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Hyaluronan/CD44-mediated VLA-4 activation in AML
which is presented by stromal cells, is thereby regulated by signaling cascades inducing increased affinity due to confor- mational changes of the integrin, the so-called inside-out signaling.30 In contrast to the well-described, classical, chemokine-induced VLA-4 inside-out activation by confor- mational affinity alterations, less is known about chemokine-independent alternative integrin activation.14,31 We have previously observed that in B-cell malignancies, signals via the B-cell receptor can induce changes not only in the affinity but also the avidity of the VLA-4 receptor.11 Here, we identified an AML-specific HA/CD44-mediated VLA-4 activation via integrin cluster formation without obvious conformational modulation, further promoting strong VLA-4/VCAM-1 binding. This chemokine bypass from CD44 towards VLA-4 is somewhat reminiscent of the previously observed E-selectin/HCELL-VLA-4 interaction in mesenchymal stem cells,32 but it clearly differs in the recep- tor-ligand couple (HA-CD44) and is observed for the first time in a leukocyte. Nevertheless, the reminiscence may point to a more general mechanism and is also interesting in light of the relevant role of E-selectin in AML and other hematologic malignancies.33-38 Notably, we did not observe CD44-VLA-4 inside-out activation in normal CD34+ mobi- lized progenitors, suggesting this is a transformation-related and tumor-acquired feature for increasing integrin-mediat- ed retention of AML cells.
SFK are crucial downstream signaling molecules of CD44 in hematopoietic cells of healthy and sick individuals.39-41 They can contribute to enrichment of CD44/b1 integrin complexes in lipid rafts42 and also function immediately downstream of integrins, in concert with focal adhesion kinases.43 Employing the SFK inhibitor PP2, we identified SFK within the CD44-mediated VLA-4 inside-out activa- tion cascade, and propose that a further stabilization of
VLA-4 clusters involves Src-FAK signaling. This could have therapeutic relevance as PP2 administration was reported to attenuate progression of a FLT3-mutated AML model.44 However, in the light of the complexity of the Src kinase family, the particular kinase responsible for the HA/CD44- mediated inside-out signaling needs to be elucidated by a genetic screening approach.
We investigated the therapeutically relevant drug midostaurin, which is approved for treatment of FLT3- mutant AML,45 and currently under clinical investigations in non-FLT3-mutated AML cases (NCT03512197). Midostaurin is a broad multikinase inhibitor, and more selective inhibitors, such as gilteritinib and quizartinib for FLT3 and dasatinib for Src kinases, recently entered the clin- ical stage.46-48 Notably, kinase signals via FLT3-ITD can increase the affinity of VLA-4 to soluble VCAM-1.49 Thus, the here reported CD44-dependent mechanism of VLA-4 activation may be an alternative pathway used by FLT3 wild-type AML cells to increase their adhesion to protective stromal cells. As FLT3 mutation status is an important prog- nostic marker in AML, it is interesting that the observed CD44/VLA-4 crosstalk was independent from the patients’ FLT3 mutation status, suggesting involvement of alternative compensatory kinases.
Further downstream of SFK, we supposed PI3K to medi- ate signaling50-52 and indeed observed diminished HA- induced CD49d cluster formation in the presence of the PI3Kδ inhibitor idelalisib. PI3K has been described to pro- mote human AML survival and BM stromal cell-mediated protection,53 giving a rationale to investigate PI3K inhibitors further in AML. As another functional consequence, the VLA-4-mediated AML adhesion triggered activation of Akt, MAPK, mTOR and NF-kB pathways, which are known important mediators of AML survival.53,54 However, in a first
Figure 7. Schematic overview of the suggested CD44-VLA-4 activation axis and downstream consequences. Binding of acute myeloid leukemia (AML) cells to the bone marrow (BM) stromal component hyaluronic acid (HA) is dependent on CD44 and enhances adhesion of AML cells to the VLA-4 substrate VCAM-1, a second important adhesion factor displayed on stromal cells. Mechanistically, this enhanced adhesion is based on inside-out activation of VLA-4, without altering the con- formation of the integrin. The signaling downstream of CD44 involves several kinases (e.g. Src family kinases [SFK]) causing clustering of the integrin, thereby sta- bilizing adhesion strength that facilitates direct interaction with stromal cells. This AML cell-stromal cell interaction leads to survival signaling involving activation of the Akt, MAPK and NF-κB pathways.
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