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F. Arruga et al.
events are time-dependent and highly reproducible, they are sensitive tools for studying immune adaptation.
This work shows that leukemia progression is accompa- nied by progressive tissue hypoxia, which in turn power- fully modulates the adenosinergic system. Modulation of the HIF-1α pathway was documented both by increased expression of the carbonic anhydrase IX protein, which is under its direct transcriptional control, and by increased staining with PIM, which binds macromolecules specifical- ly under low oxygen tension. We previously demonstrated that, under hypoxia, CLL cells undergo HIF-1α-driven reprogramming, which favors metabolic adaptation and blunting of the immune response, partly through the upregulation of the adenosinergic axis.14
Leukemia progression is also accompanied by increased expression of the enzymes that are responsible for adeno- sine conversion from ATP or ADP in the extracellular space. CD39, which is also considered a marker of memo- ry B cells, becomes expressed by the great majority of leukemic cells. The same trend is observed for CD73, which is the rate-limiting enzyme in the pathway. Immunohistochemical analysis also confirmed a general upregulation of CD73 expression with the progression of leukemia.
Extracellular adenosine can, therefore, exert both autocrine and paracrine effects, similarly to what was described in human CLL.19 On the one hand, the observa- tion that murine leukemic cells also strongly upregulate adenosine deaminase suggests that adenosine is in part recycled via conversion to inosine. On the other hand, more importantly, we found an overall increase of adenosine receptor expression and signaling in the spleens of terminal- ly ill mice, with A2A being the most expressed receptor. This was documented at the mRNA level both on leukemic B cells and on CD4+ and CD8+ T lymphocytes purified from TCL1 mice and compared to those from WT C57BL/6 mice, in line with the view that the adenosinergic axis is part of a hypoxia-driven transcriptional reprogramming. Consistently, spleen sections of leukemic mice stained much more strongly positive for A2A compared to WT spleen. The finding that A2A signaling, via PKA activation and CREB phosphorylation, was strongly increased in B and T cells from terminally ill mice, compared to control animals, argues in favor of an adenosine-mediated circuit, activated by HIF-1α, responsible, at least in part, for immunosuppression. In fact, in human B cells, PKA-mediat- ed CREB activation induces interleukin-10 production and secretion, contributing to an anti-inflammatory immune- tolerant environment and directly linking A2A signaling to immunomodulation.14,18 Consistent with this view, and with previous literature, we observed a progressive dysregula- tion of the immune system in leukemic mice, affecting T- cell functions and monocyte polarization. Restoring immune competence represents a major achievement in CLL management and here we explored the effects of tar- geting the adenosinergic axis and its potential application in a translational perspective. Importantly, we show that interfering with adenosine signaling, using a specific A2A inhibitor, significantly rescues CD8+ T-cell responses and functions while preventing the expansion of immunomod- ulatory subsets such as Treg and patrolling monocytes.
The establishment of a progressively hypoxic environ- ment promotes metabolic adaptation and reprogramming in CLL, contributing also to enhance leukemic cell aggres- siveness and to inducing drug resistance.33 Adenosine sig- naling mediates part of these effects, favoring metabolic switching of CLL cells and conferring them cytoprotection and immune regulatory functions.14 Nevertheless, our results also indicated that SCH58261 treatment in vivo had little or no effect on leukemic cells, suggesting that paracrine, rather than autocrine, effects of extracellular adenosine play an important role in disease progression, at least in this experimental setting (Figure 7), arguing in favor of using A2A inhibitors as part of combination strategies. Novel, more specific and efficient A2A inhibitors are cur- rently under development, and future studies are coming to validate their effects both as single agents and in combi- nation.
The lesson from data on solid tumors,34 and further sub- stantiated by our results, is that targeting the adenosinergic axis could be a promising strategy to re-awake the immune system and restore immune-cell functions. Applied to the field of CLL, these data might be a starting point to design and develop more effective therapies. Different possible scenarios can be envisaged: one option could be to com- bine inhibitors of the adenosinergic cascade together with drugs directly targeting central pathways of the CLL cell. Alternatively, targeting adenosine signaling may represent an adjunctive tool to potentiate chimeric antigen receptor T-cell activity against CLL cells, also considering the prom- ising results obtained with this novel therapeutic approach.35 Further studies will tell which of these avenues will be more promising.
Disclosures
No conflicts of interest to disclose.
Contributions
FA designed the study, performed experiments, analyzed and interpreted data and together with TV and SD wrote the paper; SS and AP designed the study and performed experiments; NV, GG, BBG and FT performed experiments; DE provided vital reagents; TV and SD designed the study, analyzed and interpret- ed data and wrote the paper.
Acknowledgments
We thank Dr. Denis Baev for excellent support with the design of the flow cytometry panel and with the data analysis.
Funding
This work was supported by Italian Institute for Genomic Medicine institutional funds, by the Associazione Italiana per la Ricerca sul Cancro AIRC (IG17314 to SD), by the Gilead Fellowship Program 2018 (by the ITN INTEGRATA program, grant agreement 813284 to SD), by the Italian Ministry of Health (GR-2016-02364298 to TV), by the Ministry of Education, University and Research-MIUR “Progetto Strategico di Eccellenza Dipartimentale” (#D15D18000410001 to SD in part through funds to the Department of Medical Sciences, University of Turin); and by the Ministry of Education, University and Research-MIUR PRIN project (2017CBNCYT to SD).
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