Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice

Abstract

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

Keywords: AP26113; anaplastic lymphoma kinase; brigatinib; neuroblastoma; resistant mutations.

Figure 1. Brigatinib inhibits ALK activity and proliferation of ALK addicted neuroblastoma cell lines

A, B. CLB-BAR (ALK-Δ4-11) and CLB-GE (ALK-F1174V), both ALK addicted cell lines, were treated with increasing doses of brigatinib for 6 hours. Crizotinib (250 nM) was employed as a positive control. Cells lysates were resolved on SDS/PAGE followed by immunoblotting for pALK (Y1604) and additional downstream targets as indicated. In the CLB-BAR cell line there is a genomic deletion in ALK between exon 4-11, resulting in an ALK band of approximately 170 kDa [21]. The CLB-GE cell line expresses a mutant full length version of ALK (F1174V) which is cleaved resulting in the detection of two bands with the antibody employed here. C, D. CLB-PE (ALK-WT) and IMR32 (ALK-WT) are ALK non-addicted neuroblastoma cell lines. Neuroblastoma cells were treated with increasing concentration of either brigatinib (C) and crizotinib (D) for 72 hours and cell viability was assessed by resazurin assay (Sigma, Sweden). Plotted values are means +/− SE from growth curves from at least three independent experiments performed in triplicate.

Figure 2. Brigatinib effectively blocks ALK activation and ALK-mediated neurite outgrowth in PC12 cells

A, B. PC12 cells were transfected with ALK (0.75 μg). 48 hours post-transfection cells were treated with increasing amounts of either brigatinib or crizotinib. Wild type (WT) ALK was transfected with 1.5 μg pcDNA3-ALK-WT. Whole cell lysates were blotted for pALK (Y1604) to measure ALK activation. C. PC12 cells were co-transfected with pEGFPN1 together with the indicated ALK mutant. Wild type (WT) ALK was stimulated with 1 μg/ml of mAb31. GFP-positive PC12 cells were scored for neurite outgrowth after 48 hours treatment with either brigatinib or crizotinib (control) at the indicated concentrations.

Figure 3. Effect of brigatinib on ALK gain-of-function rough eye phenotypes in a Drosophila ALK model

Ectopic expression of gain-of-function “hot spot” ALK mutations ALK-F1174L and ALK-R1275Q in Drosophila eye imaginal discs with the Gal4-UAS-system results in a rough eye phenotype that is 100% penetrant [7, 38, 40]. Offspring that were grown on food containing brigatinib displayed a concentration dependent improvement of the rough eye phenotype.

Figure 4. Effect of brigatinib in a xenograft neuroblastoma model

2.5×10⁶ CLB-BAR cells were injected into mice subcutaneously. In the first investigation mice (n=3) were employed and in the second investigation mice (n=5) were employed. A. Tumor growth curves represented as the average volume of either control group or brigatinib group (n=8 for each group, p=0.0002). In the second investigation where 5 mice were employed a subset of mice (n=2) were released from treatment after 14 days, for a further 8 days (blue curve). B. Average tumor volume in control and brigatinib treated groups after 14 days, p=0.00004. C. Average tumor weights in control and brigatinib treated groups after 14 days (p=0.01). D. Average body weight on the day at which mice were sacrificed after 14 days treatment with either brigatinib or vehicle control (p=0.9). E. Immunohistochemical staining of tumors with Ki-67 as a measure of proliferation rate as indicated, Ki-67-positive cells were counted manually per field of vision and quantitative results presented as mean ± SD (p=0.0005). Data are means +/− SE from 8 tumours, p values indicated; Student's t test. F. Immunonblotting analysis of indicated proteins from tumors collected after 8 days of treatment with vehicle or brigatinib.