Abstract 5511: Direct effect of regorafenib on tyrosine kinase activities in treatment naive colorectal cancer (CRC) primary tumor tissue
Authors: Hilhorst Maria H. , Oord Rosanne van den , Pruijt Hans , Brule Adriaan van den , Nooijen Peet , Wijn Rik de , Houkes Liesbeth 
Published: May 2015

Abstract
Background:
Regorafenib (BAY 73-4506) is the first small molecule tyrosine kinase inhibitor that has been approved for treatment of advanced CRC. In the Correct trial*, 1% of the participants showed a partial response and 42.8 % had stable disease. KRAS wild type and mutants showed equal response rates.
Regorafenib is a multityrosine kinase inhibitor with nM IC50's against CRAF, VEGFR2, KIT and RET and is about tenfold less potent for BRAF, BRAFV600E, PDGFRβ and VEGFR1 and 3. We tested the effect of regorafenib and other angiogenic inhibitors on patient-derived kinases in treatment naive primary colon tumor samples for potency and selectivity of inhibition.
Method:
Colon tumor tissue from patients who had given informed consent was fresh frozen after surgical resection. All tumor specimens were analysed for BRAF and KRAS mutation status. Tissue cryosections were lysed in buffer with phosphatase and protease inhibitors. Phosphotyrosine kinase (PTK) and serine/threonine kinase (STK) activity profiles of tumour tissue were generated on PamChip® peptide microarrays comprising hundreds of peptide sequences derived from known human phosphorylation sites. The ex vivo effect of kinase inhibitors on these kinase activity profiles was determined. Peptide phosphorylation was monitored using fluorescently labelled antibodies. Data were analysed with Bionavigator software.
Result:
Kinase activity profiles were obtained from 16 CRC patient tumors for both tyrosine as well as serine/threonine specific peptide phosphorylations. Direct enzymatic inhibition of tumor-derived PTK activity was observed in all 16 samples at low micromolar concentrations (3 µM) of spiked-in regorafenib. For the serine/threonine kinase activity, spiking in of a high concentration of regorafenib (50 µM) resulted in only low levels of inhibition of phosphorylations on a subset of peptides, including BRAF substrate peptides. No difference in inhibition was observed between KRAS and BRAF mutants and wild type tumors. Furthermore the non-inhibited kinase activity profiles did not correlate to mutation status either.
Conclusion:
For the first time the direct biochemical effect of regorafenib has been tested on primary tumor tissue from CRC patients. These data support previous findings suggesting that regorafenib, besides its angiogenic activity on vasothelial cells, can inhibit oncogenic tyrosine kinases in the tumor. These results will be a basis for biomarker analysis by correlating on-chip drug effects to clinical responses.
*Grothey et al, Lancet 2013 381(9863):303-312
Citation Format: Maria H. Hilhorst, Rosanne van den Oord, Hans Pruijt, Adriaan van den Brule, Peet Nooijen, Rik de Wijn, Liesbeth Houkes. Direct effect of regorafenib on tyrosine kinase activities in treatment naive colorectal cancer (CRC) primary tumor tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5511. doi:10.1158/1538-7445.AM2014-5511