Blocking CD70/CD27 Signaling in Combination with Hypomethylating Agents Eradicates Human CD34⁺ AML Stem and Progenitor Cells in Vitro and In Vivo

Leukemia stem cells (LSCs) are the origin of acute myeloid leukemia (AML) and are resistant to standard therapeutic regimens resulting in frequent relapse of the disease and poor prognosis especially for AML patients not fit for intensive chemotherapy. Consequently, LSCs represent a major obstacle for AML therapy. We recently identified the interaction of the TNF ligand CD70 and its receptor CD27 on LSCs as a promising therapeutic strategy to target LSCs.

In this study, we demonstrate for the first time that treatment with hypomethylating agents (HMA, azacitidine or decitabine) significantly up-regulates CD70 mRNA and protein expression on primary CD34⁺ stem/progenitor cells from newly diagnosed AML patients independent of the cytogenetic/molecular risk category in vitro (mRNA: 4.3±1.6 fold increase, FACS: 4.0±0.9 fold increase vs. vehicle treatment, n=15). Similarly, CD34⁺ stem/progenitor cells from AML patients after 5 days decitabine administration (20mg/kg by continuous intravenous infusion over 1 hour repeated daily) exhibited a significant increase in CD70 expression as analyzed by flow-cytometry (6.8±1.1 fold increase vs. diagnostic sample, n=6). In contrast, CD70 expression of CD34⁺ stem/progenitor cells from healthy donor bone marrow (n=3) was not affected by the treatment.

Consequently, co-treatment of CD34⁺ stem/progenitor cells from AML patients of each risk group (n=3) with the HMA decitabine and a blocking αCD70 monoclonal antibody strongly reduced colony-forming and re-plating capacity in vitro compared to single agent treatment. To confirm and extend our findings towards disease-initiating CD34⁺ AML LSCs in vivo, we performed patient-derived AML xenografts (PDX) from one patient with intermediate and one patient with adverse cytogenetic risk profile. Combining decitabine treatment with CD70 blockade significantly reduced AML engraftment in blood, spleen and BM compared to controls. Moreover, co-treatment significantly reduced frequency and absolute numbers of CD34⁺CD38^- and CD34⁺CD38^-CD45RA^- AML LSCs in the BM as analyzed phenotypically by FACS and eliminated human LSCs as analyzed in limiting dilution PDX experiments compared to single treatments. Importantly, CD34⁺ hematopoietic stem/progenitor cells from healthy human bone marrow (n=3) were only marginally affected by the co-treatment.

In summary, combining HMAs with blocking CD70/CD27 signaling could represent a novel strategy to eradicate human LSCs.