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A. Torossian et al.
in order to observe fluorescent signals in mRFP-EGFP-LC3 KARPAS-299 cells. Image analysis was performed using the ImageJ software (US National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
Results are presented as mean values±Standard Error of Mean (SEM) from at least three independent experiments, unless other- wise indicated. Determination of statistical significance was per- formed using the two-tailed Student t-test for side-by-side com- parison of two conditions. Statistical analyses of xenografted tumor growths were performed using the two-way analysis of variance (ANOVA) followed by the Bonferroni test using GraphPad Prism 6 software (GraphPad, San Diego, CA, USA). For all tests, statistical significance is indicated by: *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.
Results
BCL2 levels inversely correlate with ALK expression and ALK activity in anaplastic large cell lymphoma cells
Whereas overexpression of BCL2 is a classical feature in cancers, including hematopoietic tumors, previous studies reported very low levels of BCL2 in ALK-positive primary tissue samples.31,32 To investigate whether there was a link between ALK and BCL2 expression, we first checked the relative abundance of these two proteins in three ALK- positive and one ALK-negative cell lines (Figure 1A). BCL2 protein levels were found to be very low in the three ALK- positive cell lines tested (KARPAS-299, SU-DHL-1, and COST) but were much more abundant in the ALK-negative (FE-PD) cell line, suggesting an inverse cor- relation between the expression of NPM-ALK and BCL2. We then monitored BCL2 levels following treatment with the crizotinib compound for 24 hours (h) at the known plasmatic dose (500 nM) observed in patients30 (Figure 1B). Our data indicate that the abolition of ALK kinase activity resulted in a consistent and highly reproducible increase in BCL2 levels in ALK-positive cells, with no effect in ALK-negative cells (for quantification, see Online Supplementary Figure S1A). ALK knockdown, through the use of a targeted siRNA, confirmed these results, showing an ALK-dependent increase in BCL2 levels (Figure 1C and Online Supplementary Figure S1B for quantification). Finally, as crizotinib is known to inhibit both ALK and MET tyro- sine kinases,6 we then checked the effects of the specific molecular downregulation of MET, using a targeted siRNA, on BCL2 cellular levels. We did not observe any increase in BCL2 levels following MET knockdown (Online Supplementary Figure S2). Thus, this result rules out the contribution of MET inactivation in the increase of BCL2 levels following crizotinib treatment. Altogether, these data support the existence of a strictly ALK-depen- dent BCL2 repression mechanism at work. Therefore, treating ALK-positive ALCL with crizotinib impairs this mechanism and leads to the re-expression of the BCL2 oncogene.
Increased BCL2 levels limit the cytotoxic effects of crizotinib
We next asked whether crizotinib-mediated increase in BCL2 levels could limit the cytotoxic effects of the drug. We thus performed viability assays, cell cycle analyses,
and Annexin V/PI staining in cells that were knocked down or not for BCL2, and treated or not with crizotinib (Figure 2). BCL2 knockdown (confirmed by western blot analysis) (Online Supplementary Figure S3) was achieved by RNA interference using either a targeted siRNA directed against BCL2 mRNA, or miR-34a mimics, another promising BCL2 targeting strategy for clinical applica- tion.26-28 Viability assays showed that BCL2 knockdown, which restrained BCL2 elevation in crizotinib-treated cells (without up-regulating other BCL2 family isoforms, including MCL1, BCL-XL/S and BCL-W; data not shown), significantly enhanced the effects of crizotinib, as only 20% of siBCL2-transfected cells remained viable after 72 h when compared to approximately 50% of siCTL- trans- fected cells (Figure 2A). More pronounced effects on cell viability were obtained when BCL2 knockdown was achieved using miR-34a mimics, as only 6% of cells were viable at the end of the combined treatment.
We then explored whether this loss in cell viability involved a decrease in cellular growth, by analyzing the effects of BCL2 silencing and/or crizotinib treatment on cell cycle distribution (Figure 2B). We found that BCL2 downregulation using a targeted siRNA did not increase either the potent G1-S phase cell cycle arrest or the num- ber of cells in sub-G1 phase observed following crizotinib treatment. On the contrary, the use of miR-34a mimics induced per se a blockade in G1 phase and an increase in the number of cells in sub-G1 phase, which were further potentiated upon crizotinib addition (Figure 2B).
To better assess the effects of BCL2 knockdown on cell death, we performed Annexin V/PI staining. Our data first showed that crizotinib treatment (500 nM, 72 h) induced apoptosis, as reflected by a significant increase in the number of annexin V-stained cells in siCTL and miR-Neg conditions (Figure 2C). Additionally, and in agreement with the sharp loss in cell viability observed in response to combined treatments, we observed that BCL2 knock- down triggered an increase in apoptotic cell death in crizo- tinib-treated cells, as revealed by both a significant increase in the number of annexin V-stained cells and an activation of caspase 3/7 (Online Supplementary Figure S4).
Taken together, our data indicate that the cytotoxic effects of crizotinib in ALK-expressing ALCL cells are restricted through an elevation of BCL2 levels. Indeed, we demonstrate that BCL2 downregulation in addition to crizotinib treatment potentiates crizotinib-induced loss in cell viability essentially through an increase in apoptotic cell death. Of note, similar results on cell viability and apoptosis were obtained using SU-DHL-1, another ALK- positive ALCL cell line (Online Supplementary Figure S5).
BCL2 downregulation enhances crizotinib-triggered autophagic flux
Besides its role in apoptosis, BCL2 is also a known inhibitor of autophagy,19-22 a process that has been described to influence cell death mechanisms in many cancers following drug treatment.12,15,33 Since we recently showed that ALK inactivation induced autophagy endowed with pro-survival properties in ALK-positive ALCL cell lines,30 we investigated whether concomitant elevation of BCL2 could account for the cytoprotective function of the autophagic process in these conditions. To address this question, we generated a modified and clonal KARPAS-299 cell line stably transfected with a transgene encoding the LC3 protein (microtubule-associated protein
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haematologica | 2019; 104(7)