells' response to DDR inhibition. By undertaking CRISPR screens with inhibitors targeting key DDR mediators, i.e. ATR, ATM, DNAPK and CHK1, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Specifically, we identified YWHAE loss as a key determinant of sensitivity to CHK1 inhibition. We showed that KLHL15 loss protects cells from DNA damage induced by ATM inhibition. Moreover, we validated that APEX1 loss sensitizes cells to DNAPK inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results enhance the understanding of DDR pathways and will facilitate the use of DDR-targeting agents in cancer therapy.

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.

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

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.

Adverse Cerebral Cardiovascular Events Associated With Checkpoint Kinase 1 Inhibitors: A Systemic Review.

ABSTRACT: Checkpoint kinase 1 (CHK1) plays a broad role in regulating the cell cycle process and is involved in the pathogenesis of various malignant tumors. Preclinical and animal studies have shown that CHK1 inhibitors can enhance the cytotoxic effects of radiotherapy and chemotherapy. Currently, CHK1 inhibitors are actively tested in clinical trials. Nonspecific adverse cerebral cardiovascular events were reported after CHK1 inhibitor use; these events need to be monitored and managed carefully during the clinical application of CHK1 inhibitors. To get a better understanding of these, noteworthy adverse cardiovascular events, we systemically searched the PubMed, Cochrane databases, and clinicaltrials.gov, for relevant clinical trials and case reports. A total of 19 studies were identified and included in this review. Among the reported cerebral cardiovascular events, the most common is incident abnormal blood pressure fluctuations (n = 35), followed by incident QTcF prolongation (n = 15), arrhythmia (n = 13, 3 atrial fibrillation and 10 bradycardia), thromboembolic events (n = 9, 6 pulmonary embolisms, 2 stroke, and 1 cerebrovascular event), cardiac troponin T elevation (n = 2), and ischemic chest pain (n = 2). Besides, the estimated incidence for overall cardiovascular events based on the available data is 0.292 (95% confidence interval: 0.096-0.488). CHK1 inhibitors administered in tumor patients on top of conventional therapies can not only enhance the antitumor effects, but also induce adverse cerebral cardiovascular events. It is, therefore, of importance to carefully monitor and manage the CHK1 inhibitor-induced adverse effects on the cerebral cardiovascular system while applying CHK1 inhibitors to tumor patients.

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.

A phase 1 study of prexasertib (LY2606368), a CHK1/2 inhibitor, in pediatric patients with recurrent or refractory solid tumors, including CNS tumors: A report from the Children's Oncology Group Pediatric Early Phase Clinical Trials Network (ADVL1515).

BACKGROUND: Prexasertib (LY2606368) is a novel, second-generation, selective dual inhibitor of checkpoint kinase proteins 1 (CHK1) and 2 (CHK2). We conducted a phase 1 trial of prexasertib to estimate the maximum-tolerated dose (MTD) and/or recommended phase 2 dose (RP2D), to define and describe the toxicities, and to characterize the pharmacokinetics (PK) of prexasertib in pediatric patients with recurrent or refractory solid and central nervous system (CNS) tumors. METHODS: Prexasertib was administered intravenously (i.v.) on days 1 and 15 of a 28-day cycle. Four dose levels, 80, 100, 125, and 150 mg/m2 , were evaluated using a rolling-six design. PK analysis was performed during cycle 1. Tumor tissue was examined for biomarkers (CHK1 and TP53) of prexasertib activity. RESULTS: Thirty patients were enrolled; 25 were evaluable. The median age was 9.5 years (range: 2-20) and 21 (70%) were male. Twelve patients (40%) had solid tumors and 18 patients (60%) had CNS tumors. There were no cycle 1 or later dose-limiting toxicities. Common cycle 1, drug-related grade 3/4 toxicities (> 10% of patients) included neutropenia (100%), leukopenia (68%), thrombocytopenia (24%), lymphopenia (24%), and anemia (12%). There were no objective responses; best overall response was stable disease in three patients for five cycles (hepatocellular carcinoma), three cycles (ependymoma), and five cycles (undifferentiated sarcoma). The PK appeared dose proportional across the 80-150 mg/m2 dose range. CONCLUSIONS: Although the MTD of prexasertib was not defined by this study, 150 mg/m2 administered i.v. on days 1 and 15 of a 28-day cycle was determined to be the RP2D.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.