Head and neck squamous cell carcinomas (HNSCCs) coincide with poor survival rates. The lack of driver oncogenes complicates the development of targeted treatments for HNSCC. Here, we follow-up on two previous genome-wide RNA and microRNA interference screens in HNSCC to cross-examine tumor-specific lethality by targeting ATM , ATR , CHEK1 , or CHEK2 . Our results uncover CHEK1 as the most promising target for HNSCC. CHEK1 expression is essential across a panel of HNSCC cell lines but redundant for growth and survival of untransformed oral keratinocytes and fibroblasts. LY2603618 (Rabusertib), which specifically targets Chk1 kinase, kills HNSCC cells effectively and specifically. Our findings show that HNSCC cells depend on Chk1-mediated signaling to progress through S-phase successfully. Chk1 inhibition coincides with stalled DNA replication, replication fork collapses, and accumulation of DNA damage. We further show that Chk1 inhibition leads to bimodal HNSCC cell killing. In the most sensitive cell lines, apoptosis is induced in S-phase, whereas more resistant cell lines manage to bypass replication-associated apoptosis, but accumulate chromosomal breaks that become lethal in subsequent mitosis. Interestingly, CDK1 expression correlates with treatment outcome. Moreover, sensitivity to Chk1 inhibition requires functional CDK1 and CDK4/6 to drive cell cycle progression, arguing against combining Chk1 inhibitors with CDK inhibitors. In contrast, Wee1 inhibitor Adavosertib progresses the cell cycle and thereby increases lethality to Chk1 inhibition in HNSCC cell lines. We conclude that Chk1 has become a key molecule in HNSCC cell cycle regulation and a very promising therapeutic target. Chk1 inhibition leads to S-phase apoptosis or death in mitosis. We provide a potential efficacy biomarker and combination therapy to follow-up in clinical setting.
HPV-negative head and neck squamous cell carcinomas (HNSCCs) develop in precancerous changes in the mucosal lining of the upper-aerodigestive tract. these precancerous cells contain cancerassociated genomic changes and cause primary tumors and local relapses. therapeutic strategies to eradicate these precancerous cells are very limited. Using functional genomic screens, we identified the therapeutic vulnerabilities of premalignant mucosal cells, which are shared with fully malignant HNSCC cells. We screened 319 previously identified tumor-lethal siRNAs on a panel of cancer and precancerous cell lines as well as primary fibroblasts. In total we identified 147 tumor-essential genes including 34 druggable candidates. Of these 34, 13 were also essential in premalignant cells. We investigated the variable molecular basis of the vulnerabilities in tumor and premalignant cell lines and found indications of collateral lethality. Wee1-like kinase (WEE1) was amongst the most promising targets for both tumor and precancerous cells. All four precancerous cell lines were highly sensitive to Wee1 inhibition by Adavosertib (AZD1775), while primary keratinocytes tolerated this inhibitor. Wee1 inhibition caused induction of DNA damage during S-phase followed by mitotic failure in (pre)cancer cells. In conclusion, we uncovered Wee1 inhibition as a promising chemopreventive strategy for precancerous cells, with comparable responses as fully transformed HnScc cells. Head and neck squamous cell carcinomas (HNSCCs) arise in the mucosal lining of the upper-aerodigestive tract and account for around 5% of the total cancer incidence 1,2. The main risk factors for head and neck cancer are smoking and excessive alcohol consumption, infection with a high-risk type of the human papillomavirus (HPV), or genetic predisposition such as Fanconi anemia (FA) 2. FA is characterized by congenital abnormalities, progressing anemia, and cancer predisposition, particularly of oral cancers. These tumors are difficult to manage as FA-patients are sensitive to cross-linking agents such as cisplatin, hampering clinical management of tumors, and for these patients surveillance and prevention is key. Tumors in the head and neck region develop in premalignant mucosal changes, large epithelial areas characterized by cancer-associated genetic changes, also referred to as "fields". These precancerous fields can be centimeters in size, and are often macroscopically invisible. A minority manifests as visible mucosal lesions known as leukoplakia or erythroplakia, which occur with a prevalence of around 0.1-0.2% for leukoplakia 3 , and 0.02-0.2% for erythroplakia 4. Every year, 1-2% of the leukoplakia lesions progress into cancer, and erythroplakia lesions inevitably progress. Despite the occurrence and prevalence of these visible lesions, the large majority of head and neck cancers develop de novo. In resected tumor specimen, however, preceding premalignant changes can be often identified in the surgical margins by either microscopic detection of dysplasia or by ge...
Head and neck squamous cell carcinomas (HNSCC) develop in the mucosal lining of the upper-aerodigestive tract. In carcinogen-induced HNSCC, tumors emerge from premalignant mucosal changes characterized by tumor-associated genetic alterations, also coined as ‘fields’ that are occasionally visible as leukoplakia or erythroplakia lesions but are mostly invisible. Consequently, HNSCC is generally diagnosed de novo at more advanced stages in about 70% of new diagnosis. Despite intense multimodality treatment protocols, the overall 5-years survival rate is 50–60% for patients with advanced stage of disease and seems to have reached a plateau. Of notable concern is the lack of further improvement in prognosis despite advances in treatment. This can be attributed to the late clinical presentation, failure of advanced HNSCC to respond to treatment, the deficit of effective targeted therapies to eradicate tumors and precancerous changes, and the lack of suitable markers for screening and personalized therapy. The molecular landscape of head and neck cancer has been elucidated in great detail, but the absence of oncogenic mutations hampers the identification of druggable targets for therapy to improve outcome of HNSCC. Currently, functional genomic approaches are being explored to identify potential therapeutic targets. Identification and validation of essential genes for both HNSCC and oral premalignancies, accompanied with biomarkers for therapy response, are being investigated. Attentive diagnosis and targeted therapy of the preceding oral premalignant (preHNSCC) changes may prevent the development of tumors. As classic oncogene addiction through activating mutations is not a realistic concept for treatment of HNSCC, synthetic lethality and collateral lethality need to be exploited, next to immune therapies. In recent studies it was shown that cell cycle regulation and DNA damage response pathways become significantly altered in HNSCC causing replication stress, which is an avenue that deserves further exploitation as an HNSCC vulnerability for treatment. The focus of this review is to summarize the current literature on the preclinical identification of potential druggable targets for therapy of (pre)HNSCC, emerging from the variety of gene knockdown and knockout strategies, and the testing of targeted inhibitors. We will conclude with a future perspective on targeted therapy of HNSCC and premalignant changes.
Over 90% of all malignant tumors in the head and neck region are squamous cell carcinomas (HNSCC), ranking within the top 10 of most common cancers worldwide. HNSCC arise in the mucosal linings of the upper aerodigestive tract, and known risk factors are tobacco use, excessive alcohol consumption and infection with high risk types of the human papillomavirus (HPV). Despite the application of intensive treatment protocols, the 5-years survival rate of HNSCC patients has hardly increased last decades and remained at approximately 50%. Therefore, new therapeutic possibilities are urgently awaited. Previously, we identified CHEK1 as a candidate therapeutic target for HNSCC in a whole genome siRNA screen (Martens-de Kemp et al, Clin Cancer Res 2013). Additionally, a functional screen using a microRNA expressing library indicated that knockdown of ATM expression may cause a lethal effect in HNSCC cell lines as well (Lindenbergh-van der Plas et al, Clin Cancer Res 2013). The aim of the present study was to investigate whether interference with the ATM/ATR/CHEK1/CHEK2 DNA damage response pathway is indeed a promising therapeutic approach in HNSCC. RNA interference using siRNAs targeting ATM, ATR, CHEK1 and CHEK2 confirmed that particularly CHEK1 is essential for the survival of a subset of HNSCC cell lines, whereas the cell viability of primary oral keratinocytes and fibroblasts was not compromised. This was confirmed in vitro by treatment with a small molecule inhibitor targeting Chk1, and a therapeutic window between cancer and normal oral mucosal cells was observed. Functional assays indicated an S-phase delay in Chk1 inhibited HNSCC cells, followed by DNA damage. The latter was indicated by elevated phosphorylation of ATM Ser1981 and γH2Ax Ser139, as well as the appearance of chromosomal breaks. The DNA damage response initiated caspase-mediated apoptosis, resulting in programmed cell death. In summary, our data show that HNSCC cells appear to depend strongly on a functional Chk1 protein during DNA replication. Therefore, Chk1 inhibition seems an interesting therapeutic strategy for HNSCC, at least in a subset of tumors. Citation Format: Anne M. van Harten, Marijke Stigter-van Walsum, Marijke Buijze, Boudewijn Jm Braakhuis, Ruud H. Brakenhoff. Chk1 inhibition: a promising targeted treatment for head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4637.
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