Inhibition of CD38 Shows Anti-Leukemic Activity in Acute Myeloid Leukemia
Authors: Farber Meike , Arnold Lucas , Chen Yiyang , Möllmann Michael , Duehrsen Ulrich , Hanoun Maher 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is characterized by an early failure of healthy hematopoiesis and a high relapse rate, caused by persistent leukemia initiating cells in the bone marrow niche. The hematopoietic stem cell niche in myeloid malignancies shows severe structural and functional alterations with an inflammatory response upon leukemia infiltration and impaired immunosurveillance. CD38 is a glycoprotein that is expressed on various immunoregulatory cells and commonly on AML blasts. Targeting CD38 by using the monoclonal antibody daratumumab showed high efficacy in multiple myeloma which is mediated by both cell-autonomous as well as immunomodulatory mechanisms. In this study we have investigated the anti-leukemic efficacy of daratumumab as well as its underlying mechanisms in AML.
Using an in vitro co-culture model with different human AML cell lines together with a fibroblast cell line (MS-5) or human umbilical vein endothelial cells (HUVEC) revealed a significant reduction in AML cell growth upon daratumumab treatment in 5 of 8 AML cell lines. Interestingly, anti-leukemic activity of daratumumab was more pronounced when co-cultured with MS-5 stroma cells and to lower extent with HUVECs as compared to mono-cultured AML cells (mean cell reduction in MS-5 co-culture, range from 12.9 to 31%; in HUVEC co-culture, range from 1.9 to 24.1% and in mono-culture, range from 9.9 to 18%). To validate the anti-leukemic activity of daratumumab in human AML and to elaborate the microenvironment-mediated effects we created a 3D invitro model with HUVECs and human mesenchymal spheres cultured with primary AML or normal donor cells. We could demonstrate that this triple-culture model led to an increased growth support for both AML as well as normal donor samples as compared to mono-cultured cells. Adding daratumumab to mono-cultured AML cells reduced cell growth by only 7.2% (p<0.01), whereas the anti-leukemic efficacy increased to 35.6% in triple-cultured AML samples (p<0.05; n=10 replicates). Of note, daratumumab did not show any significant cytotoxicity in normal donor samples at any culture condition. The anti-leukemic efficacy of daratumumab in AML appeared to depend on the level of CD38 expression. Addition of tretinoin (ATRA) induced a ~4-fold increase of CD38 expression in AML blasts and to a lower extent in normal donor cells. In fact, combination of tretinoin and daratumumab led to an enhanced anti-leukemic activity in AML. As daratumumab has been shown to exert diverse effects in myeloma we next assessed the underlying mechanisms in AML. Upon daratumumab treatment there were no significant differences in blast proliferation and apoptosis as assessed by BrdU and Annexin V assays, nor did we observe any induction of differentiation. Given the strong immunological reactions induced by daratumumab we next performed effector function assays. While there was no increase in antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), we observed a significant induction of antibody dependent phagocytosis (ADCP) with daratumumab compared to IgG1 control. Human macrophages showed a ~1.7-fold increase in phagocytosis of AML cell lines and primary human AML cells after daratumumab treatment.
To prove the anti-leukemic activity of daratumumab in AML in vivo we intravenously transplanted primary human AML cells into immunodeficient NOD scid gamma (NSG) mice. Leukemic mice were treated with ATRA over 4 weeks and once weekly with either daratumumab or IgG1 control. Preliminary results show a reduction in circulating blasts with a significant decrease of leukemia burden in peripheral blood by ~42% (p=0.0354), while there were no significant changes in bone marrow infiltration (n=5-6 mice per group). Ongoing experiments will further elucidate the anti-leukemic efficacy of daratumumab in combination with low-dose cytarabine.
In summary, we found that targeting CD38 shows promising anti-leukemic activity with both cell-autonomous and microenvironment-mediated effects in AML. Together with tretinoin und putatively low-dose chemotherapy the efficacy of daratumumab in AML could be increased. This combination of well tolerated drugs could present a new option of niche-targeted therapies in AML.

Disclosures
Duehrsen: Janssen: Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Research Funding.