Functional Analysis of CD99 Upregulation in Acute Myeloid Leukemia

Background: Acute Myeloid Leukemia (AML) is a hematological malignancy with a 5-yr survival rate of 27%. This indicates an urgent need to identify better therapies. We previously analyzed various gene expression data sets of normal hematopoietic vs AML cells and reported that CD99 is upregulated in AML. CD99 loss of function by siRNA or monoclonal antibody decreased proliferation and migration of AML cells (Vaikari et al, ASH abstract, 2016). Recently, we have also shown that AML blasts transduced with CD99 overexpressing lentivirus exhibit a significant increase in cell proliferation (Vaikari et al, ASH abstract, 2017). Here we further expand our preclinical investigation to study the functional role of CD99 in AML.

Methods: We performed lenti-viral transduction to overexpress CD99 (CD99 OE) or empty vector (EV) in THP-1, U937, and MOLM-13 AML cell lines. Proliferation assay was performed by seeding 1X10⁵ cells/mL and measuring cell proliferation using trypan blue at 72 hours. Aggregation assay was performed by seeding 1X10⁵CD99 OE (or EV) cells in a 6 well plate and images for cell aggregation were taken 6 hours later. For the differentiation and apoptosis assays, CD99 OE (or EV) (5X10⁵ cells/mL) were starved overnight. Flow cytometry analyses for CD11b and Annexin-V PI were performed 24 hours later. To determine the effect of CD99 overexpression on cell migration, THP-1, U937, and MOLM-13 (1X10⁵ cells) CD99 OE or EV cells were seeded in a transwell chamber for 4 hours and migration towards SDF-1a was analyzed. For the THP-1 and MOLM-13 murine model, 2.5 X10⁶ cells overexpressing CD99 or EV (n=3 for each) were engrafted into NOD-scid /Il2rg^-/- (NSG) mice. Bone marrow (BM) and peripheral blood (PB) were collected to determine engraftment by hCD45 staining through flow cytometry.

Results: Transducing cells with CD99 resulted in increased cell proliferation as compared with their respective controls in THP-1 (CD99 OE vs EV: 1.78 fold, p<0.0001), U937 (CD99 OE vs EV: 1.56 fold, p<0.0001), and in MOLM-13 cells (CD99 OE vs EV: 1.87 fold, p<0.0001). THP-1, MOLM-13 and U937 cells stably overexpressing CD99 displayed higher cell aggregation capacity compared to EV cells. Cells stably overexpressing CD99 showed an increase in CD11b expression in THP-1 (CD99 OE vs EV: 2.055-fold, p=0.0027), U937 (CD99 OE vs EV: 1.56-fold, p=0.01), and MOLM-13 cells (CD99 OE vs EV: 1.89- fold, p<0.0001). Cell aggregation was also accompanied by an increase in cell apoptosis of CD99 OE cells in THP-1(CD99 OE vs EV: 3.48-fold, p=0.001), U937 (CD99 OE vs EV: 3.68-fold, p=0.11), and MOLM-13 cells (CD99 OE vs EV: 6.56-fold, p=0.0001).

Additionally, CD99 overexpression decreased cell migration compared with EV cells in THP-1(CD99 OE vs EV: 67% decrease, p<0.0001), U937 (CD99 OE vs EV: 75% decrease, p<0.0001), and MOLM-13 cells (CD99 OE vs EV: 73% decrease, p=0.0003). Based on these results, we hypothesize that homotypic interaction of CD99 could play a role in its signaling process in AML.

In both the THP-1 and MOLM-13 murine model, mice engrafted with CD99 OE cells had smaller spleens compared with EV mice. In the THP-1 murine model, CD99 OE mice had significantly less engraftment compared with the EV mice in the BM (6.69 vs 14 %, p=0.047), and PB (3.61 vs 91.67%, p<0.0001). Similarly, in the MOLM-13 murine model, hCD45 flow analysis revealed that CD99 OE mice have significantly less engraftment compared with the EV mice in the BM (39.83 vs 71.43%, p=0.0022), and PB (19.43 vs 67.13 %, p=0.018).

Conclusion: In summary, our results suggests that even though CD99 enhances AML cell proliferation, it also enhances homotypic cell interaction and cell aggregation, which results in increased cell apoptosis as well as a decrease in cell migration and possibly responsible for the decrease leukemia engraftment. Further investigations are ongoing to determine the effect of homotypic interaction of CD99 in AML.