Abstract PR10: Exploiting the G2-M cell cycle checkpoint dependency in small cell lung cancer (SCLC) using pharmacological inhibitors of CHK1 and WEE1
Authors: Sen Triparna , Tong Pan , Stewart Catherine Allison , Cristea Sandra , Hong-Fan You , Glisson Bonnie S. , Gibbons Don L. , Sage Julien , Wang Jing , Byers Lauren A. 
Published: November 2016

Background: Small cell lung cancer (SCLC), the most lethal form of lung cancer, is characterized by early metastasis and rapid development of drug resistance. SCLC is characterized by ubiquitous loss of TP53 and RB1 and mutually exclusive amplification of the MYC oncogene (CMYC, MYCN, MYCL). The loss of TP53 is thought to render SCLC cells deficient at the G1-S cell cycle checkpoint, thus making these cells completely dependent on the G2-M checkpoint for DNA repair, mainly governed by CHK1 and WEE1. CHK1 is a vital serine-threonine kinase regulating the G2-M checkpoint upon DNA damage. The WEE1 kinase is a regulator of CDK1/2 and inhibition of WEE1 leads to aberrant DNA replication and premature mitosis. In this study we investigated the effect of CHK1 (LY2606368) and WEE1 (AZD1775) inhibitors as therapeutic targets for SCLC.

Methods: Protein and gene expression was determined by immunoblot and real-time-RT-PCR respectively. Effect of the drugs on cell viability was determined by CellTiter-Glo. Efficacy of LY2606368 in H69 (chemosensitive) and H69/CR (chemoresistant) models was further determined by in vivo flank tumor regression analysis. The effect of LY2606368 monotherapy was also tested in a spontaneous SCLC genetically engineered mouse model (GEMM) model. The mechanistic studies were conducted by immunoblot analysis, apoptosis assay and flow cytometry. Pre and post-treatment cell lysates were analyzed by reverse phase protein array (RPPA) for biomarker exploration.

Result: Higher expression of CHK1 and WEE1 was observed in SCLC patient tumor samples compared to normal lung (p&lt;0.001) and in SCLC cells as compared to NSCLC cells (p&lt;0.05). LY2606368 showed efficacy in both primary and acquired cisplatin resistant in vitro and in vivo models. LY2606368 treatment showed striking single agent activity in a spontaneous GEMM model of SCLC. Proteomic analysis identified an association between LY2606368 sensitivity and high levels of the CMYC protein. The level of CMYC decreased upon LY2606368 treatment (p&lt;0.001) while pro-apoptotic pathways increased (p&lt;0.05). CHK1 was a top marker of cisplatin sensitivity and treatment of cells with LY2606368 reduced activation of the MEK/MAPK pathway which we show as markers of de novo cisplatin resistance. LY2606368 treatment caused accumulation of cells in the sub-G1 phase and increased cell death.

WEE1 inhibitor AZD1775 treatment decreased cell viability (IC50&lt;100nM in 70% SCLC cells) in majority of SCLC cells. We identified AXL (receptor tyrosine kinase) and phospho-S6K as markers of AZD1775 resistance and treatment with AXL inhibitor, TP0903, resensitized the cells to AZD1775. Cell cycle analysis revealed increased apoptosis upon treatment of AZD1775 in combination with TP0903. Western blot analysis of pre and post AZD1775 treated samples revealed sustained activation of mTOR pathway in AZD1775 primary resistant lines. Pre-treatment of the cells with the mTOR inhibitor everolimus sensitized SCLC cells to AZD1775 by causing downregulation of AKT/mTOR pathway.

Significance: The study establishes CHK1 and WEE1 as therapeutic targets in SCLC, a disease for which the standard of care has remained unchanged for &gt;30 years. Together, these results strongly indicate the potency of G2-M cell cycle checkpoint inhibitors as effective targeted therapies in both chemosensitive and chemoresistant patient population. The CHK1 inhibitor, LY2606368, and the WEE1 inhibitor, AZD1775, are both in current Phase 1 clinical trials. The correlative biomarker data from this study will not only indicate the subset of the patient population who are most likely to respond to these treatments, but it will also guide the selection of most effective combination strategies with CHK1 and WEE1 inhibition.

This abstract is also being presented as Poster A25.

Citation Format: Triparna Sen, Pan Tong, Catherine Allison Stewart, Sandra Cristea, You Hong-Fan, Bonnie S. Glisson, Don L. Gibbons, Julien Sage, Jing Wang, Lauren A. Byers. Exploiting the G2-M cell cycle checkpoint dependency in small cell lung cancer (SCLC) using pharmacological inhibitors of CHK1 and WEE1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR10.