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Hyaluronan/CD44-mediated VLA-4 activation in AML
the interactions between AML cells and the supportive microenvironment may pave the way for novel combina- tion therapies antagonizing residual disease.
The glycoprotein CD44 functions by binding to its major ligand hyaluronic acid (HA), which is expressed by BM stromal cells and endothelial cells.2 In AML, targeting CD44 reduced leukemic repopulation in serial transplantations by eradication of leukemic stem cells.3
A second key orchestrator of leukemic cell-BM microen- vironment interactions is the integrin VLA-4, a CD49d/CD29 heterodimer. The binding of VLA-4 to its lig- and VCAM-1 is strengthened by inside-out signaling. This means that external stimuli mediate intracellular signaling triggered by other cell surface receptors, resulting in a change of either the avidity or the affinity of the integrin for its ligands.4 Avidity changes occur due to cluster formation of the integrin, whereas affinity is increased by conforma- tional changes.5 Cooperativity of CD44 and VLA-4 has pre- viously been suggested, but little is known about the mech- anism.6-8 To elucidate the mechanistic crosstalk between the two key homing factors, CD44 and VLA-4, to the BM in AML cell lines and primary AML cells, we used adoptive transplantations as well as static and shear flow adhesion assays in combination with immunofluorescence microscopy approaches. We uncovered a novel HA/CD44- induced inside-out activation of the integrin VLA-4. This activation leads to increased avidity due to VLA-4 clusters but no alterations in affinity between VLA-4 and its ligand VCAM-1. This elevated adhesion is important for AML cell retention in the stromal niche.
Methods
Study approvals and processing of patients’ samples
Following written informed consent, BM aspirates from patients with newly diagnosed AML were collected at the Third Medical Department, Paracelsus Medical University Salzburg, Austria (Salzburg ethics committee approval number: 415- E/2009/2-2016). Normal CD34+ progenitor cells from patients with myeloma or non-Hodgkin lymphoma who underwent hematopoietic stem/progenitor cell mobilization were used as non-myeloid controls (Salzburg ethics committee approval num- ber: 415-E/1177/8-2010). Mononuclear cells were isolated using density gradient centrifugation and the viable cells were frozen until further usage. The patients’ characteristics are shown in Online Supplementary Table S1.
The approval number for the animal experiments is BMWF- 66.012/0032-WF/V/3b/2017.
Adoptive transfers
For blocking experiments, primary AML cells or OCI-AML3 cells were pretreated with αCD44 Fab fragments (clone 515, 5 mg/mL) or αCD49d (clone HP2/1, 5 mg/mL) antibodies for 15 min at 37°C, where indicated. The specificity of the blockade was con- firmed by isotype control experiments in representative experi- ments. For homing versus engraftment assays (3 h and 3 days), cells were stained using the CellTraceTM Violet Cell Proliferation Kit (Thermo Fisher). Cells (0.3-1.3x106) were injected intravenously into NOD scid gamma (NSG) mice. After 3 h or 3 days, the mice were sacrificed, and the number of human cells that had homed to BM, spleen and peripheral blood was determined using αCD44 (clone J.173)- and αCD49d (clone 9F10)-specific antibodies. Homing rate was calculated as the number of CD44 and CD49d double-positive cells divided by the number of total measured
cells divided by the number of injected AML cells.9,10 Proliferation after 3 days was determined on the basis of CellTraceTM dye dilu- tion rates.10 For long-term engraftment (28 days) shCont or shCD49d OCI-AML3 cells were injected intravenously into NSGS mice. After 28 days, the mice were sacrificed, and the number of human CD15 and CD45 double-positive cells per million meas- ured BM cells, spleen cells or per microliter of blood was deter- mined.
Clusteringassay
VLA-4 clustering assays were performed as described else- where,11 using 7.5 mg/mL VCAM-1/Fc. AML cells were pretreated for 10 min with 10 mg/mL HA, 60 min with 1 mM midostaurin, 30 min with 10 mM PP2 and 30 min with 10 mM cobimetinib (APExBIO, Houston, USA), where indicated. Cells were allowed to adhere for 30 min at 37°C before fixation with 4% paraformaldehyde. Slides were stained with αCD49d (clone AHP1225), αCD29 (clone 12G10) primary antibodies or isotype control (not shown) followed by a secondary antibody. For CD49d cluster analysis of normal progenitor cells from patients with non-myeloid malignancies, cells were additionally stained with αCD34 antibody (clone QBEND-10). For quantification, high-resolution images were acquired on a Leica TCS SP5 II laser- scanning microscope using a 63×/1.4-NA oil-immersion objective (Leica, Wetzlar, Germany). The number of clusters was analyzed using ImageJ software by particle analysis setting the size of the particle at >2 pixels.12
Stroma binding
Falcon culture slides were left either uncoated or coated with 20 mg/mL fibronectin for 1 h at 37°C and then 70,000 M2 stromal cells were seeded and cultured overnight. Primary AML (1x106 cells) or OCI-AML3 cells (0.5x106 cells) were seeded on M2 stro- mal cells and co-cultured for 30 min at 37°C. Cells were washed, fixed with 4% paraformaldehyde, and stained using DAPI Antifade Reagent. Images were taken with an Olympus IX81 microscope (UPLSAPO 20xO/0.85 objective). Numbers of cells were determined in 12 pictures for each treatment with ImageJ software.
Additional experimental procedures are described in the Online Supplementary Methods.
Results
CD44 reflects and mediates leukemic infiltration of bone marrow
We measured CD44 and CD49d surface expression of BM-derived primary AML patients’ samples and the AML cell line OCI-AML3 by flow cytometry. All AML patient- derived blasts, identified via CD45/side scatter gating,13 and OCI-AML3 cells, expressed CD44 and CD49d (Figure 1A). We also screened various other AML cell lines, which cover most of the AML subtypes, i.e., MV4-11, KG-1a, HL-60, MOLM-13 and MOLM-14, and found a similar expression pattern (Online Supplementary Figure S1A).
We next determined the in vivo contribution of CD44 and CD49d to homing of AML cells by performing short-term adoptive transfer experiments of primary human AML cells as well as OCI-AML3 cells in immunodeficient NSG mice. In five independent experiments, total mononuclear cells from BM aspirates of five different AML patients (2 with wild-type FLT3, 3 with FLT3-ITD mutations) with a blast content of over 75% were either left untreated or treated with αCD44 Fab fragment (clone 515) or αCD49d antibody
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