Daratumumab Inhibits AML Metabolic Capacity and Tumor Growth through Inhibition of CD38 Mediated Mitochondrial Transfer from Bone Marrow Stromal Cells to Blasts in the Leukemic Microenvironment
Authors: Mistry Jayna J , Hellmich Charlotte , Moore Jamie A , Marlein Christopher R , Pillinger Genevra , Collins Angela , Bowles Kristian M , Rushworth Stuart A 
Published: November 2019

Background
Acute myeloid leukemia (AML) is dependent on the bone marrow microenvironment, where bone marrow stromal cells (BMSCs) are an important tumor supporting cell type. We have previously demonstrated that, contrary to the Warburg hypothesis, AML blasts rely on oxidative phosphorylation for survival and are dependent on increased mitochondrial levels compared to non-malignant CD34+ progenitor cells. Moreover, we found that AML blasts meet their high metabolic demands by transferring in mitochondria from surrounding BMSC. We have also recently described how mitochondria are transferred from BMSC to myeloma cells in a pro-tumoral, CD38 dependent, mechanism. As the leukemia-initiating cells in AML may reside within the CD34+/CD38+ compartment, we examined the mitochondrial transfer and the resultant metabolic and functional consequences of inhibiting CD38 using daratumumab in the setting of the AML microenvironment.
Methods
Primary AML blasts and primary AML BMSC were isolated from patients bone marrow in accordance with the Declaration of Helsinki. BMSC were separated by adherence and then characterised using flow cytometry for expression of CD90+, CD73+, CD105+ and CD45-. Mitochondrial transfer was assessed in vitro using qPCR and MitoTracker staining based methods. In vivo experiments using an NSG AML xenograft model were carried out with darartumumab (or control) treatment given on days 9 and 16 post AML transplant. Tumor engraftment and growth were monitored weekly by live animal in vivo imaging. Post transplantation, AML mitochondrial content and transfer were assessed by evaluation of murine mitochondrial DNA in human AML blasts by species specific PCR analysis. Post transplantation mitochondrial function was measured by TMRM and Seahorse analysis.
Results
In-vitro experiments using MitoTracker Green demonstrate that daratumumab inhibits the transfer of mitochondria from BMSC to AML. In-vivo, daratumumab treatment significantly reduced tumor growth in human xenograft mouse model. Furthermore, we found that two doses of daratumumab resulted in reduced mitochondrial potential and oxygen consumption rate in the AML cells derived from the BM microenvironment of the AML engrafted NSG mice. Finally, examination of human AML cells sorted from NSG mouse bone marrow confirmed that mouse mitochondrial DNA content in the human AML blasts was reduced from animals treated with daratumumab compared to animals with AML treated with vehicle control.
Conclusion
Daratumumab treatment inhibits mitochondrial transfer from BMSC to AML in the BM microenvironment, resulting in a reduction of pro-tumoral oxidative phosphorylation in the blasts and subsequent reduced leukemia growth, which is associated with improved animal survival. While it is likely that daratumumab functions through a number of mechanisms of action, here we show in the NSG mouse model (which lacks functional B cells, T cells and NK cells and where macrophages and dendritic cells are defective) that inhibition of mitochondrial transfer in AML can be added to the list of mechanisms of action for daratumumab. These data support the further investigation of daratumumab as a therapeutic approach for the treatment of this mitochondrial dependent tumor.

Disclosures
Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.