Abstract 1651: The use of ‘X-MAN’ isogenic cell lines to define PI3-kinase inhibitor activity profiles
Authors: Goodall John , Foster Rebecca E. , Mead Suzanne , Grimshaw Kyla , Torrance Chris 
Published: February 2011

Abstract
The tumour microenvironment promotes selective outgrowth of cells carrying mutations in oncogenes or tumour suppressors, enabling the activated pathways to drive tumour growth. Two such pathways are the PI3-K/AKT and RAS/RAF/MEK signaling pathways, which demonstrate a high frequency of mutations in human cancer. These pathways represent important therapeutic targets, and a number of targeted agents are now in clinical trials. Despite being pertinent to the clinical situation, the role that the tumour microenvironment plays in modifying responsiveness to therapeutic agents targeted against these pathways is poorly understood.
A major challenge faced during the development of PI3-K/AKT and RAS/RAF/MEK targeted agents is to demonstrate inhibitor selectivity and efficacy in a patient-relevant cell based system. Isogenic ‘X-MAN’ paired cell lines help address this by providing a system in which the endogenous wild-type or mutant alleles of the parental cell line are selectively activated or silenced via targeted homologous integration, providing a pair of human cell-lines in which the only difference between the cells is the mutation of interest. This approach therefore overcomes the issues that may occur when over-expression methods are employed.
We therefore set out to investigate how changes in the tumour microenvironment altered cell responses to PI3-K and MEK inhibitors. We employed isogenic HCT116 human colon cancer cells where the parental cells harbor pre-existing H1047R PIK3CA and G13D KRAS alleles and compared these to isogenic clones that retain either the PIK3CA or KRAS wild-type alleles. Inhibitors were profiled under a range of defined conditions chosen to model the tumour microenvironment, including low glucose, low serum and hypoxia, and the activity of the compounds investigated.
Under standard culture conditions, we found that PI3-K and MEK inhibitors showed minimal selectivity for cells with activating mutations compared to wild-type cells. When grown under defined culture conditions which more closely mimic the tumour microenvironment, the effects of the driving mutations became dominant and a differential between wild-type and mutant cell responses to inhibitors was observed. The selective PI3-K inhibitors GDC-0941 and PI-103 showed a &gt;10-fold increase in potency when tested in anti-proliferative assays in mutant vs, wild-type cell lines. However, the dual PI3-K/mTOR inhibitor BEZ-235 did not show selectivity in this system.
We show that by using isogenic cell lines under conditions which model the tumour microenvironment, differences in activity profiles between specific inhibitors can be quickly and definitively identified, highlighting the advantages of using this system in profiling molecularly targeted agents.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1651.