Abstract 5451: Over-expression of NPM-ALK drives resistance to TKIs in ALK+ ALCL but is toxic upon drug withdrawal, permitting prolonged tumour control through discontinuous dosing
Authors: Sundara Rajan Soumya , Amin Amit Dipak , Groysman Matthew , Pongtornpipat Praechompoo , Schatz Jonathan 
Published: August 2015

Abstract
Activated Anaplastic Lymphoma Kinase (ALK) is seen in several cancers, including 2-7% of non-small cell lung cancer and ∼ 70% of Anaplastic Large Cell Lymphoma (ALCL). The most common ALK fusion in ALCL is a translocation, t(2;5)(p23;35), placing the kinase domain of ALK before the constitutively active promoter of nucleophosmin (NPM). Activated ALK drives oncogenesis by turning on multiple proliferative pathways. The success of tyrosine kinase inhibitors (TKIs) in ALK+ lung cancer has prompted evaluation of their use in ALK+ ALCL as a therapeutic strategy for patients who fail combination chemotherapy. Clinical studies in lung cancer show resistance to these drugs limits progression-free survival. We aim to identify resistance mechanisms against approved TKIs, crizotinib and ceritinib, in ALK+ ALCL.
We selected for resistance in 3 patient-derived cell lines through propagation in gradually increasing drug concentrations. We assessed resistant subclones with long-insert whole-genome sequencing, viability and proliferation assays, qPCR and western blots. Similar profiling was carried out upon drug withdrawal. We validated our findings in cytokine-dependent murine pro-B cells transformed with NPM-ALK during ceritinib incubation. We xenografted resistant clones to SCID mice and assessed tumour burden over time.
Resistant clones showed viability stimulation by TKIs and when washed out of drug, these clones underwent apoptosis due to hyper-stimulation of ALK signalling. Genomic amplification of NPM-ALK was seen leading to an increased expression at the mRNA and protein levels. TKI-dependence therefore was co-selected with resistance, showing for the first time toxicity to cancer cells due to an overdose of ALK signalling. We validated our findings in IL3-dependent FL/5.12 cells, which also showed co-selection for drug resistance and dependence due to ALK over-expression. Resistant ALCL lines selected for ability to grow without TKI behaved similarly to parent lines, showing no stimulation of viability in presence of drug. ALK mRNA and protein also return to baseline in these cells. SCID mice injected with resistant cells required TKI treatment for tumour engraftment and tumour burden decreased when drug dosing was stopped. When tumours re-grew, drug treatment was reinitiated, generating a second response demonstrating in vivo that NPM-ALK up-regulation results in both resistance and dependence on the TKI and resistant cells die from an overdose of ALK signalling in the absence of drug.
Our results reveal that up-regulation of the fusion-ALK drug target is a resistance mechanism, previously un-reported in any ALK+ cancers. Up-regulation of NPM-ALK provides cells means to acquire resistance but also results in ALK overdose when drug is withdrawn, revealing intermittent dosing as a potential therapeutic strategy to prolong tumour control in ALK + patients.
Citation Format: Soumya Sundara Rajan, Amit Dipak Amin, Matthew Groysman, Praechompoo Pongtornpipat, Jonathan Schatz. Over-expression of NPM-ALK drives resistance to TKIs in ALK+ ALCL but is toxic upon drug withdrawal, permitting prolonged tumour control through discontinuous dosing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2015-5451