Contributions
Abstract: PB1657
Type: Publication Only
Background
The bone marrow niche is critical for maintenance and retention of normal and malignant stem and progenitor cells. Key factors of this process are the adhesion molecule CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer. CD44 comprises a glycoprotein family, which is tightly regulated by alternative splicing of up to 10 exons, thereby influencing the binding capacity of the ligands, particularly hyaluronan. In AML, CD44 has early been suggested as a marker for poor prognosis and leukemia initiating cells but how CD44 standard and variant forms interact with other adhesion molecules and shape the tumor environment is still not understood well.
Aims
Here we aimed to identify the functional interplay between the homing receptors CD44 and VLA-4 in AML primary patient samples as well as AML cell lines and elucidate the influence of therapeutic kinase inhibitors on the signaling cascades involved in the CD44/VLA-4 interaction, which may influence drug resistance and AML engraftment.
Methods
We employed flow cytometry for surface phenotyping of CD44, CD44v6, and CD49d on primary patient samples, and several AML cell lines. Composition of all CD44 variants was further screened by reverse transcription-PCR. Hyaluronan binding capacity was determined by flow cytometric assays and videomicroscopic adhesion assays under shear flow. VLA-4 binding capacity was determined in adhesion assays under shear flow on VCAM-1 substrates. VLA-4 activation was further studied by cytometrical assays determining integrin affinity in real-time and integrin clustering was analyzed by immunofluorescence and quantified using a confocal microscope. Signaling cascades were approached by using a range of src kinase, CD45, PI3K and other kinase inhibitors, among them midostaurin. Short-term adoptive transfer experiments of AML cells in immunodeficient donor mice were used to investigate the individual as well as synergistic role of CD44 and VLA-4.
Results
We demonstrate that CD44, CD44v6 and CD49d are expressed on primary AML cells and on AML cell lines OCI-AML3 and KG-1a at comparable amounts. Hyaluronan binding of AML cells was constitutive, suggesting an activated phenotype, dependent on functional CD44, which correlates with CD44v6 expression. Stimulation of CD44 via hyaluronan induced VLA-4 activation in a specific manner involving CD45, Src kinases, PI3-kinase, and could also be inhibited by midostaurin. CD44-induced inside-out activation of VLA-4 resulted in enhanced adhesion of AML cells on VCAM-1, which was mechanistically based on integrin clustering but not affinity regulation. Pathophysiological consequences are currently investigated using murine models engrafted with primary AML samples.
Conclusion
Based on our findings we propose that CD44-hyaluronan binding interactions promote clustering of VLA-4, which increases its adhesiveness to VCAM-1, resulting in supportive bone marrow lodgment of leukemia cells. Collectively, our investigations provide a mechanistical description of a novel CD44 function in acute myeloid leukemia, i.e. activation of VLA-4, required for leukemia cell recirculation and engraftment in the bone marrow environment.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, CD44, Integrin activation, Microenvironment
Abstract: PB1657
Type: Publication Only
Background
The bone marrow niche is critical for maintenance and retention of normal and malignant stem and progenitor cells. Key factors of this process are the adhesion molecule CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer. CD44 comprises a glycoprotein family, which is tightly regulated by alternative splicing of up to 10 exons, thereby influencing the binding capacity of the ligands, particularly hyaluronan. In AML, CD44 has early been suggested as a marker for poor prognosis and leukemia initiating cells but how CD44 standard and variant forms interact with other adhesion molecules and shape the tumor environment is still not understood well.
Aims
Here we aimed to identify the functional interplay between the homing receptors CD44 and VLA-4 in AML primary patient samples as well as AML cell lines and elucidate the influence of therapeutic kinase inhibitors on the signaling cascades involved in the CD44/VLA-4 interaction, which may influence drug resistance and AML engraftment.
Methods
We employed flow cytometry for surface phenotyping of CD44, CD44v6, and CD49d on primary patient samples, and several AML cell lines. Composition of all CD44 variants was further screened by reverse transcription-PCR. Hyaluronan binding capacity was determined by flow cytometric assays and videomicroscopic adhesion assays under shear flow. VLA-4 binding capacity was determined in adhesion assays under shear flow on VCAM-1 substrates. VLA-4 activation was further studied by cytometrical assays determining integrin affinity in real-time and integrin clustering was analyzed by immunofluorescence and quantified using a confocal microscope. Signaling cascades were approached by using a range of src kinase, CD45, PI3K and other kinase inhibitors, among them midostaurin. Short-term adoptive transfer experiments of AML cells in immunodeficient donor mice were used to investigate the individual as well as synergistic role of CD44 and VLA-4.
Results
We demonstrate that CD44, CD44v6 and CD49d are expressed on primary AML cells and on AML cell lines OCI-AML3 and KG-1a at comparable amounts. Hyaluronan binding of AML cells was constitutive, suggesting an activated phenotype, dependent on functional CD44, which correlates with CD44v6 expression. Stimulation of CD44 via hyaluronan induced VLA-4 activation in a specific manner involving CD45, Src kinases, PI3-kinase, and could also be inhibited by midostaurin. CD44-induced inside-out activation of VLA-4 resulted in enhanced adhesion of AML cells on VCAM-1, which was mechanistically based on integrin clustering but not affinity regulation. Pathophysiological consequences are currently investigated using murine models engrafted with primary AML samples.
Conclusion
Based on our findings we propose that CD44-hyaluronan binding interactions promote clustering of VLA-4, which increases its adhesiveness to VCAM-1, resulting in supportive bone marrow lodgment of leukemia cells. Collectively, our investigations provide a mechanistical description of a novel CD44 function in acute myeloid leukemia, i.e. activation of VLA-4, required for leukemia cell recirculation and engraftment in the bone marrow environment.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, CD44, Integrin activation, Microenvironment