Abstract 3593: Raptor upregulation contributes to maintenance of 4EBP1 phosphorylation and TORC kinase resistance in renal cancer cells
Authors: Earwaker Philip , Willenbrock Frances , Protheroe Andrew , Macaulay Valentine 
Published: August 2015

Abstract
Advanced renal cell cancer (RCC) is incurable, but recent advances in targeted treatment have improved the prognosis. Rapalogue inhibitors of the mammalian target of rapamycin (mTOR) are licensed for treatment of advanced RCC, although clinical benefit is modest. Novel mTOR kinase inhibitors show more complete blockade of mTOR downstream targets, and greater in vitro anti proliferative activity, although resistance remains a problem. The aim of this study was to create a model of in vitro resistance to PI3K-mTOR kinase inhibitor BEZ235, in order to identify biomarkers and mediators of resistance.
After 5 months continuous culture in BEZ235, resistance to 20nM BEZ235 was induced in the RCC4 cell line, generating subline RCC4B20. These cells had 14 fold higher BEZ235 growth inhibitory 50 (GI50) concentration compared to sensitive cells (99 nM vs. 7 nM). There was cross-resistance to alternative mTOR kinase inhibitor AZD2014 (GI50 1131 vs. 166 nM), but not to PI3K, AKT or allosteric mTORC1 inhibitors, suggesting that resistance was primarily directed at mTOR kinase. RCC4B20 cells were screened at the protein level by antibody array, and RNA level by microarray, and hits were validated by western blotting and qRT-PCR.
Proteins and pathways found to be up-regulated in RCC4B20 cells included MET, pMET, ABL, and MEK/ERK by antibody array, and NOTCH signalling by microarray. These were validated as showing altered regulation, but BEZ235 resistance was not blocked by depletion or inhibition of these targets, suggesting that these changes were correlates, but not mediators of resistance. An hypothesis-led approach directed attention to the mTORC1 pathway. In BEZ235 resistant cells, the mTORC1 target 4E-BP1 was re-phosphorylated, despite on-going mTOR blockade as evidenced by suppressed S6 phosphorylation. Recovery of 4E-BP1 phosphorylation was associated with 4.2 ± 0.3 fold increase in levels of RAPTOR, a scaffolding protein for mTOR substrates. Phosphorylation of 4E-BP1 was suppressed by rapamycin, or by RAPTOR depletion, both interventions partially reversing BEZ235 resistance. Finally, RCC4B20 cells lost resistance after 8 weeks of drug withdrawal, suggesting that resistance may have been associated with reversible changes in gene expression due to histone modification. Indeed, histone deacetylase inhibitor panobinostat partially reversed resistance to BEZ235.
In summary, the acquisition of resistance to mTOR kinase inhibition was accompanied by complex molecular changes, many of which were passengers, not drivers of resistance. Resistance was partially suppressed by interventions directed at raptor up-regulation and histone modification, suggesting that these changes do mediate resistance. The combination of BEZ235 with rapamyin or panobinostat warrant further investigation to evaluate potential to overcome resistance to mTOR kinase inhibition in RCC.
Citation Format: Philip Earwaker, Frances Willenbrock, Andrew Protheroe, Valentine Macaulay. Raptor upregulation contributes to maintenance of 4EBP1 phosphorylation and TORC kinase resistance in renal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3593. doi:10.1158/1538-7445.AM2015-3593