Page 275 - 2021_02-Haematologica-web

Letters to the Editor
Daratumumab inhibits acute myeloid leukemia metabolic capacity by blocking mitochondrial transfer from mesenchymal stromal cells
Acute myeloid leukemia (AML) proliferation is depend- ent on a complex multi-faceted interplay between the blasts and the bone marrow (BM) microenvironment.1 We and others have previously demonstrated that func- tional mitochondria are transferred from the mesenchy- mal stem cells (MSC) to the AML blasts facilitating pro- gression of the disease,2,3 in a process which is hijacked from hematopoietic stem cell response to infection.4 Clinical observation trials using venetoclax, which tar- gets BCL2 (which in turn is a key regulator of the mito- chondrial apoptotic pathway), in combination with hypomethylating agents showed tolerable safety and favorable overall response rate in elderly patients with AML.5 Taken together, these data imply that mitochon- dria represent an attractive and biologically plausible drug target in the treatment of AML.
CD38 is a transmembrane glycoprotein which is expressed on many cells of the hematopoietic system including malignant plasma cells, red blood cells, myeloid cells, lymphoid cells and subsets of leukemia-initiating AML blasts.6-8 Furthermore, we have recently shown that
CD38 mediates pro-tumoral mitochondrial transfer from MSC to malignant plasma cells in myeloma,9 which in part explains why daratumumab (an anti-CD38 mono- clonal antibody) is clinically effective in treating patients with myeloma.10 More recently, daratumumab has been shown to have preclinical activity in AML.11 Therefore, taken together, we hypothesize that daratumumab treat- ment would impair AML metabolic capacity and conse- quently inhibit tumor proliferation, via a mechanism which blocks mitochondrial transfer from BMSC to the blasts.
Initially, to determine if CD38 inhibition blocks mito- chondrial transfer from MSC to AML blasts, we used an in vitro co-culture system. MitoTracker Green FM stain (MTG) was used to quantify mitochondria in AML after co-culture with MSC. We incubated both MSC and AML with MTG. The cells were washed twice in phosphate buffered saline (PBS) and incubated for 4 hours (h). The cells were then co-cultured for 24 h with and without daratumumab. Using flow cytometry, AML was shown to have less MTG fluorescence when treated with dara- tumumab (Figure 1A-C). Figure 1D shows the presence of mouse mtDNA in human AML after co-culture with mouse MSC, and the transfer of mouse mtDNA to human AML was inhibited by the addition of daratu-
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Figure 1. CD38 inhibition blocks mitochondrial transfer from mesenchymal stem cells (MSC) to acute myeloid leukemia (AML) blasts. (A) Primary AML and MSC were pre-stained with MitoTracker green (MTG) fluorescent mitochondrial dye (FM) for 1 hour (h) and then cultured alone or together for 24 h. Flow cytom- etry was used to detect MTG FM in the AML blasts (n=8). (B) Primary AML and MSC were pre-stained with MTG FM for 1 h and then cultured together for 24 h in the presence of vehicle or daratumumab (Dara) (100 ng/mL). Flow cytometry was used to detect MTG FM in the AML blasts (n=6). (C) Primary AML were pre- stained with MTG FM for 1 h and then treated with vehicle or daratumumab for 24 h (100 ng/mL). Flow cytometry was used to detect MTG FM in the AML blasts (n=5). (D) Human AML was cultured with mouse MSC in the presence of vehicle or daratumumab for 24 h (100 ng/mL) for 24 h. AML were isolated and meas- ured for mouse mitochondrial DNA content. Primary AML were transduced with CD38 targeted shRNA for 48 h. (E) RNA was then extracted and examined for CD38 mRNA expression. (F) AML and MSC were then pre-stained with MTG FM for 1 h and then cultured together for 24 h. Flow cytometry was used to detect MTG FM in the AML blasts (n=4). We used the Mann-Whitney U test and Wilcoxon matched pairs signal rank test to compare results between groups. MFI: mean fluorescence intensity. *P<0.05; **P<0.01.
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