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BCL2 limits crizotinib efficiency in ALk+ ALCL
ly based on short-pulse chemotherapy courses, reach event-free survival rates of 70%.1 However, some patients still fail therapy and continued therapeutic improvements with reduced toxicity are being pursued. Recently, target- ed therapy against ALK using the dual ALK/MET tyrosine kinase inhibitor crizotinib has been shown to be effective in relapsed/resistant ALK-positive ALCL.6,7 However, as reported for other tyrosine kinase inhibitors, escape mech- anisms which allow cancer cells to overcome the effects of crizotinib have already been described in ALK-positive non-small cell lung carcinoma (NSCLC), inflammatory myofibroblastic tumors (IMT), and ALCL patients.8,9
Recently, several studies performed in ALK-associated cancers, including ours in ALCL, demonstrated that macroautophagy (hereafter referred to as “autophagy”) is induced following treatments with ALK tyrosine kinase inhibitors and acts as a cell survival-promoting mechanism restraining the cytotoxic effects of the drugs.10 Autophagy is a highly-conserved catabolic pathway and a dynamic process (autophagic flux) that is responsible for double membrane autophagosome synthesis, delivery of autophagic substrates to the lysosomes, and degradation of autophagic substrates inside lysosomes.11 In cancer, autophagy is often described as a cell survival-promoting mechanism, but cell death-promoting roles have also been reported according to the stage of cancer development and treatments administered.12-14 Thus, autophagy is considered to be a multifaceted regulator of cell death and outstanding questions remain as to how it interacts with other forms of cell death, including apoptosis and necro- sis/necroptosis.15
Indeed, the inter-relationship between these different forms of cell death is extremely complex and the common underlying molecular machinery makes it difficult to dis- tinguish one form from another.15-17 BCL2-family proteins regulate all major types of cell death, including apoptosis, necrosis and autophagy, thus operating as nodal points at the convergence of multiple oncological pathways.18 The pro-survival BCL2 family members, BCL2 and BCL2L1 (BCL-XL/S) have been reported to directly inhibit autophagy by binding to a BH3-like domain of the BECN1 autophagy protein.19 Lindqvist et al. have, however, recently proposed that pro-survival BCL2 family members indirectly inhibit components of the autophagy pathway by inhibiting the activation of BAX and BAK.20,21
Deregulation of BCL2 and other anti-apoptotic proteins has been demonstrated to be an important resistance mechanism to treatments in solid tumors and hematologic malignancies.18 It has been proposed that the oncogenic properties of BCL2 could originate not only from its ability to block apoptosis but also from its capacity to inhibit BECN1-dependent autophagy, thus preventing BECN1-dependent autophagic cell death.22 This further supports BCL2 as a critical target for cancer treatment, and numerous BCL2 targeting strategies are being developed for therapeutic applications. These include pharmacologi- cal inhibitors, such as the highly selective BCL2 inhibitor venetoclax (ABT-199)23,24 and, more recently, BCL2-target- ed DNAi25 or microRNA mimics like miR-34a.26-28
Here, we show for the first time that ALK inactivation in ALK-positive ALCL induces an increase in BCL2 levels, which could contribute to therapeutic failures of current ALK-targeted therapies. We found that BCL2 downregula- tion strongly potentiates the cytotoxic effects of crizotinib both in vitro and in vivo, by increasing the intensity of the
autophagic flux as a support for subsequent cell death. Our data strongly suggest that the BCL2 protein, acting at the crossroads between different forms of cell death, is the keystone of an escape mechanism in therapeutically-chal- lenged ALK-positive ALCL. Therefore, BCL2 downregula- tion in combination with crizotinib treatment could significantly improve clinical outcome in ALK-positive ALCL patients.
Methods
Human cell lines
KARPAS-299, COST, and SU-DHL-1 ALK-positive ALCL cell lines which express the NPM-ALK fusion protein were originally obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany) or established in our labo- ratory.29 The ALK-negative ALCL cell line FE-PD was a kind gift from Dr. K. Pulford (Oxford University, Oxford, UK). Cells were cultured as previously described.30 The mRFP-EGFP-LC3 KARPAS- 299 cell line (clonal cell population) was established in our labora- tory (Online Supplementary Methods). This study was carried out in accordance with protocols approved by the institutional review board, and the procedures followed were in accordance with the Declaration of Helsinki of 1975, as revised in 2000.
Chemicals
Crizotinib (PF-2341066) was synthesized and purchased from @rtMolecule (Poitiers, France). Chloroquine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin was provided with the Cyto-ID Autophagy detection kit (Enzo Life Sciences, Switzerland). Stock solutions of crizotinib (500 μM), chloroquine (50 mM) were prepared in phosphate buffered saline (PBS). Stock solutions of rapamycin (500 μM) were prepared in dimethyl sul- foxide (DMSO).
Cell viability determination by MTS colorimetric measurements
The CellTiter 96AQueus One Solution Cell Proliferation assay (Promega, Fitchburg, WI, USA) was used according to the manu- facturer’s instructions to follow the growth of the cells and deter- mine the number of viable cells.
Cell death measurements using annexin V/propidium iodide staining
Analysis of dying cells was carried out using annexin V (Annexin V-PE) and propidium iodide (PI) staining (BD Biosciences), according to standard protocols. This was followed by flow cytometry using a MACSQUANT MQ10 (Miltenyi Biotec, Santa Barbara, CA, USA). Results were analyzed using FlowJo software (v.10, BD Biosciences).
Flow cytometric quantification of autophagic flux
Autophagic flux was either assessed on a stable clonal popula- tion of mRFP-EGFP-LC3-expressing KARPAS-299 cells, recently generated in our laboratory (Online Supplementary Methods) or on whole cell population of KARPAS-299, SU-DHL-1 and COST cells using the Cyto-ID- based procedure, according to the manufactur- er’s instructions (Enzo Life Sciences, Switzerland).
Confocal microscopy
Cells were fixed (20 min, 4% PFA) on polylysine-coated slides (0.01%) and stained with DAPI using ProLong® Gold Antifade Mountant with DAPI (Thermofisher, Waltham, MA, USA). Analysis by confocal microscopy (LSM-780, Zeiss) was performed
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