Adenosine pathway as a therapeutic target in diffuse large B cell lymphoma Free

Purine metabolites have emerged as important mediators of B cell function and tumor immunity. However, their effect on B-cell lymphomagenesis and lymphoma microenvironment (LME) has not been characterized. In stressful environments such as cancer, high extracellular ATP (eATP) concentrations are generated. Then, eATP is hydrolyzed to ADP and AMP by the ectonucleotidase diphosphohydrolase (CD39) and subsequently converted to extracellular adenosine (eADO) by the ecto-5´-nucleotidase (CD73). In solid tumors, eADO has been shown to bind the A2AR receptors in T cells, NK cells, macrophages and DC cells causing an inhibitory effect on these cells. Importantly, eADO also exerts an inhibitory function in B cells through its binding to its primary receptor, A2AR. Thus, the immunosuppressive function of eADO in solid tumors has led to the emergence of purinome inhibitors as a therapeutic strategy, but their potential for lymphoma treatment remains to be determined.

To examine the expression of A2AR, CD39 and CD73 in DLBCL, we used RNA sequencing, immunohistochemistry (IHC) staining and immunoblotting in DLBCL patient samples and cell lines. RNA seq revealed higher expression of CD39 in ABC-DLBCL (N=58) than in GC-DLBCL (N=35). A2AR was similar in all cases and there was a lower and variable expression of CD73. By IHC, CD39 staining was positive in all 10 cases, while 5/10 (50%) cases were positive for CD73. Among the CD73 positive cases, 2/5(40%) express CD73 in the membrane of tumor cells while 3/5 (60%) express CD73 in the endothelial cells or other cells in the LME. We then evaluated a DLBCL cell line panel that included GC (N=4) and ABC-DLBCLs (N=3), only the latter express CD39 (Ly3, HBL-1 and Ly10). In contrast, none of them expressed CD73.

To assess expression of functional A2AR in DLBCL, we did functional testing in the cell line panel. Short stimulation with A2AR agonist (CGS-21680) causes an increase in intracellular cAMP in some cell lines (HBL-1, Ly7, Ly1) but not in others (Ly19, Ly10). This increase in cAMP can be reverted with A2AR antagonists (ciforadenant and istradefylline). To explore downstream signaling we performed phosphokinase arrays in Ly1 and HBL-1 cell lines. We found an increase in phospho-CREB at Ser133 (2.6-fold), phospho-p53 at Ser15 (1.6-fold) and phospho-AMPK at Thr172 (3-fold) in comparison to vehicle-treated cells. Viability assays showed that in contrast to normal B cells, DLBCL cells are resistant to inhibition of survival by ADO signaling through A2AR. There was also no consequence of ADO agonist stimulation in B cell plasmacytic differentiation based on FACs for IgM, CD38, CD138, HLA-ABC, HLA-DR.

To evaluate the tumor-intrinsic and immune mediated effects of adensoine signaling in vivo, we used adoptive transfer of tumors from H1c^+/-e^+/-;VavP-Bcl2 mice, reflecting a subtype of MCD DLBCL with linker histone alterations and BCL2 overexpression. These tumors cells were able to engraft in both immunodeficient (NOD-SCID) and syngeneic immunocompetent (B6) mice, and they express A2AR, CD39 and CD73. NOD-SCID (N=9) and B6 mice (N=15) were subcutaneously implanted with 2x10e6 cells in both flanks. Five days post-transplantation, mice were randomized based on their tumor size into treatment groups: vehicle, 10 mg/kg and 50 mg/kg. The A2AR inhibitor ciforadenant was administered by daily oral gavage. In NOD-SCID mice, tumor size measurements at day 6, revealed no significant difference in treated vs. vehicle groups. Moreover, the time to death of the vehicle group was very similar to the 10 mg/kg and slightly higher than 50 mg/kg (Log rank (Mantel-Cox) test p=0.28). In contrast, B6 mice showed tumor responses to ciforadenant, showing no increase in tumor size in the 10 mg/kg arm (day 0 vs day 8 p= 0.97; day 0 vs day 14 p=0.26) when compared to tumor growth in the vehicle arm (day 0 vs day 8 p=0.0008; day 0 vs day 14 p=0.0062). Also, no significant tumor increase was observed in the 50 mg/kg arm (day 0 vs day 8 p>0.99; day 0 vs day 14 p=0.42). Ciforadenant at both doses increased survival in B6 but not in NOD-SCID mice, with time of death considered when tumors reached 2 cm and mice were euthanized (Log rank (Mantel-Cox) test p=0.019). Taken together our results demonstrate that DLBCL cells are resistant to the inhibitory effects of eADO seen in normal B cells, and that A2AR antagonists are effective for the treatment DLBCL via an immune mediated mechanism in a preclinical model of DLBCL.