MP39-04 IL-6 RECEPTOR ANTIBODY ENHANCES THE EFFECT OF TKI AGAINST RENAL CELL CARCINOMA

Abstract

INTRODUCTION AND OBJECTIVES: Abnormal chromosome segregation during mitosis causes aneuploidy, a hallmark of cancers associated with high risk for tumorigenesis. Mitotic checkpoint, or spindle assembly checkpoint prevents missegregation of chromosomes by arresting cells in metaphase until all the chromosomes are properly aligned. Mps1 kinase activity is essential for spindle checkpoint signaling. High levels of Mps1 serine/threonine kinase are found in colon cancer tissues and several cancer cell line. We found that Mps1 is over-expressed in ccRCC. We have further shown that Mps1 requires the molecular chaperone Hsp90 for its activity. Hsp90 protects an array of mutated, over-expressed and/or chimeric kinases that are vital for tumor growth and survival. The Hsp90 inhibitors are currently undergoing evaluation in human trials. We have previously shown that Hsp90 is a post-translationally modified protein and this regulates its chaperone activity. We hypothesize that Mps1 kinase targets and phosphorylates Hsp90 and therefore regulates its chaperone activity. This in turn impacts the tumor selectivity of Hsp90 inhibitors. METHODS: Functional genomics and quantitative proteomic (stable isotope labeling by amino acids in cell culture) was used to identify the Mps1 kinase that targets and phosphorylates Hsp90. ccRCC and adjacent normal tissue was obtained with written informed consent through the Upstate Department of Urology. 8-mm core of tissue was dissected into 3mm pieces and cultured in 24-well plates containing 1 mL RPMI-1640 with 10% FBS, antibiotic/antimycotic solution. Tissues were cultured at 37 C for 24 hours with or without Mps1 inhibitor NMS-P715 and for further 24 hours in the presence of fluorescently labeled Hsp90 inhibitor ganetespib (FL-ganetespib), then formalin-fixed and paraffin embedded. The tissues were immunostained for Hsp90. RESULTS: Mps1 kinase is over-expressed in ccRCC compared to normal adjacent tissue. Mps1 targets and phosphorylates T115 in the molecular chaperone Hsp90 and this regulates the chaperone activity. Ex vivo data showed that FL-ganetespib selectively accumulates in ccRCC and not in the normal tissue. Pharmacologic inhibition of Mps1 reverts this drug uptake by ccRCC. CONCLUSIONS: Mps1 protein kinase is an Hsp90 client and it is involved in post-translational regulation of the chaperone function. Over-expression of Mps1 in the ccRCC hyperphosphorylates Hsp90 and this is the molecular basis of the tumor selectivity of Hsp90 inhibitors.