Neonatal Lethality in Knockout Mice Expressing the Kinase-Dead Form of the Gefitinib Target GAK Is Caused by Pulmonary Dysfunction

PLOS ONE, Oct 2011

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd+/+ mice but not in GAK-kd-/- pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.

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Neonatal Lethality in Knockout Mice Expressing the Kinase-Dead Form of the Gefitinib Target GAK Is Caused by Pulmonary Dysfunction

et al. (2011) Neonatal Lethality in Knockout Mice Expressing the Kinase-Dead Form of the Gefitinib Target GAK Is Caused by Pulmonary Dysfunction. PLoS ONE 6(10): e26034. doi:10.1371/journal.pone.0026034 Neonatal Lethality in Knockout Mice Expressing the Kinase-Dead Form of the Gefitinib Target GAK Is Caused by Pulmonary Dysfunction Hiroe Tabara 0 Yoko Naito 0 Akihiko Ito 0 Asako Katsuma 0 Minami A. Sakurai 0 Shouichi Ohno 0 Hiroyuki Shimizu 0 Norikazu Yabuta 0 Hiroshi Nojima 0 Hiroaki Matsunami, Duke University, United States of America 0 1 Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University , Osaka , Japan , 2 Department of Pathology, Kinki University Faculty of Medicine , Osaka , Japan Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd+/+ mice but not in GAK-kd-/- pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients. - Funding: This work was supported in part by grants-in-aid for Scientific Research S (No. 15101006), Scientific Research B (No. 20370081) and Exploratory Research (No. 21651085) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp/english/) to Hiroshi Nojima. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. EGFR is a membrane receptor tyrosine kinase that is activated by ligand binding and dimerization, resulting in the activation of a signaling pathway that controls cell proliferation, differentiation, and survival [1]. Constitutively active EGF-EGFR signaling due to overexpression of mutated or wild-type EGFR is found in a broad range of human carcinomas, leading to the activation of antiapoptotic pathways and uncontrolled cell proliferation [2], [3]. EGFR selective tyrosine kinase inhibitors (TKIs) such as gefitinib (Iressa) and erlotinib (Tarceva) that bind to the adenosine triphosphate (ATP)-binding site of the enzyme have been used as successful treatments for NSCLC patients, particularly in the presence of activating mutations within the EGFR gene [4], [5]. Although occurring at low frequency, progressive respiratory dysfunction, including acute interstitial pneumonia (IP) is the most severe adverse effect of gefitinib [6], which has limited the therapeutic benefit of this drug. Tumor regression in gefitinib treated NSCLC patients is at least partly due to apoptotic death of tumor cells. Shutdown of the EGFR-MEK-ERK signaling cascade induces activation of the proapoptotic BH3-only protein BIM, causing gefitinib-induced tumor cell apoptosis [7]. Moreover, induction of another BH3-only protein, p53 up-regulated modulator of apoptosis (PUMA), by p73, is also involved in EGFR inhibitor-induced apoptosis [8], [9]. However, the molecular mechanisms underlying the development of IP in response to gefitinib treatment and the selectivity of the drug for its cellular targets are not fully understood. Two protein kinases were identified by liquid chromatography (LC)-MS/MS as novel gefitinib targets [10], namely a negative regulator of EGFR signaling, GAK [11] and Rip2/RICK (receptor-interacting caspase-like apoptosis-regulatory kinase), a signal transducer and integrator of signals for both the innate and adaptive immune systems that functions through the promotion of nuclear factor kappa B and caspase activation [12], [13]. Both targets are affected by gefitinib as potently as the tyrosine kinase activity of wild-type EGFR in vitro [10]. Although the physiological significance of these phenomena needs to be elucidated for the selection of EGFR-directed drugs with minimal side effects, there is little data presently available. The ubiquitously expressed kinase GAK was first identified as a (...truncated)


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Hiroe Tabara, Yoko Naito, Akihiko Ito, Asako Katsuma, Minami A. Sakurai, Shouichi Ohno, Hiroyuki Shimizu, Norikazu Yabuta, Hiroshi Nojima. Neonatal Lethality in Knockout Mice Expressing the Kinase-Dead Form of the Gefitinib Target GAK Is Caused by Pulmonary Dysfunction, PLOS ONE, 2011, Volume 6, Issue 10, DOI: 10.1371/journal.pone.0026034