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J.C. Gutjahr et al.
treatment with αCD44 blocking antibody (clone 515) (Figure 2A). We further confirmed this HA-binding capacity by flow cytometry using fluorescein-labeled HA (HA-FITC) (Online Supplementary Figure S3A). Next, we tested whether AML cells were capable of tethering to the VLA-4 ligand VCAM-1 under shear flow conditions. Unstimulated OCI- AML3 and primary AML cells from five different patients bound immobilized VCAM-1 at low adhesive strength, which was evident by the low frequency of firm adhesion on this substrate (Figure 2Bi+ii). Treatment with a blocking αCD49d antibody abrogated all interactions of the OCI- AML3 and primary cells with the immobilized VCAM-1, confirming the VLA-4 dependency of this process (Figure 2Bi, Online Supplementary Figure S3B). Notably, pre-treating AML cells with the CD44 ligand HA and then perfusing the cells over a VCAM-1 substrate increased adhesion, without changing CD44 or CD49d surface expression (Figure 2Bi+ii, Online Supplementary Figure S3C), which suggests HA/CD44-induced inside-out VLA-4 activation. Co-immo- bilization of both ligands HA and VCAM-1 resulted in strong CD49d-dependent adhesive capacity of the leukemia cells, suggesting inside-out activation rather than mere additive effects in adhesion (Figure 2C). CD44-medi- ated inside-out signaling is well known to vary depending on the molecular weight of the HA trigger.16 Indeed, CD44- induced VLA-4 activation was only achieved by high molecular weight HA, but not by low molecular weight HA (Figure 2D). Using shCD44-transduced OCI-AML3 cells confirmed that the increased VLA-4/VCAM-1 binding upon HA treatment is CD44-dependent (Figure 2E). These data indicated an unusual integrin activation, different from the well-described, classical CXCL12/CXCR4-induced VLA-4 activation,17 which led us to investigate the nature of this molecular crosstalk .
Hyaluronic acid-induced inside-out signaling to VLA-4 results in CD49d cluster formation but not in VLA-4 affinity modulation
VLA-4-dependent adhesion is controlled by either affini- ty changes, gained by several conformational states of the integrin,18 or avidity changes due to clustering of the mole- cule on the cell surface19 (Online Supplementary Figure S4A). To investigate the alterations in VLA-4 conformational states upon HA treatment, we used the αCD29 antibody (clone HUTS-21) that binds solely to the ligand-occupied state of VLA-4.20 To mimic VLA-4 ligand binding, we used a probe containing the conserved Leu-Asp-Val (LDV) sequence, specific for the VLA-4 binding site. Manganese was used as a positive control as it induces the maximal extent of VLA-4 activation, which is not achieved under physiological conditions.20 Surprisingly, OCI-AML3 cells expressed VLA-4 in an inactive conformation irrespectively of whether the cells were treated with HA or not and bound its ligand with comparable affinity (Online Supplementary Figure S4B).15,20 This unexpected finding prompted us to elucidate whether the HA-induced AML cell arrests on the substrate are based on increased avidity rather than affinity of VLA-4 to VCAM-1. We performed immunofluorescence microscopy and found increased CD49d cluster formation on AML cells upon treatment with HA. This was quantified by counting the number of the clusters on the individual OCI-AML3 and patient AML cells (1 representative of 6 patients shown) (Figure 3Ai+ii). The mean number of clusters per cell was compared between untreated and HA-treated cells from all six
patients (Figure 3Aiii). HA treatment also induced clustering of the VLA-4 b subunit CD29 (Online Supplementary Figure S4C). Pretreatment with blocking αCD44 (clone 515) inhib- ited cluster formation on OCI-AML3 as well as primary AML cells (Figure 3B). Using CD44 knockdown and control transduced OCI-AML3 cells, we confirmed that HA- induced CD49d clustering only occurred in cells that expressed CD44 (Figure 3C). This cluster formation trans- lated into enhanced adhesive capacity, as we confirmed in an additional static cell adhesion assay, using an alternative colorimetric method for cell counting (Online Supplementary Figure S4D). We also performed an avidity-detecting shear flow assay, as described by Alon et al.,21 by perfusing OCI- AML3 cells and primary cells over an αCD49d (clone HP2/1) substrate, further confirming our observations (Figure 3D, Online Supplementary Figure S4E). To get an insight into the lateral organization of the VLA-4 clusters on the membrane, we used methyl-beta-cyclodextrin (MßcD), which interferes with lipid structures. Although MßcD did not significantly reduce the number of HA-induced VLA-4 clusters, it abrogated their function to support cell tethering to VCAM-1 (Online Supplementary Figure S4F).
Next, we studied whether CD44-mediated inside-out activation is a general mechanism that also occurs in non- transformed progenitor cells. Using CD34+ cells from four different patients harboring a non-myeloid, i.e. lymphoid malignancy occurring at later differentiation states (non- Hodgkin-lymphoma [n=3] and multiple myeloma [n=1]), we did not find HA-induced CD49d clustering, suggesting that induced cluster formation is a specific feature of trans- formed myeloid progenitor cells (Figure 3E).
Transformed myeloid progenitors may differ in their CD44variant (CD44v) composition, with an impact on the clinical outcome of AML patients.22 We have analyzed the CD44v composition of several cell lines as well as primary AML samples and normal CD34+ cells by reverse transcrip- tion PCR and found differences in the length of CD44v6 containing transcripts among the different primary samples (Online Supplementary Figure S5A). Interestingly, in OCI- AML3 CD44v6 cells, co-immunoprecipitated with CD49d, with a slight pull down increase when cells were preincu- bated with HA (Online Supplementary Figure S5B). In conclu- sion, our data demonstrate that HA/CD44 binding induces CD49d cluster formation in AML, but not normal CD34+ progenitor cells, without changing the conformation of the VLA-4 heterodimer.
Src family kinase inhibition and midostaurin treatment interfere with the CD44-VLA-4 activation axis
Src family kinases (SFK) are important downstream mol- ecules of HA/CD4423 and likely candidates for integrin acti- vation.24 To confirm Src and PI3K activation upon HA treat- ment, we analyzed Src and Akt phosphorylation by west- ern blot in native, control and shCD44-transduced OCI- AML3 cells (Online Supplementary Figure S6A). Remarkably, treating cells with the pan-SFK inhibitor PP2 abrogated the formation of HA-induced CD49d clusters on the surface of OCI-AML3 cells (Figure 4Ai) and six different AML patients’ samples (Figure 4Aii + Ci), providing evidence that CD49d clustering was Src family-dependent. To start from broad but therapeutically relevant kinase inhibition, we used the multikinase inhibitor midostaurin, approved for the treatment of FLT3-mutated AML. We found that midostaurin is highly potent in antagonizing HA-induced CD49d cluster formation of OCI-AML3 cells (Figure 4Bi)
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