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P.-Y. Dumas et al.
found in acute myeloid leukemia (AML).2 Although the FLT3-ITD mutation is a late event in leukemogenesis,3 it is an important target for the disease.4 Indeed, FLT3-ITD mutation is associated with a poor prognosis,5-7 and its frequent occurrence at relapse suggests that FLT3-ITD AML-initiating cells are key targets for long-lasting remis- sion.
The FLT3-ITD mutation induces constitutive activity of the receptor and a distinct pattern of activated signaling pathways, the principal change being the activation of the transcription factor STAT5.8 FLT3 tyrosine kinase inhibitors (FLT3-TKI), which were developed as ATP- competitive inhibitors, were initially tested in clinical tri- als and produced variable benefits according to the dis- ease heterogeneity. Among these treatments, quizartinib (AC220), a FLT3-TKI specifically designed for FLT3, induces a hematologic improvement in monotherapy associated with approximately 50% of response.9 However, bone marrow (BM) blasts show little noticeable cell apoptosis, but are associated with cell-cycle arrest and terminal differentiation.10 Remissions are of short duration, with the emergence of resistance related to sev- eral mechanisms. Intrinsic mechanisms include the acti- vation of bypass signaling pathways11 and activation loop or gatekeeper mutations.4 Extrinsic mechanisms include cell-to-cell interactions and secretion of cytoprotective factors.12
AXL belongs to the TAM receptor family, which also includes TYRO3 and MER.13 This RTK is activated by homodimerization upon binding of its major ligand growth arrest-specific 6 (GAS6).14 The GAS6/AXL path- way contributes to cell growth, survival, invasiveness, chemotaxis, apoptotic body clearance and immunity.15 AXL is ectopically- or over-expressed in a wide variety of cancers and has always been associated with a poor prog- nosis.16 We have reported resistance mechanisms involv- ing AXL in chronic myeloid leukemia.17 In AML, AXL and GAS6 levels of expression have been related to poor out- comes.18,19 Paracrine AXL activation has been shown to induce AML resistance to conventional chemotherapies but also to FLT3-targeted therapy.20-23 However, no infor- mation is available concerning the regulation of AXL expression in the context of the AML-supportive hematopoietic niche, which sustains AML resistance in vivo.
Indeed, AXL expression and function have been shown to be modulated by stress, nutrient deprivation and low oxygen (O2) concentration in various solid tumors.14,24 These characteristics are quite similar to those within the hematopoietic microenvironment of AML-initiating cells. We show here that the hematopoietic microenvironment educates AML cells to over-express AXL through a cytokine-dependent STAT5 activation and low O2 con- centration.
Methods
Cell culture, apoptosis and AXL, GAS6 and STAT5 gene expression knock-down using shRNA
All cell lines (MV4-11, MOLM-13, MOLM-14, UT7-mpl, K562, MS5, OP9, HS27a) were cultured in RPMI1640 or αMEM medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. Hypoxia was induced by incubating cells in a specific O2 cham-
ber (BioSpherix). The UT7-mpl cell medium was supplemented with granulocyte-macrophage colony-stimulating factor (GM- CSF, 2.5ng/mL, Diaclone, France), as previously described.25 UT7-mpl cells were pre-incubated for 18 hours (h) in the absence of GM-CSF, before being activated by cytokines. Where indicated, cells were cultured with vehicle or thrombopoietin peptide (Sigma), interleukin (IL)-3 (Miltenyi Biotec), AXL-Fc chimeric proteins (R&D Systems), AC220 (quizartinib) (LC lab- oratories), R428 (Selleckchem), Ly294002, pimozide, JAK inhibitor-I (Calbiochem/Merck). AC-4-130, a selective STAT5 inhibitor, was provided by JANPIX Ltd. (UK) under the license from Prof. Patrick Gunning’s group (University of Toronto, Canada).26 Cell apoptosis was assessed using an APC-conjugated Annexin V labeling detection kit coupled to flow cytometry and BDFACSDIVATM software (BD Bioscience). For shRNA, viral supernatants were titrated by serial dilutions and a FACS analy- sis of GFP co-expressed marker in transduced K562 cells 72 h later (Accuri C6, Beckton Dickinson). Lentiviruses were added once, at a multiplicity of infection of 5-20 according to the cells. Transduced cells were sorted 2-3 days after transduction using GFP. Two different shRNA were used for all targets (Online Supplementary Table S1). To confirm silencing, AXL, GAS6 or STAT5 protein expressions were analyzed by immunoblotting.
Acute myeloid leukemia patient and cord blood donor biological samples
Acute myeloid leukemia samples were obtained from patients at the University Hospital of Bordeaux and University Hospital Paris Centre who gave written informed consent for the use of biological samples for research, in accordance with the Declaration of Helsinki. This allowed the collection of clini- cal and biological data in an anonymized database, registered at the “Commission Nationale de l’Informatique et des Libertés” (authorization n. 915285) and “Comité de Protection des Personnes” (authorization n. 2015-08-11D). For FLT3-ITD AML patient samples, selection criteria were high leukemic burden (blast cells level > 70%) and high FLT3-ITD/WT ratio (ITD/WT > 0.5) (Online Supplementary Table S2). Bone marrow mononu- clear cells were co-cultured with MS5 stromal cells in H5100 medium (Stem Cell Technologies), unless otherwise stated. When indicated, AML cells were preincubated for 18 h in serum- free IMDM medium in the absence of MS5 before being incu- bated for 7 h in the presence of a cytokine cocktail that included IL-3 (20 ng/mL), GM-CSF (10 ng/mL) and TPO (20 nM). Human cord blood (CB) units were collected according to institutional guidelines. Cord blood CD34+ were purified and cultured in a four-cytokine-supplemented serum-free IMDM medium, as pre- viously described.27
Further details about the methods used are available in the
Online Supplementary Appendix.
Results
Stromal protection of FLT3-ITD AML cells to quizartinib correlates with STAT5 activation
We first confirmed the protective effect of three stro- mal cell lines on MV4-11, MOLM-13 and MOLM-14 FLT3-ITD AML cell lines exposed to quizartinib (AC220), an ATP-competitive FLT3- inhibitor. Co-culture of AML cells with one human (HS27a) and two murine (OP9 or MS5) stromal cells decreased apoptosis triggered by FLT3- TKI treatment compared to AML cells cultured without stroma (Figure 1A and Online Supplementary Figure S1A and B). This decrease persisted at high AC220 concentra-
2018
haematologica | 2019; 104(10)