CD39, CD73 and the Adenosine Pathway Mediate Immune Escape in Juvenile Myelomonocytic Leukemia (JMML)

Introduction: Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of early childhood, driven by somatic and/or germline mutations in the genes PTPN11, KRAS, NRAS, NF1 or CBL. Patients with JMML can be cured through allogeneic HSCT; however, the risk of relapse is high, particularly when a mutation in the PTPN11 gene (which codes for the SHP2 tyrosine phosphatase) is present, as this mutation is often linked to a high-risk form of the disease. Relapse risk is higher in the absence of chronic GvHD indicating that immune cells are crucial to prevent leukemia relapse. We suggest that immune escape of JMML cells contributes to both, leukemia emergence and relapse.

Aims: We aim to investigate the mechanisms by which JMML cells protect themselves and escape immune recognition. Further, we test the ability of targeting these mechanisms to initiate immune response.

Methods: We used two model systems: (i) human naïve JMML cells with PTPN11 mutation harvested from bone marrow (BM) or spleen and (ii) genetically modified mice expressing Ptpn11^D61Y in their hematopoietic system. Total mRNA isolated from splenic bulk JMML cells was analyzed for different immune checkpoints (ICPs). ICPs were analyzed on a single-cell and protein level by mass cytometry in human JMML patients and by flow cytometry in MxCre;Ptpn11^D61Y/+ mice. We investigated the link between oncogenic SHP2 signaling and ICP expression in leukemic cells by treating cells with granulocytes-macrophage colony-stimulating factor (GM-CSF) and/or inhibitors of MEK and PI3K for 24 hours. Functional effects of leukemic cells on T cell activation and proliferation were tested in vitro by co-culture of PTPN11-mutated myeloid cells and CD3/28 stimulated wildtype (WT) T cells, both in the presence and absence of checkpoint inhibitors. Checkpoint inhibition was also applied in MxCre;Ptpn11^D61Y/+ mice. In vivo treatment was preceded by depletion of PTPN11-mutated T cells and transfer of syngeneic WT T cells.

Results: We detected high mRNA levels of genes encoding for CD39/ENTPD1, CD73/NT5E in human splenic JMML cells. CyTOF analysis indicated increased expression of CD39 on PTPN11 JMML cells when compared to spleens of control patients.

Significantly increased protein levels of CD39, CD73, two ectonucleotidases relevant in hydrolyzing ATP to adenosine were found in murine monocytic and granulocytic cells isolated from spleens but not BM of MxCre;Ptpn11^D61Y/+ mice. In vitro expression of CD73 but not CD39 was increased in mouse leukemic monocytic and granulocytic cells upon GM-CSF treatment and reduced upon inhibition of both MEK and PI3K. When WT T cells stimulated with CD3/28 beads were co-cultured with myeloid cells isolated from leukemic mice, an almost complete repression of T cell proliferation and activation was observed. This effect was reproduced by exposure of T cells with adenosine. The inhibitory effect on T cells could be fully reversed by addition of CD39 inhibitor POM-1 (50 µM concentration) to the co-culture. POM-1 treatment resulted in significantly upregulated ATP levels in the supernatant of JMML cells indicating reduced hydrolysis. MxCre;Ptpn11^D61Y/+ mice presenting with myeloproliferation were subjected to T cell depletion and transfer of WT T cells and subsequently treated with POM-1 (25 mg/kg) for 14 days (7 doses). In a preliminary set of experiments we observed a trend towards reduced spleen size after POM-1 treatment.

Conclusions: We provide evidence for an immunosuppressive role of the adenosine pathway in JMML. CD39 and CD73 are expressed on human JMML cells and on the respective murine cells. CD73 appears to be directly regulated by the oncogenic SHP2 signaling. Adenosine produced by JMML cells inhibits T cell function mediating tumor evasion. Enzymatic inhibition of CD39 on JMML cells restored T cell proliferation and activation in vitro and reduced leukemia burden in vivo. Our data indicate the potential use of CD39 inhibitors to increase the anti-leukemia activity of T lymphocytes, both before HSCT and to prevent relapse.