Evidence Suggesting That Discontinuous Dosing of ALK Kinase Inhibitors May Prolong Control of ALK+ Tumors

Abstract

The anaplastic lymphoma kinase (ALK) is chromosomally rearranged in a subset of certain cancers, including 2% to 7% of non-small cell lung cancers (NSCLC) and ∼70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors crizotinib and ceritinib are approved for relapsed ALK(+) NSCLC, but acquired resistance to these drugs limits median progression-free survival on average to ∼10 months. Kinase domain mutations are detectable in 25% to 37% of resistant NSCLC samples, with activation of bypass signaling pathways detected frequently with or without concurrent ALK mutations. Here we report that, in contrast to NSCLC cells, drug-resistant ALCL cells show no evidence of bypassing ALK by activating alternate signaling pathways. Instead, drug resistance selected in this setting reflects upregulation of ALK itself. Notably, in the absence of crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested or killed cells, allowing a prolonged control of drug-resistant tumors in vivo with the administration of discontinuous rather than continuous regimens of drug dosing. Furthermore, even when drug resistance mutations were detected in the kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We confirmed these findings derived from human ALCL cells in murine pro-B cells that were transformed to cytokine independence by ectopic expression of an activated NPM-ALK fusion oncoprotein. In summary, our results show how ALK activation functions as a double-edged sword for tumor cell viability, with potential therapeutic implications.

Figure 1

TKI resistance-dependence in ALK+ ALCL. A, initial sensitivities to ALK inhibition (see also Figure S1 for timelines acquired resistance). B and C, stimulation of viability by both ALK inhibitors in cells with acquired resistance. D and E, proliferation of parent and sub-clonal cells seeded at 10⁵/mL in media with DMSO, crizotinib, or ceritinib. F, Hoechst stained cells from D on Day 4 (scale bars 15μm). G, combined early/late apoptotic cells from D–E at day 4 by flow cytometry. Means of technical quadruplicates (A–C) or independent triplicates (D, E, G) ±SEM. *p=0.05 (Mann-Whitney).

Figure 2

NPM-ALK up-regulation drives resistance-dependence. A, LI-WGS reads indicate NPM-ALK amplification in resistant-dependent subclone. B, copy-number assay confirms gain specifically of the fusion kinase-encoding regions of NPM and ALK (see also Supplementary Fig. S2 for additional resistant cells). C and D, increased NPM-ALK mRNA expression in subclones. E, immunoblotting with and without crizotinib in parent and sub-clones. Means of technical triplicates (B–D) ±SEM. ** p<0.01, *p<0.05, NS p 0.05 (t-test).

Figure 3

ALK up-regulation drives resistance-dependence in transformed pro-B cells. A, retroviral introduction of NPM-ALK in a GFP co-expressing vector transformed FL5.12 cells to cytokine independence with simultaneous ceritinib incubation. (*Cells did not survive.) B, GFP intensity by ceritinib concentration. C and D, NPM-ALK mRNA and protein expression with increasing ceritinib concentration. E, Proliferation in cytokine-free media of FL5.12/NPM-ALK cells transformed with 50 nM ceritinib co-incubation, with and without drug. Means of technical quadruplicates (C) and independent triplicates (A, D, E and F) ±SEM. ** p<0.01, *p<0.05, NS p 0.05 (t-test).

Figure 4

Proliferation with TKIs requires return of ALK to baseline. A, RS cell viability in response to ALK inhibition, with original parent lines for comparison. B and C, RS proliferation (B) and apoptosis (C) on day 4 after seeding at 10⁵/mL in media containing DMSO, crizotinib, or ceritinib. D and E, LI-WGS (D) and qPCR copy-number assays (E) show no amplification of NPM-ALK in K299 RS sub-clones. F, mRNA expression relative to original parent. See also Supplementary Fig. S3 (for protein analysis). Means of technical quadruplicates (A) or technical triplicates (B, C, E and F) ±SEM.

Figure 5

ALK resistance-dependence in vivo. A, mean tumor burden of mice injected with 10⁶ K299-CR1000 cells and treated with vehicle or ceritinib (n=5 per group). B, individual tumor burden of tumor-bearing mice following ceritinib discontinuation and subsequent resumption. C–F, Viability assays (C), proliferation (D), copy-number (E) and mRNA expression (F) using cells taken from mice 1 and 3 before resumption of ceritinib show the RS phenotype. Cells from mouse 2 were not successfully cultured. Means of technical quadruplicates (C) or independent triplicates (D) or technical triplicates (E–F) ±SEM.

Figure 6

Schematic of ALK dosage and cell viability. A narrow range of ALK activity promotes cell viability. Addition of TKI to ALK-addicted parent cells or withdrawal from resistant cells surviving inhibitor due to ALK up-regulation drives cells into dynamic states of lost viability ending death but for opposite reasons of ALK signaling dosage.