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Targeting A2A in CLL
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The first observation was that CD39, the enzyme that converts ATP or ADP to AMP, and CD73, the enzyme that removes the last phosphate generating adenosine, were significantly upregulated in leukemic B cells from eight different TCL1 leukemias. This was evident by flow cytometry (Figure 2A and B) and confocal microscopy (for CD73 only) (Figure 2C) performed at early and late time points, and confirmed by qRT-PCR performed on cells col- lected from spleens of terminally ill animals (Figure 2A and B). Cells from peripheral blood and bone marrow showed the same behavior (Online Supplementary Figure S1A and B). Conversely, only CD39 was increased both at the mRNA and protein levels on CD4+ and CD8+ T cells col- lected from spleen, peripheral blood and bone marrow of leukemic mice, compared to WT mice. In these cells, CD73 was transcriptionally stable although we observed slight but significant downregulation of surface protein expression (Figure 2A and B, Online Supplementary Figure S1A and B).
Upregulation of ATP-dismantling enzymes is consistent with an enhanced adenosine-producing capability of the leukemic microenvironment, which was in fact docu- mented using high-performance liquid chromatography to determine adenosine production by leukemic B cells. For
Figure 3. Upregulation of adenosine-producing capacity during leukemia pro- gression. (A) Top panels. A high-performance liquid chromatography profile of inosine (INO) and adenosine (ADO) peaks at baseline or upon AMP supply in the presence or absence of the indicated doses of the adenosine deaminase inhibitor EHNA. Bottom panels. Cumulative graphs of INO and ADO concentra- tions in the indicated experimental conditions in five independent experiments. Statistics were calculated using the Wilcoxon test for paired data. (B) Quantitative real-time polymerase chain reaction analysis of Ada expression in B cells and CD4+ or CD8+ T lymphocytes of TCL1 mice (n=20) compared to the expression of wild-type (WT) animals (n=20). Statistics were calculated with the Mann-Whitney test for unpaired data. RE: relative expression.
these studies, we used purified leukemic cells from termi- nally ill mice and incubated them with AMP, the substrate of CD73, which controls the last step in the reaction and measured adenosine and inosine accumulation. Results showed rapid and efficient consumption of AMP, with conversion to inosine (Figure 3A), the final degradation product, indicating that adenosine is metabolized by adenosine deaminase into inosine. In agreement, (i) the use of inhibitors of adenosine deaminase – erythro-9-(2- hydroxy-3-nonyl)adenine (EHNA) – prevented inosine generation, leading to the appearance of an adenosine peak following incubation with AMP (Figure 3A) and (ii) qRT-PCR data indicated increased adenosine deaminase expression by the leukemic subset (Figure 3B). These results are in line with what was observed for human CLL in which hypoxia induced accumulation of inosine by leukemic cells, suggesting that under hypoxia this cell population actively scavenges extracellular nucleotides.
Upregulation of the adenosine-sensing ability during leukemia progression
According to our working hypothesis, hypoxia would boost not only adenosine production, but also adenosine sensing in the leukemic microenvironment. To investigate
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