The Membrane Scaffold CD82 Regulates Acute Myeloid Leukemia Dormancy and Bone Marrow Niche Interactions

Acute Myeloid Leukemia (AML) patients respond favorably to induction chemotherapy treatment, however, due to a high rate of minimal residual disease, a significant majority of patients fatally relapse. The bone marrow niche can provide a protective environment for leukemic stem cells (LSCs) and is often the primary site for minimal residual disease after chemotherapy. Therefore, in order to improve long-term patient outcomes, it is critical to expand our focus to the development of therapies that are specifically directed towards the AML cell population that is residing within the bone marrow. Previous studies from our lab have established the membrane-scaffold protein, CD82, as a positive regulator of AML adhesion and signaling within the bone marrow. In this study, we test the hypothesis that specific bone marrow niche interactions and signaling mediated by CD82 overexpression promote AML cell dormancy and contribute to minimal residual disease.

To assess the effects of CD82 expression on AML cell dormancy, we first measured leukemia engraftment using luciferase engineered AML cells and preclinical mouse models. Luciferase engineered cells differentially expressing CD82 were intravenously injected into immunocompromised NOD-scid IL2rgnull (NSG) mice, and bioluminescence intensity (BLI) was longitudinally assessed as a measure of tumor burden using the IVIS® Spectrum in vivo imaging system. At 4 weeks of tumor growth, animals were sacrificed and AML cells were isolated from the blood and bone marrow and quantified by flow cytometry (hCD45+,GFP+). Our results suggest that CD82 overexpressing cells have a reduced tumor engraftment potential when compared to CD82 knockdown and control AML cells. To confirm that the reduced engraftment observed was not due to disrupted bone marrow trafficking, we completed homing experiments where bone marrow was harvested 16 hours post intravenous injection. Homing studies demonstrated that CD82 overexpressing cells display comparable bone marrow homing to control cells and increased bone marrow homing when compared to the CD82 knockdown cells. Interestingly, CD82 knockdown cells demonstrate a disruption in homing. Next, we went on to investigate how CD82 expression contributes to AML cell cycle activation in the bone marrow. Following engraftment, mice were injected with 100mg/kg of BrdU as a measure of proliferation and additional BrdU was also supplemented in the drinking water untill the time of harvest. Three days post injection, the animals were sacrificed and AML cells were isolated from the bone marrow and identified by flow cytometry (hCD45+,GFP). BrdU incorporation was measured with anti-BrdU specific antibodies. We find that over 60% of CD82 knockdown and control AML cells displayed increased BrdU incorporation, whereas only 30% of CD82 overexpressing cells were BrdU positive. These results suggest that CD82 overexpression may promote a more quiescent phenotype in AML cells localized within the bone marrow.

Recognizing that specific bone marrow regions can play a critical role in providing cues for AML cell survival and growth, we investigated how CD82 expression impacts AML cell localization within the bone marrow. Femoral bones from NSG mice with established leukemia were fixed, cryoembedded and cryosectioned. The thick femur sections were immunostained and analyzed using confocal microscopy to assess AML cell (CD45+,GFP+) localization within the bone marrow with respect to endothelial and osteoblastic niches. Our preliminary analysis suggests that while CD82 knockdown cells infiltrate the entire bone marrow, CD82 overexpressing AML cells distribute to the marrow periphery near osteoblastic regions. Future studies will be focused on elucidating the specific interacting partners of AML cells that are promoted by CD82 and the impact of the N-linked glycosylation pattern of CD82. Collectively, our findings demonstrate a role of CD82 in AML cell dormancy, potentially by fostering specific interactions within bone marrow niche, which may contribute to increased minimal residual disease and reoccurrence.