Hematopoietic stem and progenitor cell (HSPC) transplantation represents a treatment option for patients with malignant and nonmalignant hematological diseases. Initial steps in transplantation involve the bone marrow homing and engraftment of peripheral blood-injected HSPCs. In recent work, we identified the tetraspanin CD82 as a potential regulator of HSPC homing to the bone marrow, although its mechanism remains unclear. In the present study, using a CD82 knockout (CD82KO) mouse model, we determined that CD82 modulates HSPC bone marrow maintenance, homing, and engraftment. Bone marrow characterization identified a significant decrease in the number of long-term hematopoietic stem cells in the CD82KO mice, which we linked to cell cycle activation and reduced stem cell quiescence. Additionally, we demonstrate that CD82 deficiency disrupts bone marrow homing and engraftment, with in vitro analysis identifying further defects in migration and cell spreading. Moreover, we find that the CD82KO HSPC homing defect is due at least in part to the hyperactivation of Rac1, as Rac1 inhibition rescues homing capacity. Together, these data provide evidence that CD82 is an important regulator of HSPC bone marrow maintenance, homing, and engraftment and suggest exploiting the CD82 scaffold as a therapeutic target for improved efficacy of stem cell transplants.

Hematopoietic stem and progenitor cell (HSPC) mobilization into the blood occurs under normal physiological conditions and is stimulated in the clinic to enable the isolation of HSPCs for transplantation therapies. In the present study, we identify the tetraspanin CD82 as a novel regulator of HSPC mobilization. Using a global CD82 knockout (CD82KO) mouse, we measure enhanced HSPC mobilization after granulocyte-colony stimulating factor (G-CSF) or AMD3100 treatment, which we find is promoted by increased surface expression of the sphingosine 1-phosphate receptor 1 (S1PR 1 ) on CD82KO HSPCs. Additionally, we identify a disruption in S1PR 1 internalization in CD82-deficient HSPCs, suggesting that CD82 plays a critical role in S1PR 1 surface regulation. Finally, combining AMD3100 and anti-CD82 treatments, we detect enhanced mobilization of mouse HSPCs and human CD34+ cells in animal models. Together, these data provide evidence that CD82 is an important regulator of HSPC mobilization and suggests exploiting the CD82 scaffold as a therapeutic target to enhance stem cell isolation.

The significant cellular demand of the hematopoietic system is maintained by a rare pool of tissue-specific, hematopoietic stem and progenitor cells (HSPCs) that are primarily found in a quiescent state. Upon hemopoietic stresses, such as significant bleeding, overwhelming infection, and myelosuppressive therapy, HSPCs undergo rapid cell cycle activation, but ultimately must return to quiescence to prevent exhaustion of the hematopoietic system. Emerging evidence from our laboratory suggests that the tetraspanin CD82 plays a critical role in the regulation of HSPC quiescence and activation. Tetraspanins are membrane scaffold proteins with the ability to modulate signaling through the formation of tetraspanin-enriched microdomains, which organize membrane signaling receptors and intracellular signaling molecules critical for propagating downstream signaling. Previous data from our laboratory identified a role for CD82 in HSPC quiescence, where we find a reduction in long term-HSCs in global CD82KO mice, resulting from increased HSPC activation and a reduction of quiescent G 0 cells. In the present study, we test the hypothesis that CD82 expression promotes HSPC return to quiescence following hematopoietic stress, by regulating the activation of TGF-β signaling. To investigate the impact of CD82 expression on hematopoietic regeneration under stress, we treated WT and CD82KO mice with 2 doses of 200mg/kg of chemotherapy agent 5-FU and measured overall survival. Interestingly, we find that CD82KO mice have significantly longer overall survival compared to their WT counterparts. Moreover, we find an increase in peripheral blood HSPCs in CD82KO mice during the early recovery period from 5FU treatment suggestive of enhanced cell activation upon stress and effective regeneration post injury. Mechanistically, the multifunctional cytokine TGF-β plays an essential role in supporting HSPC quiescence and activation, as one of the most potent inhibitors of HSPC growth both in vitroand in vivo. To investigate whether CD82 plays a role in TGF-β signaling, we stimulated WT and CD82KO HSPCs with TGF-β and measured nuclear translocation of SMAD2/3, a downstream effector of TGF-β activation. Confocal imaging demonstrates that CD82KO HSPCs have decreased nuclear translocation of SMAD2/3 upon TGF-β activation, consistent with reduced TGF-β signaling. Similarly, primary human CD34 +HSPCs sorted for low and high CD82 expression also have decreased and increased SMAD2/3 nuclear translocation, respectively. Moreover, gene expression analysis of cell cycle regulators identified the reduced expression of p57, a downstream gene target of TGF-β signaling in HSPCs harvested from CD82KO mice. Currently, we are investigating the impact of the CD82 scaffold on TGF-β signaling of HSPCs localized within the context of the bone marrow niche, by analyzing bone marrow sections from mice injected with HSPC-like cells differentially expressing CD82. Together, these data suggest that CD82 regulates HSPC quiescence and activation through modulation of TGFβ signaling. Future studies will focus on investigating how CD82 modulates local TGFβ signaling within the niche. Disclosures No relevant conflicts of interest to declare.

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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. Disclosures No relevant conflicts of interest to declare.