Abstract B42: Combinatorial CDK4/6 and ALK inhibition demonstrates on-target synergy against neuroblastoma
Authors: Wood Andrew C. , Krytska Kateryna , Ryles Hannah , Sano Renata , Hansel Theodore , Li Nanxin , King Frederick , Smith Timothy , Tuntland Tove , Kim Sunkyu , Caponigro Giordano , He You-Qun , Harris Jennifer , Mosse Yael 
Published: May 2015

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 of critical importance to improve clinical response rates. We hypothesize that simultaneous targeting of ALK and additional oncogenic networks is crucial for rational design of alternative treatment strategies.
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 further investigated the combination of LDK378 and LEE011 on in vitro proliferation, viability, caspase activation, and the Cyclin D/CDK4/CDK6/RB and pALK signaling networks in 3 cell lines with representative ALK status. Additionally, we performed in vivo trials in 3 neuroblastoma CB17 xenograft models comparing LDK378 alone, LEE011 alone, and combination 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. Synergy was confirmed in the neuroblastoma-derived cell lines NB1643 (ALK R1275Q) and SHSY5Y (ALK F1174L) with combination indices of 0.3 – 0.7 at high effect sizes, while EBC1 (ALK wild-type) showed additivity. Compared to either drug alone the combination of LEE011 and LDK378 increased dose dependent abrogation of pALK and pRb (Rb hypophosphorylation is the result of CDK4 inhibition), enhanced caspase 3/7 activation and decreased cell viability. In wild-type (NB1691) and mutated ALK (F1174L) neuroblastoma xenografts combination therapy significantly prolonged survival compared to either drug alone (P&lt;0.0001), with complete and sustained regressions over the fourth week of dosing. Combination therapy increased tumor Cmax and AUC 2-3 fold for both LEE011 and LDK378 over monotherapy, but plasma concentrations were unaltered.
Conclusions: LDK378 and LEE011 demonstrate on-target synergy with augmented abrogation of respective molecular targets resulting in increased caspase activation 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.
Citation Format: Andrew C. Wood, Kateryna Krytska, Hannah Ryles, Renata Sano, Theodore Hansel, Nanxin Li, Frederick King, Timothy Smith, Tove Tuntland, Sunkyu Kim, Giordano Caponigro, You-Qun He, Jennifer Harris, Yael Mosse. Combinatorial CDK4/6 and ALK inhibition demonstrates on-target synergy against neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B42.