King, Frederick, Ainscow, Edward, Tuntland, Tove, Kim, Sunkyu, Grandinetti, Kathryn, Caponigro, Giordano, He, You Qun, Krupa, Shiva, Li, Nanxin, Harris, Jennifer, Wood, Andrew C., Krytska, Kateryna, Ryles, Hannah, Infarinato, Nicole, Sano, Renata, Hansel, Theodore, Hart, Lori, Mosse, Yael P. and Smith, Timothy R. (2016) Combination CDK4/6 and ALK inhibition demonstrates on-target synergy against neuroblastoma. Cancer Research, 74 (19). pp. 2856-2868. ISSN 00085472

Abstract

BACKGROUND: Activated ALK by mutation or amplification is a validated therapeutic target in neuroblastoma, and identifying therapeutic strategies to overcome primary resistance to direct ALK kinase inhibition will be critical to improve clinical responses. We hypothesized that simultaneous targeting of ALK and additional oncogenic networks would improve efficacy. METHODS: We performed a synergy screen combining molecularly targeted compounds (n=14) and standard-of-care chemotherapy agents (n=8) in extensively characterized human neuroblastoma cell lines (n=14). We investigated the combination of LDK378 and LEE011 on in vitro proliferation, cell cycle, viability, caspase activation, and the Cyclin D/CDK4/CDK6/RB and pALK signaling networks in neuroblastoma-derived cell lines with representative ALK status. Transport inhibitor studies were performed in Caco2 cells. We performed in vivo trials in neuroblastoma CB17 xenograft models comparing LDK378 alone, LEE011 alone, and the combination of LDK378 and LEE011, with plasma and tumor pharmacokinetics to evaluate for drug-drug interactions. RESULTS: Pairwise combination screening for in vitro cell viability identified synergistic interactions of LDK378, an ALK inhibitor, with LEE011, a CDK4 and CDK6 inhibitor. In mutant and wild-type ALK cell lines there was moderate to strong synergy with combination indices of 0.2 - 0.8 at clinically relevant high effect sizes with the fraction of cells affected ≥ 0.8. Compared to either drug alone, combination LEE011 and LDK378 increased dose dependent abrogation of pALK and pRb, inhibition of proliferation, apoptosis and decreased cell viability. LDK378 did not show significant cellular accumulation after P-gp, BCRP, and MRP2 inhibition or after LEE011 1uM and 10uM. LEE011 showed only modest accumulation with MRP2 inhibition, but not after BCRP or P-gp inhibition. In NB1691 (ALK wild-type) and SHSY5Y (ALK-F1174L, resistant mutation) neuroblastoma xenografts, combination therapy significantly prolonged survival compared to either drug alone (P<0.0001). In SHSY5Y xenografts, combination therapy achieved complete regressions using murine doses that achieved plasma drug exposures comparable to the adult recommended doses for LEE011 and LDK378. Combination therapy increased tumor LEE011 and LDK378 maximum concentration (Cmax) and area under curve (AUC) 2-3 fold over monotherapy, but plasma concentrations were unaltered. CONCLUSION: Dual ALK and CDK4/6 inhibition in neuroblastoma models demonstrates potent on-target in vitro synergy and in vivo activity with augmented abrogation of respective molecular targets, resulting in inhibition of proliferation and cell death. While mechanisms for altered intratumoral drug distribution require further investigation, these data support the development of a combination ALK and CDK4/6 inhibitor clinical trial with eligibility not restricted to cases with somatic ALK mutations.

Item Type:	Article
Date Deposited:	06 Jul 2017 00:45
Last Modified:	25 Jan 2019 00:45
URI:	https://oak.novartis.com/id/eprint/31196