RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma

Abstract

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-offunction screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC 50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC 50 values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease. N euroblastoma is an embryonal tumor of early childhood thought to arise from fetal sympathetic neuroblasts (1). Children with localized neuroblastoma can be cured with surgery and/or chemotherapy. About half of children with neuroblastoma have high-risk disease, however, characterized by widespread disease dissemination at diagnosis. For these children, current treatment consists of chemotherapy, surgery, external beam radiation therapy, myeloablative chemotherapy with stem cell rescue, and a maintenance therapy regimen combining retinoids and anti-GD2-based immunotherapy (2). Despite the intense multimodality therapy, at least half of high-risk patients will experience relapse that is almost always fatal and survivors show significant morbidity (1).To address the unmet need of identifying bona fide molecular targets for drug development in neuroblastoma, we and others have undertaken comprehensive characterization of the neuroblastoma genome, leading to the identification of mutations in the anaplastic lymphoma kinase gene (ALK) in 10% of newly diagnosed cases (3, 4). Ongoing comprehensive resequencing efforts will likely discover other potential drug targets in this disease. Although these data may identify the critical genes and pathways for drug development efforts, it is also possible that ot...

Chk1-induced CCNB1 overexpression promotes cell proliferation and tumor growth in human colorectal cancer

“…[22][23][24][25][26] In contrast to the well-known utility of Chk1 inhibitors in sensitizing tumors to chemotherapy agents, 23,27,28 Chk1 overexpression or downexpression also occurs in some types of tumors, including breast cancer, ovarian cancer, cervical cancer, and neuroblastoma. 22,[29][30][31][32][33] In our study, we found Chk1 mRNA and protein levels were upregulated in colorectal cancer tissues compared with paired normal tissues, and positively correlated with CCNB1 expression. We hypothesized that CCNB1 might serve as a Chk1-induced oncogene in colorectal cancer.…”

CHK1 Inhibition Radiosensitizes Head and Neck Cancers to Paclitaxel-Based Chemoradiotherapy

“…High p-CHK1 expression in the nucleus or cytoplasm also significantly correlated with recurring HPV þ tumors. Although this is a small sample set, high CHK1 expression and/or activity has previously been correlated with therapy-resistant cancer cells (35,36) and associated with high-grade, high-risk tumors often resistant to therapy and with a poor prognosis (36)(37)(38)(39)(40). Confirmation of our findings in a larger head and neck cancer dataset will be required.…”

Overview and recent advances in the treatment of neuroblastoma

“…Studies have demonstrated that RET is required for retinoic acid-induced neuroblastoma differentiation [158], and that RET inhibition is effective in neuroblastoma preclinical models [159]. Other recent studies have identified the polo-like kinase 1 (PLK1) as a potential target for neuroblastoma therapy, based on screens of a library of kinase inhibitors in neuroblastoma preclinical models [160], while a screening study using an siRNA library identified the checkpoint kinase 1 (CHK1) as a potential target [161]. Transcriptome analysis of neuroblastoma tumor formation in the MYCN transgenic mouse model identified the centromereassociated protein E (CENP-E) as an additional potential therapeutic target [162].…”