CD44 engagement enhances acute myeloid leukemia cell adhesion to the bone marrow microenvironment by increasing VLA-4 avidity.

Adhesive properties of leukemia cells shape the degree of organ infiltration and the extent of leukocytosis. CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer, are important factors of progenitor cell adhesion in bone marrow (BM). Here, we report their cooperation in acute myeloid leukemia (AML) by a novel non-classical CD44-mediated way of inside-out VLA-4 activation. In primary AML BM samples from patients and the OCI-AML3 cell line, CD44 engagement by hyaluronan induced inside-out activation of VLA-4 resulting in enhanced leukemia cell adhesion on VCAM-1. This was independent from VLA-4 affinity regulation but based on ligand-induced integrin clustering on the cell surface. CD44-induced VLA-4 activation could be inhibited by the Src family kinase inhibitor PP2 and the multikinase inhibitor midostaurin. In further consequence, the increased adhesion on VCAM-1 allowed AML cells to strongly bind stromal cells. Thereby VLA-4/VCAM-1 interaction promoted activation of Akt, MAPK, NF-kB and mTOR signaling and decreased AML cell apoptosis. Collectively, our investigations provide a mechanistic description of an unusual CD44 function in regulating VLA-4 avidity in AML, supporting AML cell retention in the supportive BM microenvironment.

VLA-4 Expression and Activation in B Cell Malignancies: Functional and Clinical Aspects.

Lineage commitment and differentiation of hematopoietic cells takes place in well-defined microenvironmental surroundings. Communication with other cell types is a vital prerequisite for the normal functions of the immune system, while disturbances in this communication support the development and progression of neoplastic disease. Integrins such as the integrin very late antigen-4 (VLA-4; CD49d/CD29) control the localization of healthy as well as malignant B cells within the tissue, and thus determine the patterns of organ infiltration. Malignant B cells retain some key characteristics of their normal counterparts, with B cell receptor (BCR) signaling and integrin-mediated adhesion being essential mediators of tumor cell homing, survival and proliferation. It is thus not surprising that targeting the BCR pathway using small molecule inhibitors has proved highly effective in the treatment of B cell malignancies. Attenuation of BCR-dependent lymphoma-microenvironment interactions was, in this regard, described as a main mechanism critically contributing to the efficacy of these agents. Here, we review the contribution of VLA-4 to normal B cell differentiation on the one hand, and to the pathophysiology of B cell malignancies on the other hand. We describe its impact as a prognostic marker, its interplay with BCR signaling and its predictive role for novel BCR-targeting therapies, in chronic lymphocytic leukemia and beyond.

Rac GTPases in Hematological Malignancies.

Emerging evidence suggests that crosstalk between hematologic tumor cells and the tumor microenvironment contributes to leukemia and lymphoma cell migration, survival, and proliferation. The supportive tumor cell-microenvironment interactions and the resulting cellular processes require adaptations and modulations of the cytoskeleton. The Rac subfamily of the Rho family GTPases includes key regulators of the cytoskeleton, with essential functions in both normal and transformed leukocytes. Rac proteins function downstream of receptor tyrosine kinases, chemokine receptors, and integrins, orchestrating a multitude of signals arising from the microenvironment. As such, it is not surprising that deregulation of Rac expression and activation plays a role in the development and progression of hematological malignancies. In this review, we will give an overview of the specific contribution of the deregulation of Rac GTPases in hematologic malignancies.

The VLA-4 integrin is constitutively active in circulating chronic lymphocytic leukemia cells via BCR autonomous signaling: a novel anchor-independent mechanism exploiting soluble blood-borne ligands.

In chronic lymphocytic leukemia (CLL), survival of neoplastic cells depends on microenvironmental signals at lymphoid sites where the crosstalk between the integrin VLA-4 (CD49d/CD29), expressed in ~40% of CLL, and the B-cell receptor (BCR) occurs. Here, BCR engagement inside-out activates VLA-4, thus enhancing VLA-4-mediated adhesion of CLL cells, which in turn obtain pro-survival signals from the surrounding microenvironment. We report that the BCR is also able to effectively inside-out activate the VLA-4 integrin in circulating CD49d-expressing CLL cells through an autonomous antigen-independent BCR signaling. As a consequence, circulating CLL cells exhibiting activated VLA-4 express markers of BCR pathway activation (phospho-BTK and phospho-PLC-γ2) along with higher levels of phospho-ERK and phospho-AKT indicating parallel activation of downstream pathways. Moreover, circulating CLL cells expressing activated VLA-4 bind soluble blood-borne VCAM-1 leading to increased VLA-4-dependent actin polymerization/re-organization and ERK phosphorylation. Finally, evidence is provided that ibrutinib treatment, by affecting autonomous BCR signaling, impairs the constitutive VLA-4 activation eventually decreasing soluble VCAM-1 binding and reducing downstream ERK phosphorylation by circulating CLL cells. This study describes a novel anchor-independent mechanism occurring in circulating CLL cells involving the BCR and the VLA-4 integrin, which help to unravel the peculiar biological and clinical features of CD49d+ CLL.

Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation.

Integrin-mediated interactions between hematopoietic cells and their microenvironment are important for the development and function of immune cells. Here, the role of the integrin adaptor Kindlin-3 in B cell homeostasis is studied. Comparing the individual steps of B cell development in B cell-specific Kindlin-3 or alpha4 integrin knockout mice, we found in both conditions a phenotype of reduced late immature, mature, and recirculating B cells in the bone marrow. In the spleen, constitutive B cell-specific Kindlin-3 knockout caused a loss of marginal zone B cells and an unexpected expansion of follicular B cells. Alpha4 integrin deficiency did not induce this phenotype. In Kindlin-3 knockout B cells VLA-4 as well as LFA-1-mediated adhesion was abrogated, and short-term homing of these cells in vivo was redirected to the spleen. Upon inducible Kindlin-3 knockout, marginal zone B cells were lost due to defective retention within 2 weeks, while follicular B cell numbers were unaltered. Kindlin-3 deficient follicular B cells displayed higher IgD, CD40, CD44, CXCR5, and EBI2 levels, and elevated PI3K signaling upon CXCR5 stimulation. They also showed transcriptional signatures of spontaneous follicular B cell activation. This activation manifested in scattered germinal centers in situ, early plasmablasts differentiation, and signs of IgG class switch.

An Updated Perspective on Current Prognostic and Predictive Biomarkers in Chronic Lymphocytic Leukemia in the Context of Chemoimmunotherapy and Novel Targeted Therapy.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course. Novel biomarkers discovered over the past 20 years have revolutionized the way clinicians approach prognostication and treatment especially in the chemotherapy-free era. Herein, we review the best established prognostic and predictive biomarkers in the setting of chemoimmunotherapy (CIT) and novel targeted therapy. We propose that TP53 disruption (defined as either TP53 mutation or chromosome 17p deletion), unmutated immunoglobulin heavy chain variable region gene status (UM IGHV), NOTCH1 mutation, and CD49d expression are the strongest prognosticators of disease progression and overall survival in the field of novel biomarkers including recurrent gene mutations. We also highlight the predictive role of TP53 disruption, UM IGHV, and NOTCH1 mutation in the setting of CIT and TP53 disruption and CD49d expression in the setting of novel targeted therapy employing B-cell receptor (BCR) and B-cell lymphoma-2 (BCL2) inhibition. Finally, we discuss future directions in the field of biomarker development to identify those with relapsed/refractory disease at risk for progression despite treatment with novel therapies.

Functional and clinical relevance of VLA-4 (CD49d/CD29) in ibrutinib-treated chronic lymphocytic leukemia.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.