Abstract 3192: Cooperation between adenosinergic and hypoxic signals in shaping chronic lymphocytic leukemia microenvironment
Authors: Serra Sara , Brusa Davide , Buonincontri Roberta , Audrito Valentina , Vaisitti Tiziana , Robson Simon , Deaglio Silvia 
Published: August 2015

Abstract
Extracellular adenosine generated from ATP/ADP through the concerted action of the ectoenzymes CD39 and CD73 elicits potent cytoprotective and immunosuppressive effects mediated by type-1 purinergic receptors.
Chronic lymphocytic leukemia (CLL) patients expressing the ectoenzymes CD39 and CD73 can actively produce adenosine. This condition activates an autocrine adenosinergic axis that supports engraftment of leukemic cells in a growth-favorable environment. These effects are mediated by the A2A adenosine receptor, which inhibits chemotaxis and limits spontaneous and drug-induced apoptosis of CLL cells.
Following the reported cross-talk between hypoxia and adenosine, we tested the hypothesis of a functional interplay between the adenosinergic axis and hypoxic signals in the CLL microenvironment.
Results confirmed that CLL cells robustly increase HIF-1α expression when cultured under hypoxia. Under these conditions a significant increase in the mRNA and protein levels of CD73, CD26 and A2A was observed. An HPLC assay confirmed that hypoxic CLL cell cultures produce higher extracellular adenosine levels compared to normoxia. The increase is apparent upon inhibition of adenosine deaminase and nucleoside transporters, suggesting that under hypoxia scavenging mechanisms are activated.
Attention was then focused on the stromal and T cell compartments, which are critical to the formation and maintenance of the leukemic niche. Hypoxia enhanced differentiation of circulating monocytes into nurse-like cells, macrophages of the M2 type playing an essential role in nurturing leukemic cells. During hypoxia, differentiated NLC overexpressed A2A, which was also functional. Indeed, an increased AKT and ERK1/2 phosphorylation was observed following pharmacological activation. The enhancement of NLC differentiation under hypoxic conditions relied, at least in part, on the activation of A2A: its engagement by the agonist favored NLC generation in normoxia, with overexpression of IDO, CD163 and CD206. Moreover, activation of A2A induced secretion of immunomodulatory cytokines, such as IL-6, IL-10 and CCL18, while pharmacological blockade of A2A under hypoxia prevented NLC growth.
In the T cell compartment, hypoxic cultures were followed by the sharp up-regulation of the A2A, without significantly affecting the enzymes that generate adenosine. Co-cultures of T lymphocytes and CLL cells under hypoxia resulted in a dramatic decrease of T cell proliferation, partially rescued by antagonists of A2A. Furthermore, expression of the PD-1 immunoinhibitory receptor was enhanced in hypoxic T cells, suggesting that there are multiple inhibitory mechanisms.
Together, these results indicate that the adenosinergic and hypoxic axes synergize in shaping the CLL niche, suggesting that the pharmacological inhibition of adenosinergic signals may counteract some of the effects mediated by an hypoxic environment.
Citation Format: Sara Serra, Davide Brusa, Roberta Buonincontri, Valentina Audrito, Tiziana Vaisitti, Simon Robson, Silvia Deaglio. Cooperation between adenosinergic and hypoxic signals in shaping chronic lymphocytic leukemia microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3192. doi:10.1158/1538-7445.AM2015-3192