Oncogene addiction and radiation oncology: effect of radiotherapy with photons and carbon ions in ALK-EML4 translocated NSCLC

Abstract

Background: Patients with Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) positive lung cancer are sensitive to ALK-kinase inhibitors. TAE684 is a potent second generation ALK inhibitor that overcomes Crizotinib resistance. Radiotherapy is an integral therapeutic component of locally advanced lung cancer. Therefore, we sought to investigate the effects of combined radiotherapy and ALK-inhibition via TAE684 in ALK-positive vs. wild type lung cancer cells.

Methods: Human non-small cell lung cancer (NSCLC) cell lines harboring wild-type ALK (A549), EML4-ALK translocation (H3122) and murine Lewis Lung Cancer (LLC) cells were investigated. Cells were irradiated with 1-4 Gy X-Rays (320 keV) and carbon ions (Spread-out Bragg Peak, SOBP (245.4-257.0 MeV/u)) at Heidelberg Ion Therapy center. TAE684 was administered at the dose range 0-100 nM. Clonogenic survival, proliferation and apoptosis via caspase 3/7 expression level were assessed in all three cell lines using time-lapse live microscopy.

Results: TAE684 inhibited the proliferation of H3122 cells in a dose-dependent manner with a half maximal inhibitory concentration (IC₅₀) of ~ 8.2 nM. However, A549 and LLC cells were relatively resistant to TAE684 and IC₅₀ was not reached at concentrations tested (up to 100 nM) in proliferation assay. The antiproliferative effect of TAE684 was augmented by radiotherapy in H3122 cells. TAE684 significantly sensitized H3122 cells to particle therapy with carbon ions (sensitizer enhancement ratio ~1.61, p < 0.05). Caspase 3/7 activity was evidently enhanced after combination therapy in H3122 cells.

Conclusions: This is the first report demonstrating synergistic effects of combined TAE684 and radiotherapy in EML4-ALK positive lung cancer cells. In addition to conventional photon radiotherapy, ALK-inhibition also enhanced the effects of particle irradiation using carbon ions. Our data indicate beneficial effects of combined ALK-inhibition and radiotherapy in treatment of this distinct subpopulation of NSCLC that warrant further evaluation.

Keywords: ALK inhibitors; Carbon ions; EML4-ALK-fusion; Non-small-cell lung cancer; Radiotherapy.

Fig. 1

Selective antiproliferative effect of TAE684 in ALK-positive NSCLC. The kinase inhibition profile by TAE684 (IC₅₀, mapped by TREE spot) as well as its 2D and 3D molecular structures (adapted from PubChem) are shown (a). Cell proliferation was assessed by counting viable cells 72 post treatment with a cell-permeant DNA-binding fluorescent dye (CyQuant-Direct) (b). Alternatively, cell proliferation was monitored longitudinally by live microscopy and the confluence levels as well as representative photomicrographs are presented (c and d). TAE684 potently inhibited cell proliferation in ALK-positive H3122 NSCLC but was less or not effective in A549 and LLC cells, respectively. Bars represent mean ± SD. TK: tyrosine kinase; Ctrl: control

Fig. 2

TAE684 selectively augments radiotherapy-induced antiproliferative effects in ALK positive NSCLC. Cell proliferation after incubation with vehicle or TAE684 (40 nM) alone or in combination with irradiation (4 Gy) was evaluated in H3122 (a), LLC (b) and A549 (c) cells (left panel). Only in ALK-positive H3122 cells dual treatment with TAE684 and radiotherapy reduced cell proliferation by 56% as compared to radiotherapy alone (p < 0.01). Photomircographs of representative fields after 72 h (H3122 and A549) or 36 h (LLC) are shown on the right panel. Cell numbers were normalized to that of non-treated groups for each cell line. Bars indicate mean ± SD. * p < 0.05 and ** p < 0.01

Fig. 3

Synergistic effects of combined TAE684 and radiotherapy on NSCLC survival and apoptotic activity. The survival fraction after 4 nM TAE684 monotherapy revealed high sensitivity of H3122 cells and moderate response of LLC cells instead of A549 cells (a). In parallel, TAE684 induced apoptosis in H3122 cells and the addition of radiotherapy enhanced Caspase 3/7 level in a synergistic manner (b). H3122 (c), LLC (d) and A549 (e) cells were treated with vehicle or TAE684 (4 nM) and irradiated with 0, 1, 2 or 4 Gy. TAE684 selectively sensitized ALK-positive H3122 to radiotherapy. In contrast, a trend toward antagonistic effects was found in LLC cells treated with this dual combination. Bar represent mean ± SD

Fig. 4

TAE684 sensitizes ALK positive NSCLC cells to carbon ions. The surviving fraction of H3122(a), LLC (b) and A549 (c) cells was determined after vehicle or TAE684 (4 nM) treatment and carbon ion irradiation (0, 1, 2 or 4 Gy). TAE684 treatment potently sensitized ALK-positive H3122 to carbon ion radiotherapy, while a moderate radiosensitivity was induced in LLC cells and no response was observed in A549 cells. Bars indicate mean ± SD

Fig. 5

Selective radiosensitization of EML4-ALK oncogene addicted tumors exemplified by crizotinib. A schematic overview of another study conducted in frame of the KFO-214 evaluating the impact of the first generation ALK inhibitor crizotinib on tumor response to radiotherapy [30]. Different tumor cell lines were profiled for their addiction to ALK signaling by western (ALK activation) and FISH (ALK translocation). Crizotinib was originally intended to target cMET, hence cMET signaling was also investigated in all evaluated cell lines. Comprehensive in-vivo tumor growth delay studies revealed selective radiosensitzation of ALK addicted NSCLC cell lines. This was in line with synergistic effects of dual combination observed after photon orcarbon irradiation, respectively, by clonogenic survival assay and isobologram analysis. Together with data reported on second-generation ALK inhibitor TAE684 here, our data support the combination of this class of agents with radiotherapy in ALK addicted NSCLC