Genetic inhibition of the atypical kinase Wee1 selectively drives apoptosis of p53 inactive tumor cells

Pappano et al. BMC Cancer 2014, 14:430 http://www.biomedcentral.com/1471-2407/14/430 RESEARCH ARTICLE Open Access Genetic inhibition of the atypical kinase Wee1 selectively drives apoptosis of p53 inactive tumor cells William N Pappano*†, Qian Zhang†, Lora A Tucker, Chris Tse and Jieyi Wang Abstract Background: Tumorigenesis is the result of genomic or epigenomic insults and subsequent loss of the proper mechanisms to respond to these alterations leading to unscheduled growth. Tumors arising from these mutations often have altered cell cycles that offer proliferative advantages and lead to the accumulation of additional mutations that can lead to more aggressive phenotypes. Nevertheless, tumor cells must still adhere to the basic tenets of the cell cycle program to ensure their survival by DNA duplication, chromosomal segregation and cytokinesis. The atypical tyrosine kinase Wee1 plays a key role in regulating the cell cycle at the DNA synthesis and mitotic checkpoints via phosphorylation and subsequent inactivation of cyclin-dependent kinases (CDKs) in both healthy and tumorigenic cells. Methods: To assess the role of Wee1 in tumor cell proliferation we performed small interfering RNA (siRNA) experiments in a panel of diverse cell lines derived from various tissue origins. We also tested the hypothesis that any potential effects would be as a result of the kinase activity of Wee1 by siRNA rescue studies with wild-type or kinase-dead versions of Wee1. Results: We find that, in general, cells with wild-type p53 activity are not susceptible to loss of Wee1 protein via siRNA. However, Wee1 siRNA treatment in tumor cells with an inherent loss of p53 activity results in a deregulated cell cycle that causes simultaneous DNA synthesis and premature mitosis and that these effects are kinase dependent. These cumulative effects lead to potent inhibition of cellular proliferation and ultimately caspase-dependent apoptosis in the absence of co-treatment with cytotoxic agents. Conclusions: These results suggest that, while Wee1 acts as a tumor suppressor in the context of normal cell growth and its functional loss can be compensated by p53-dependent DNA damage repairing mechanisms, specific inhibition of Wee1 has deleterious effects on the proliferation and survival of p53 inactive tumors. In total, targeting the atypical kinase Wee1 with an siRNA-based therapeutic or a selective ATP competitive small molecule inhibitor would be a feasible approach to targeting p53 inactive tumors in the clinic. Keywords: Wee1, p53, Apoptosis, CDK1, CDK2, DNA damage * Correspondence: † Equal contributors Discovery Research, AbbVie Inc., 1 N. Waukegan Road, North Chicago, IL 60064, USA © 2014 Pappano et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pappano et al. BMC Cancer 2014, 14:430 http://www.biomedcentral.com/1471-2407/14/430 Background Proper maintenance of the cell cycle is essential for the development and homeostasis of all living organisms. Neoplasms arising within these organisms also rely on a coordinated cell cycle to facilitate their rapid growth rate in response to external sources of nutrients and signaling stimuli. Progression through the cell cycle in both naïve and tumor tissue is monitored at checkpoints that sense possible defects in DNA synthesis and chromosomal segregation. Activation of these checkpoints results in cell cycle arrest and allows cells to rectify any negative perturbations that may be transmitted to their resulting daughter cells. Tumor cells with defective checkpoints rely more heavily on other checkpoints within the cell cycle to ensure their survival. Thus, deregulation of cell cycle control can have catastrophic results and therefore has led to a concerted effort towards generation of novel therapeutic agents targeting the cell cycle in tumors [1]. Transition through the cell cycle is dependent on the cyclin-dependent kinase (CDK) family of regulatory proteins. CDK activity is tightly monitored through several complex mechanisms including modulation of CDK stability by binding partner cyclins and CDK inhibitors [2,3]. However, only a subset of CDK-cyclin complexes are directly involved in progression of the cell cycle through these respective checkpoints. The active CDK2-Cyclin E complex is essential to drive the G1/S transition of the cell cycle after the restriction checkpoint and the CDK1-Cyclin B complex (also known as the M-phase-promoting-factor) is the master regulator that initiates the G2/M transition after the mitotic checkpoint [4]. In addition to their association with cyclins and CDK inhibitors, CDK1 and CDK2 activity are modulated both negatively and positively by phosphorylation and de-phosphorylation events [4]. Wee1 is an atypical tyrosine kinase that most closely resembles serine/threonine kinases in both sequence and structure [5] and acts directly upon CDK1 and CDK2. Wee1 phosphorylation of tyrosine 15 (Y15) of CDK1 and CDK2 results in CDK inactivation and inhibition of S-phase and mitotic entry [6,7]. Wee1 kinase activity therefore serves as a master regulator of cell cycle checkpoints by inactivating the CDK1-Cyclin B and CDK2-Cyclin E complexes until it can be assured that genomic integrity will be maintained and that the appropriate genetic information will be passed on to daughter cells. Previous reports of Wee1 inhibition by small molecule kinase inhibitors and siRNA demonstrate that loss of Wee1 activity sensitizes p53 inactive cells to DNA damaging agents and radiosensitization [8-12]. We hypothesized that loss of Wee1 in the absence of cytotoxics should be able to affect tumor cell proliferation because all metazoan cells, including cancer cells, rely on at least partially functioning checkpoints to insure their survival. To elucidate the roles of Wee1 in cancer cell cycle Page 2 of 11 progression we have utilized siRNA knockdown and rescue. We find that loss of Wee1 kinase activity results in dramatic cell cycle events including simultaneous mitosis and DNA synthesis that ultimately lead to apoptosis in a sub-set of p53 deficient cells. The anti-proliferative and apoptotic effects of Wee1 siRNA treatment can be circumvented by expression of a wild-type Wee1 rescue construct but not a kinase-defective version in affected target cells. This work provides new insights into the development of cancer therapeutics, suggesting that a small molecule inhibitor of Wee1 kinase should be efficacious against a large number of p53 inactive solid tumors as a single agent and provide a safe the (...truncated)