ATR/CHK1 inhibitors and cancer therapy

The cell cycle checkpoint proteins ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (CHK1) prevent the entry of cells with damaged or incompletely replicated DNA into mitosis when the cells are challenged by DNA damaging agents, such as radiation therapy (RT) or chemotherapeutic drugs, that are the major modalities to treat cancer. This regulation is particularly evident in cells with a defective G1 checkpoint, a common feature of cancer cells, due to p53 mutations. In addition, ATR and/or CHK1 suppress replication stress (RS) by inhibiting excess origin firing, particularly in cells with activated oncogenes. Those functions of ATR/CHK1 make them ideal therapeutic targets. ATR/CHK1 inhibitors have been developed and are currently used either as single agents or paired with radiotherapy or a variety of genotoxic chemotherapies in preclinical and clinical studies. Here, we review the status of the development of ATR and CHK1 inhibitors. We also discuss the potential mechanisms by which ATR and CHK1 inhibition induces cell killing in the presence or absence of exogenous DNA damaging agents, such as RT and chemotherapeutic agents. Lastly, we discuss synthetic lethality interactions between the inhibition of ATR/CHK1 and defects in other DNA damage response (DDR) pathways/genes.     

PARP and CHK inhibitors interact to cause DNA damage and cell death in mammary carcinoma cells

The present studies examined viability and DNA damage levels in mammary carcinoma cells following PARP1 and CHK1 inhibitor drug combination exposure. PARP1 inhibitors [AZD2281 ; ABT888 ; NU1025 ; {"type":"entrez-nucleotide","attrs":{"text":"AG014699","term_id":"3649917","term_text":"AG014699"}}AG014699] interacted with CHK1 inhibitors [UCN-01 ; AZD7762 ; LY2603618] to kill mammary carcinoma cells. PARP1 and CHK1 inhibitors interacted to increase both single strand and double strand DNA breaks that correlated with increased γH2AX phosphorylation. Treatment of cells with CHK1 inhibitors increased the phosphorylation of CHK1 and ERK1/2. Knock down of ATM suppressed the drug-induced increases in CHK1 and ERK1/2 phosphorylation and enhanced tumor cell killing by PARP1 and CHK1 inhibitors. Expression of dominant negative MEK1 enhanced drug-induced DNA damage whereas expression of activated MEK1 suppressed both the DNA damage response and tumor cell killing. Collectively our data demonstrate that PARP1 and CHK1 inhibitors interact to kill mammary carcinoma cells and that increased DNA damage is a surrogate marker for the response of cells to this drug combination.     

DNA damage response inhibitors: Mechanisms and potential applications in cancer therapy

Over the last decade the unravelling of the molecular mechanisms of the DNA damage response pathways and of the genomic landscape of human tumors have paved the road to new therapeutic approaches in oncology. It is now clear that tumors harbour defects in different DNA damage response steps, mainly signalling and repair, rendering them more dependent on the remaining pathways. We here focus on the proteins ATM, ATR, CHK1 and WEE1, reviewing their roles in the DNA damage response and as targets in cancer therapy. In the last decade specific inhibitors of these proteins have been designed, and their potential antineoplastic activity has been explored both in monotherapy strategies against tumors with specific defects (synthetic lethality approach) and in combination with radiotherapy or chemotherapeutic or molecular targeted agents. The preclinical and clinical evidence of antitumor activity of these inhibitors emanating from these research efforts will be critically reviewed. Lastly, the potential therapeutic feasibility of combining together such inhibitors with the aim to target particular subsets of tumors will be also discussed.     

CHK1和CHK2 shRNA转染对食管癌细胞照射后G2期阻滞的影响

目的 观察RNA干扰细胞周期检测点激酶CHK1和CHK2表达对食管癌细胞照射后G2期阻滞的影响。方法 CHK1和CHK2基因各选择4段序列分别设计合成短发卡状RNA（shRNA）,分别与pENTR^TM／U6质粒连接后转染Eca109食管癌细胞。采用Western blotting检测CHK1和CHK2蛋白表达,RT-PCR检测其mRNA表达,流式细胞仪检测5Cy照射后细胞周期变化,克隆法检测5Cy照射后细胞存活率。结果 CHK1和CHK2基因各成功建立4个序列的shRNA连接质粒,转染Eca109细胞后均使其蛋白表达降低。用抑制效果最好的CHKI和CHK2shRNA转染Eca109细胞后,其mRNA表达下降;在转染后72h,子一代细胞CHK1和CHK2蛋白仍明显降低,并使5Cy照射后1h的CHK2-T68磷酸化水平降低,而对CHK1-S345磷酸化水平无明显影响。CHK1 shRNA转染的Ecal09细胞在5Gy照射后12h时,明显减轻G2期阻滞程度;转染后72h和5Cy照射后12h,子一代Eca109细胞G2期阻滞仍明显低于单纯5Gy照射组;而CHK2shRNA转染不影响照射后C2期阻滞。CHK1和CHK2shRNA转染可降低5Gy照射后Eca109细胞存活率。结论 shRNA转染对CHK1和CHK2蛋白表达的抑制效应至少可以持续3d,且可传递给子代细胞;CHK1 shRNA转染可以减轻Eca109细胞照射后G2期阻滞,增加放射敏感性。     

DNA repair inhibitors in cancer treatment

Chemotherapy and radiation are two important modalities for cancer treatment. Many agents in clinical used have the ability to induce DNA damage, however they may be highly cytotoxic as a secondary effect. Different mechanisms are involved both, in detection and repair of DNA damage. The modulation of these pathways, has a great impact on clinical outcome and is frequently responsible of therapeutic resistance. Therefore, pharmacological inhibition of DNA damage repair pathways has been explored as a useful strategy to enhance chemo and radiosensitivity, thus it could be used for reversing drug resistance. Different agents have shown excellent results in preclinical studies in combination with radiation or chemotherapy. Early phase clinical trials are now being carried out using different DNA repair inhibitors targeting several enzymes such as PARP, DNA-PK or MGMT. Supported by an unrestricted educational grant from Pfizer.     

Absence of Germline CHK2 Mutations in Familial Gastric Cancer

Recently, the CHK2 gene was identified as being a candidate gene responsible for Li-Fraumeni syndrome (LFS). Gastric cancer is often clustered in families with LFS, so it is possible that germline CHK2 mutation is also present in familial gastric cancer (FGC). We therefore defined the genomic structure of the CHK2 gene, designed intronic primers, and searched for germline CHK2 mutations in 25 FGC cases by polymerase chain reaction-single strand conformational polymorphism analysis of the entire coding region. In all of the 25 cases, at least two siblings had histories of gastric cancer. There were no FGC cases that showed germline CHK2 mutations. Thus, it was indicated that germline CHK2 mutations do not contribute to the familial clustering of gastric cancer.     

Targeting ATR in cancer medicine

DNA damage occurs continually through various intrinsic and extrinsic mechanisms such as ultraviolet radiation, smoking, reactive oxygen species, and errors during replication. The cellular DNA damage response (DDR) comprises signaling networks that regulate a spectrum of processes, including cell cycle progression, which enable DNA repair to occur. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and rad3-related (ATR) kinase are 2 key regulators of the DDR cell cycle checkpoints. ATR plays an essential role in the repair of replication-associated DNA damage, while ATM is activated by DNA double-strand breaks. The investigation of cell cycle checkpoint signaling through ATR and ATM, as well as the relevant pathways involved in oncogenesis and cancer progression, has led to the discovery and development of potent and selective ATR inhibitors (ATRi). Preclinical data have demonstrated that ATR inhibition leads to tumor synthetic lethality in specific molecular contexts, and it exhibits synergy in combination with different antitumor therapies, including chemotherapy, radiotherapy, and poly(ADP-ribose) polymerase inhibitors. ATRi are now being assessed in early-phase clinical trials as single agents and in combinatorial regimens, including platinum and other chemotherapies, radiotherapy, poly(ADP-ribose) polymerase inhibitors, and immune checkpoint inhibitors. This article details the preclinical biology leading to the discovery and development of novel ATRi and discusses the rationale for monotherapy and combination antitumor strategies. We focus on the clinical development of ATRi and discuss the progress made in identifying putative predictive biomarkers of response for patient selection, such as p53, ATM, ARID1A, and other DDR aberrations.     

The CHK1 inhibitor SRA737 synergizes with PARP1 inhibitors to kill carcinoma cells

Inhibitors of PARP1 are approved therapeutic agents in ovarian carcinomas. We determined whether the novel clinically relevant CHK1 inhibitor SRA737 interacted with PARP1 inhibitors to kill carcinoma cells. In multiple mammary and ovarian cancer lines SRA737 synergized with the PARP1 inhibitors olaparib and niraparib to cause cell death. The [SRA737 + niraparib] drug combination activated an ATM-AMPK-ULK1-mTOR pathway which resulted in the formation of autophagosomes, temporally followed by autolysosome formation. Phosphorylation of ULK1 S317 was essential for kinase activation against ATG13. The drug combination elevated eIF2α phosphorylation which was causal at increasing Beclin1 and ATG5 expression, reducing MCL-1 and BCL-XL levels, and causing CD95 activation. Knock down of CD95, eIF2α, ATM, AMPKα, ULK1, Beclin1 or ATG5 reduced drug combination lethality. Blockade of either caspase 9 function or that of AIF each partially prevented cell death. Expression of activated mTOR or of c-FLIP-s or of BCL-XL reduced cell killing. In vivo, SRA737 and niraparib interacted in an additive fashion to suppress the growth of mammary tumors. Multiplex analyses revealed that drug combination treated tumors had reduced their plasma levels of sERBB1, sERBB2, sVEGFR1, sVEGFR2, sIL-6R, HGF, PDGFAB/BB and CXCL16 and enhanced the levels of CCL26, IL-8 and MIF. Surviving tumors had activated ERK1/2 and AKT. This finding argues that IL-8/ERK/AKT signaling may be an evolutionary survival response to [SRA737 + niraparib].     

Targeting the DNA damage response for patients with lymphoma: Preclinical and clinical evidences

The DNA damage response (DDR) is a well-coordinated cellular network activated by DNA damage. The unravelling of the key players in DDR, their specific inactivation in different tumor types and the synthesis of specific chemical inhibitors of DDR represent a new hot topic in cancer therapy. In this article, we will review the importance of DDR in lymphoma development and how this can be exploited therapeutically. Specifically, we will focus on CHK1, WEE1, ATR, DNA-PK and PARP inhibitors, for which preclinical data as single agents or in combination has been accumulating, fostering their clinical development. The few available clinical data on these inhibitors will also be discussed.     

Mutation analysis of the CHK2 gene in families with hereditary breast cancer.

Recently CHK2 was functionally linked to the p53 pathway, and mutations in these two genes seem to result in a similar Li-Fraumeni syndrome (LFS) or Li-Fraumeni-like syndrome (LFL) multi-cancer phenotype frequently including breast cancer. As CHK2 has been found to bind and regulate BRCA1, the product of one of the 2 known major susceptibility genes to hereditary breast cancer, it also more directly makes CHK2 a suitable candidate gene for hereditary predisposition to breast cancer. Here we have screened 79 Finnish hereditary breast cancer families for germline CHK2 alterations. Twenty-one of these families also fulfilled the criteria for LFL or LFS. All families had previously been found negative for germline BRCA1, BRCA2 and TP53 mutations, together explaining about 23% of hereditary predisposition to breast cancer in our country. Only one missense-type mutation, Ile(157)-->Thr(157), was detected. The high Ile(157)--> Thr(157)mutation frequency (6.5%) observed in healthy controls and the lack of other mutations suggest that CHK2 does not contribute significantly to the hereditary breast cancer or LFL-associated breast cancer risk, at least not in the Finnish population. For Ile(157)--> Thr(157)our result deviates from what has been reported previously.     

Targeting ATR in DNA damage response and cancer therapeutics

The ataxia telangiectasia and Rad3-related (ATR) plays an important role in maintaining genome integrity during DNA replication through the phosphorylation and activation of Chk1 and regulation of the DNA damage response. Preclinical studies have shown that disruption of ATR pathway can exacerbate the levels of replication stress in oncogene-driven murine tumors to promote cell killing. Additionally, inhibition of ATR can sensitise tumor cells to radiation or chemotherapy. Accumulating evidence suggests that targeting ATR can selectively sensitize cancer cells but not normal cells to DNA damage. Furthermore, in hypoxic conditions, ATR blockade results in overloading replication stress and DNA damage response causing cell death. Despite the attractiveness of ATR inhibition in the treatment of cancer, specific ATR inhibitors have remained elusive. In the last two years however, selective ATR inhibitors suitable for in vitro and – most recently – in vivo studies have been identified. In this article, we will review the literature on ATR function, its role in DDR and the potential of ATR inhibition to enhance the efficacy of radiation and chemotherapy.     

Mutation analysis of the CHK2 gene in breast carcinoma and other cancers

Background

Mutations in the CHK2 gene at chromosome 22q12.1 have been reported in families with Li-Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage and is involved in cell-cycle pathways and p53 pathways.

Methods

We screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using seven microsatellite markers, and screened 119 breast tumors with single-strand conformation polymorphism and DNA sequencing for mutations in the CHK2 gene.

Results

Seventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5' splice site mutation at site +3 in intron 8 (TTgt [a → c]atg) was also detected.

Conclusion

We conclude that somatic CHK2 mutations are rare in breast cancer, but our results suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor growth.     

Ataxia Telangiectasia Mutated Protein Loss and Benefit From Oxaliplatin-based Chemotherapy in Colorectal Cancer

Background

Loss of ataxia telangiectasia mutated (ATM), a key protein regulating DNA repair signaling, has been suggested to increase sensitivity to DNA damaging agents. We conducted a study analyzing the loss of ATM protein expression in colorectal cancer and correlated this with clinical outcomes.

Enhancing CHK1 inhibitor lethality in glioblastoma

The present studies were initiated to determine whether inhibitors of MEK1/2 or SRC signaling, respectively, enhance CHK1 inhibitor lethality in primary human glioblastoma cells. Multiple MEK1/2 inhibitors (CI-1040 (PD184352); AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01, AZD7762) to kill multiple primary human glioma cell isolates that have a diverse set of genetic alterations typically found in the disease. Inhibition of SRC family proteins also enhanced CHK1 inhibitor lethality. Combined treatment of glioma cells with (MEK1/2 + CHK1) inhibitors enhanced radiosensitivity. Combined (MEK1/2 + CHK1) inhibitor treatment led to dephosphorylation of ERK1/2 and S6 ribosomal protein, whereas the phosphorylation of JNK and p38 was increased. MEK1/2 + CHK1 inhibitor-stimulated cell death was associated with the cleavage of pro-caspases 3 and 7 as well as the caspase substrate (PARP). We also observed activation of pro-apoptotic BCL-2 effector proteins BAK and BAX and reduced levels of pro-survival BCL-2 family protein BCL-XL. Overexpression of BCL-XL alleviated but did not completely abolish MEK1/2 + CHK1 inhibitor cytotoxicity in GBM cells. These findings argue that multiple inhibitors of the SRC-MEK pathway have the potential to interact with multiple CHK1 inhibitors to kill glioma cells.     

The SKP2‐p27 axis defines susceptibility to cell death upon CHK1 inhibition

Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under‐replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B‐Cell CLL/Lymphoma 2 (BCL2)‐regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53‐deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F‐box protein S‐phase Kinase‐Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin‐dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1‐S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1‐inhibitor treatment and reduced cell death in S‐phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1‐inhibitor treatment.     

Anti‐tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells

Ataxia telangiectasia and Rad3‐related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)‐dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3‐(4,5‐dimethylthiazol‐2‐yl)?2,5‐diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti‐proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC₅₀ values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK‐BR‐3 and BT‐474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)‐positive breast cancer cells. Sensitive SK‐BR‐3 but not the less sensitive BT‐474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check‐point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2‐positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2‐positive breast cancer treatment.     

The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo

ATR and ATM are DNA damage signaling kinases that phosphorylate several thousand substrates. ATR kinase activity is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). ATM kinase activity is increased at DSBs. ATM has been widely studied since ataxia telangiectasia individuals who express no ATM protein are the most radiosensitive patients identified. Since ATM is not an essential protein, it is widely believed that ATM kinase inhibitors will be well-tolerated in the clinic. ATR has been widely studied, but advances have been complicated by the finding that ATR is an essential protein and it is widely believed that ATR kinase inhibitors will be toxic in the clinic. We describe AZD6738, an orally active and bioavailable ATR kinase inhibitor. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 potentiates the cytotoxicity of cisplatin and gemcitabine in NSCLC cell lines with intact ATM kinase signaling, and potently synergizes with cisplatin in ATM-deficient NSCLC cells. In contrast to expectations, daily administration of AZD6738 and ATR kinase inhibition for 14 consecutive days is tolerated in mice and enhances the therapeutic efficacy of cisplatin in xenograft models. Remarkably, the combination of cisplatin and AZD6738 resolves ATM-deficient lung cancer xenografts.