CD73-Generated Extracellular Adenosine Creates Microenvironmental Conditions Favoring Growth and Survival of Chronic Lymphocytic Leukemia Cells

Abstract 621

CD39 (ecto-nucleoside-triphosphate-diphosphohydrolase-1) and CD73 (5'-nucleotidase) are surface enzymes with extracellular catalytic sites. CD39 hydrolyses ATP/ADP to AMP, which is then converted to adenosine (ADO) by CD73. Once ADO is released in the extracellular milieu, it may re-enter the cell or engage different types of purinergic receptors, eliciting potent autocrine and paracrine cytoprotective, anti-inflammatory and immunosuppressive effects. Several lines of evidence suggest that the tumor microenvironment is marked by increased turnover of extracellular nucleotides and nucleosides, as well as by upregulation of ecto-enzymes that dismantle them. These alterations are believed to contribute to increasing local concentrations of ADO, creating local conditions that favor tumor expansion. We have investigated expression of CD39/CD73 by chronic lymphocytic leukemia (CLL) cells and evaluated the functional significance of the autocrine ADO production in supporting the survival of the leukemic cells and their expansion.

Immunohistochemical analyses of lymph node biopsies indicated that CD39 was widely expressed by neoplastic cells, as well as by surrounding non-neoplastic stroma. On the contrary, CD73 was largely restricted to CLL proliferation centers and to cells with a prolymphocyte / para-immunoblast morphology, suggesting that ADO generation is confined to specific tissue niches and to specific cell subpopulations. The highest intensity of expression was scored by Ki-67⁺ CLL cells, in close association with T lymphocytes and localized to perivascular areas, where recirculation to and from lymphoid organs occurs.

When analyzed in the peripheral blood, CD39 was constantly expressed, while CD73 was present in approximately 30% of cases and associated with markers of aggressive disease. CD39⁺/CD73⁺ CLL cells could selectively produce ADO from ADP, in a time- and concentration-dependent way. RT-PCR data indicated that CLL express high levels of A2A ADO receptor, further elevated in proliferating leukemic cells. Ligation of the A2A receptor by extracellular ADO or by the selective agonist CGS21680 increased cytoplasmic cAMP levels. The functional consequence was the inhibition of CXCL12-mediated chemotaxis, suggesting that the adenosinergic axis contributes to keeping CLL cells in a growth-favorable environment where they were attracted by CXCL12. Furthermore, activation of the axis conferred significant protection from spontaneous and/or fludarabine-induced apoptosis. The contribution of CD73 in regulating ADO production was confirmed by using a selective enzyme inhibitor.

These results are consistent with the existence of an adenosinergic loop, active in selected CLL patients, creating conditions in the lymph node microenvironment that favor the development, maintenance and progression of the leukemia.