Identification of the KIF and MCM protein families as novel targets for combination therapy with CDK4/6 inhibitors in bladder cancer

Abstract

CDK4/6 inhibitors have proven their potency for the treatment of cancer but only in combination with hormone or targeted therapies. The aim of this study was the identification of molecules that are involved in response mechanisms to CDK4/6 inhibitors and the development of novel combination therapies with corresponding inhibitors in bladder cancer. Genes of response to therapy and genes that confer resistance to the CDK4/6 inhibitor palbociclib were identified by performing an analysis of published literature and own published data using a CRISPR-dCas9 genome wide gain of function screen. Genes that were down-regulated upon treatment were compared with genes that confer resistance when up-regulated. Two of the top 5 genes were validated by quantitative PCR and western blotting upon treatment with palbociclib in the bladder cancer cell lines T24, RT112 and UMUC3. As inhibitors for combination therapy, we used ciprofloxacin, paprotrain, ispinesib and SR31527. Analysis of synergy was done using the "zero interaction potency" model. Cell growth was examined using sulforhodamine B staining. A list of genes that met the requirements for inclusion in the study was generated from 7 publications. Of the 5 most relevant genes, MCM6 and KIFC1 were chosen and their down-regulation upon treatment with palbociclib was confirmed by qPCR and immunoblotting. The combination of inhibitors against both, KIFC1 and MCM6 with PD resulted in a synergistic inhibition of cell growth. We have identified 2 molecular targets whose inhibition has promising potential for effective combination therapies with the CDK4/6 inhibitor palbociclib.

Keywords: Bladder cancer; CDK4/6 inhibitor; KIFC1; MCM protein complex; Palbociclib; combination therapy.