Abstract B30: Colorectal cancer HCT116 cells resistant toward a novel Aurora kinase inhibitor CYC116: Molecular hallmarks of drug resistance and pharmacologic interventions restoring the drug sensitivity
Authors: Kollareddy Madhusudhan Reddy , Zheleva Daniella , Srovnal Josef , Radova Lenka , Dzubak Petr , Brahmkshatriya Pathik Subhashchandra , Hajduch Marian 
Published: March 2013

Abstract
CYC116 is a novel pan-Aurora kinase and VEGFR2 inhibitor, which has been discovered and developed by Cyclacel Ltd. In preclinical studies it displayed broad anticancer activity and it has been recently evaluated in a Phase I clinical study. Emergence of drug resistance towards CYC116 as well as other Aurora kinase inhibitors in the clinic is highly likely.
Our work was focused on identification and characterization of potential cancer cell resistance mechanisms towards CYC116, alongside with ZM447439, an experimental Aurora kinase inhibitor. We generated several HCT116 resistant clones on both p53+/+ and −/− backgrounds by exposing cells to cytotoxic concentrations of CYC116 and ZM447439. HCT116:CYC116, HCT116p53−/−:CYC116, HCT116:ZM447439, HCT116p53−/−:ZM447439 clones were 9 to 82 folds, 36 to 64 folds, 18 to &gt;83 folds and 33 to 39 folds more resistant to selecting agents, respectively. Resistant clones also displayed cross-resistance to other clinical Aurora kinase inhibitors and multidrug resistance to some anticancer agents tested. CYC116 clones, but not ZM447439 acquired polyploidy during the selection. As expected, all the resistance clones did not show up-regulation of common drug transporters including PgP and MRP1. Also we did not observe significant changes in Aurora kinases expression. ZM447439, but not CYC116 induced three novel mutations in Aurora B, namely I216L, L152S, and N76V. Structural modeling studies revealed that L152S may significantly affect the ZM447439 binding.
Pangenomic microarray expression studies revealed that 885, 1085, 224, and 212 number of gene sets were differentially expressed (ANOVA p&lt;0.001) in p53+/+:CYC116, p53−/−:CYC116, p53+/+:ZM447439, and p53−/−:ZM447439 groups compared to paternal cell lines, respectively. 23 most relevant genes were selected from all the groups for qRT-PCR validation studies on human primary tumors in vitro sensitive/resistant to CYC116. Interestingly, majority of cell line findings were confirmed also on primary human cells, suggesting validity of these genes as biomarkers of drug susceptibility or resistance.
An apoptotic gene Bcl-xL was found to be significantly up-regulated in CYC116 resistant clones, particularly in p53+/+ cells. Knock-down of Bcl-xL using RNAi technology partially reversed the resistance to CYC116. Moreover, Bcl-xL overexpressing p53+/+ CYC116 clones were highly sensitive to a synthetic Bcl-xL inhibitor, ABT-263, compared to the parent cells.
Our data cumulatively provide a genetic basis of resistance to Aurora kinase inhibitors, which could be used to predict and monitor clinical response and also to select patients who might benefit from Aurora kinase inhibition. Moreover, our study suggest a role of bcl-2 protein family inhibitors for reversal of drug resistance against Aurora kinase inhibitors and their possible significance for therapy of tumors primarily or secondary resistant to these drugs.