Small Molecule Therapeutics The CDK4/6 Inhibitor LY2835219 Overcomes Vemurafenib Resistance Resulting fromMAPKReactivation and Cyclin D1 Upregulation

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanomapatientswithB-RAFV600Emutation.However,most patients treatedwith vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently,we found thatMAPK is reactivated and cyclinD1 is elevated invemurafenib-resistant tumors, aswell as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1–CDK4/6 signaling by LY2835219 is an effective strategy to overcomeMAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma. Mol Cancer Ther; 13(10); 2253–63. 2014 AACR.