Alectinib, an Anaplastic Lymphoma Kinase Inhibitor, Abolishes ALK Activity and Growth in ALK-Positive Neuroblastoma Cells

Abstract

Oncogenic receptor tyrosine kinases including anaplastic lymphoma kinase (ALK) are implicated in numerous solid and hematologic cancers. ALK mutations are reported in an estimated 9% of neuroblastoma and recent reports indicate that the percentage of ALK-positive cases increases in the relapsed patient population. Initial clinical trial results have shown that it is difficult to inhibit growth of ALK positive neuroblastoma with crizotinib, motivating investigation of next generation ALK inhibitors with higher affinity for ALK. Here, alectinib, a potent next generation ALK inhibitor with antitumor activity was investigated in ALK-driven neuroblastoma models. Employing neuroblastoma cell lines and mouse xenografts we show a clear and efficient inhibition of ALK activity by alectinib. Inhibition of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. The results suggest that alectinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma and should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

Keywords: ALK inhibitors; alectinib; anaplastic lymphoma kinase (ALK); crizotinib; neuroblastoma; resistant mutations; xenograft.

Figure 1

Alectinib inhibits signaling in ALK addicted neuroblastoma cell lines. (A,B) ALK addicted cell lines, CLB-BAR (amplified MYCN/ALK, ALKΔexon 4-11), and CLB-GE (amplified MYCN/ALK, ALK-F1174V) were treated with alectinib at the concentrations indicated for 6 h and lysate immunoblotted for pALK-Y1604 and the ALK downstream targets ERK1/2, AKT, and MYCN. Crizotinib (250 nM) was used as a positive control. ALK runs at approximately 170 kDa in CLB-BAR cells due to a genomic deletion in ALK between exon 4-11 (22, 48). In CLB-GE cells, the mutant full-length ALK-F1174V mutant is cleaved and is detected as two bands. (C,D) CLB-PE and IMR-32 harbor a wildtype ALK and are ALK-wt and non-ALK addicted neuroblastoma cell lines. All four neuroblastoma cell lines were grown in increasing concentrations of either alectinib (C) or crizotinib (D) for 72 h after which cell viability was monitored by resazurin assay (Sigma, Sweden). Mean ± SD values were plotted from three independent experiments, performed in triplicate. (E) IC₅₀ values from the cell viability analysis in C and D were generated in GraphPad Prism 7.0. The results were analyzed as log (inhibitor) vs. normalized response and are expressed as mean ± SD.

Figure 2

Alectinib inhibits activity of ALK mutants in PC12 cells. (A,B) PC12 cells transiently transfected with either ALK-wt (1.5 μg) or ALK mutant variants as indicated (0.75 μg), were treated with increasing concentrations of either alectinib (A) or crizotinib (B) at 48 h post-transfection. ALK activation was measured by immunoblotting for pALK-Y1604. (C,D) Shows the total ALK levels (as a loading control) for (A,B), respectively.

Figure 3

Cell cycle analysis and apoptosis analysis upon treatment of neuroblastoma cells with alectinib. (A) CLB-BAR, CLB-GE, and SK-N-AS cells were grown on six-well plates with complete growth medium and treated with alectinib (100 nM) for 24 h. Cell lysates were immunoblotted with antibodies against PARP. ERK was used as a loading control. Three independent experiments were performed (biological replicates). Values were analyzed by normalizing, the fraction of full-length PARP vs. cleaved PARP and calculated in GraphPad Prism 7.0, and are expressed as mean ± SD. P-values were calculated by Student paired t-test, ^**p-value > 0.005; ns, non-significant. (B) Cell cycle analysis of CLB-BAR, CLB-GE, and SK-N-AS cells treated with alectinib 75 and 150 nM for 24 h. The data was normalized to the respective control. Three independent experiments were performed (biological replicates). Control vs. alectinib (75 nM) and control vs. alectinib (150 nM) in G0/G1 phase (blue bar) and S phase (orange bar) showed significant difference in both CLB-BAR and CLB-GE, but not in SK-N-AS. P-values were calculated by Student paired t-test, ^*p-value <0.05; ns, non-significant.

Figure 4

Alectinib inhibits ALK mediated neurite outgrowth efficiently in PC12 cells. ALK mutant variants were co-transfected with pEGFPN1 in PC12 cells. Neurite outgrowth was measured in GFP-positive cells 48 h post-treatment with either alectinib or crizotinib at the specified concentration. Those GFP cells having neurite outgrowth double the size of cells were determined as a positive neurite-carrying cells. Data shows the percentage of GFP neurites-carrying cells Graph shows the mean of three independent experiment ± SD.

Figure 5

Alectinib inhibits tumor growth in a neuroblastoma xenograft model. 1 × 10⁶ CLB-BAR cells were injected subcutaneously in female BALB/cAnNRj-Foxn1nu mice. Mice were treated with vehicle control (n = 10), crizotinib (80 mg/kg once daily, n = 10) or alectinib (20 mg/kg once daily, n = 10) upon tumor growth. (A) Curves indicate tumor volume with vehicle control, crizotinib or alectinib treatment over 14 days. Red dotted line indicates the last day of treatment (day 14). The samples after red dotted line indicate the tumor growth after release from alectinib treatment (n = 5). Values were calculated with a two-way ANOVA, with Sidak's multiple comparison test employing GraphPad Prism 7.0, ^*p = 0.0222 and ^****p < 0.0001. (B) Average tumor volume in either control or alectinib treated groups after 14 days (n = 5). (C) Average tumor weight in either control (n = 5) or alectinib treated groups after 14 days (n = 5). (D) Body weight of either control or alectinib treated mice during treatment. (B,C) values were calculated in GraphPad Prism 7.0, and are expressed as mean ± SD, P-values were calculated with an unpaired two-tailed Student t-test, ^***p-value < 0.0001 (C) and ^**p-value = 0.0072 (B).

Figure 6

(A) Representative images of tumors treated with either alectinib, crizotinib, or vehicle and stained with anti-Ki-67, anti-CD31, phosphor-Histone H3, and cleaved caspase 3. (B) Bar graphs show mean values ± SD; P-values were calculated by Mann-Whitney test, ^**p-value ≤ 0.008, ^*p-value < 0.05; ns, non-significant. Scale bars indicate 100 μm.