Fig 2: The proposed model of action of NPM-ALK and RAF1 inhibitions on autophagy flux and cells outcome. The pharmacological inactivation of the NPM-ALK oncogene in NPM-ALK+ ALCL was previously shown to induce a cytoprotective autophagic flux. This status was shown to be associated with the downregulation of miR-7-5p levels and the increased expression of one of its targets: RAF1. When pharmacological or molecular tools allowing RAF1 inactivation were used in combination with NPM-ALK inhibition, the autophagy flux was potentiated and associated with increased cell death, therefore, highlighting the superiority of the co-treatment to kill NPM-ALK+ lymphoma cells.

Fig 3: ALK inhibitors delay M phase progression. (A) Whole cell lysates were analyzed by western blotting with anti-ALK and anti-Lamin B (loading control) antibodies. (B) SH-SY5Y cells were treated with inhibitors for 2 days, and viable cells were determined by WST-8 assay. Relative values of absorbance to solvent control (dimethyl sulfoxide, DMSO) are shown as mean ± SD, calculated from three independent experiments. (C) SH-SY5Y cells were synchronized with 4 µM RO-3306 for 20 h. After release, cells were treated with 1 µM crizotinib, 0.5 µM ceritinib, and 0.3 µM TAE684 for 1 h and stained for α-tubulin and DNA. Based on microtubule and chromosome morphology, M phase cells were classified into four groups: prophase/prometaphase (P/PM), metaphase (M), anaphase/telophase (A/T), and cytokinesis (Cyto). The percentages of cells of each group (n > 246 in each experiment) and associated mitotic indices (n > 453 in each experiment) are plotted as the mean ± SD, calculated from three independent experiments. (D) The number of cells with misaligned chromosomes was counted under a microscope, and their percentages are plotted as the mean ± SD of three independent experiments (n > 246 in each experiment). Representative images of cells with misaligned chromosomes are shown; scale bar = 5 µm. The Tukey–Kramer multiple comparison test was used to calculate p values. ** p < 0.01; N.S., not significant. (E) SH-SY5Y cells were treated with inhibitors in the same concentrations as in (C) for 1 h, and whole cell lysates were analyzed by Western blotting using anti-pALK-Y1507 and anti-ALK antibodies. Lamin B was incorporated as a loading control.

Fig 4: ALK inhibition delays both prophase/prometaphase and metaphase. SH-SY5Y cells were transfected with ALK-targeting siRNA (siALK #1) or nontargeting siCtrl. At 52 h after siRNA transfection, cells were synchronized with 4 µM RO-3306 and mitotic progression was monitored every 3 min for 5 h by time-lapse imaging. (A) Representative images of siCtrl-transfected cells and ALK knockdown cells. Scale bar = 10 µm. (B) Duration of prophase and prometaphase (P/PM, green), metaphase (M, red), and anaphase and telophase (A/T, blue) for 30 individual control cells and 31 individual siALK–transfected cells, was measured as described in material and methods. Line graphs show percentages of total mitotic cells (black), cells in P/PM (green), M (red), and A/T (blue) plotted against time.

Fig 5: ALK is responsible for ALK-inhibitor-induced M phase delay. (A) SH-SY5Y cells were transfected with ALK-targeting siRNAs (siALK #1, #2, and #3) or nontargeting siCtrl. At 72 h after transfection, whole cell lysates were prepared and analyzed by western blotting. (B) At 52 h after siRNA transfection, SH-SY5Y cells were synchronized with 4 µM RO-3306 and then cultured in fresh media for 90 min. Based on microtubule and chromosome morphology, M phase cells were classified into two groups: before (P/PM/M) or after (A/T/Cyto) anaphase onset. Percentages of cells (n > 243 in each experiment) and mitotic indices (n > 503 in each experiment) are plotted as the mean ± SD calculated from three independent experiments. (C,D) SH-SY5Y/ALK-HA cells were transfected with siRNA targeting the 3’-UTR region of ALK (siALK #3) or nontargeting siCtrl and were simultaneously treated with 5 µg/mL Dox. (C) Fifty-two hours after transfection, the cells were synchronized with RO-3306 and M phase progression was analyzed as described in (B). Percentages of cells (n > 193 in each experiment) and mitotic indices (n > 413 in each experiment) are plotted as the mean ± SD of three independent experiments. The Tukey–Kramer multiple comparison test was used to calculate p values. ** p < 0.01. (D) At 72 h after transfection, whole cell lysates were analyzed by Western blotting with anti-HA, anti-ALK, and anti-lamin B antibodies.