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V.P. Vaikari et al.
fold, P=0.03), and AML-7 (0.36-fold, P=0.01) (Figure 7B). Treatment with mAbCD99 (5 μg/mL) caused a 50% decrease in cell viability at 48 h in THP-1 and MOLM-13 cells but not in healthy donor PBMC (P<0.0001 and P<0.001, respectively) (Figure 7C). The decrease in viabil- ity was accompanied by an increase in Annexin-V apopto- sis stain in THP-1 (7.35-fold, P=0.04) (Figure 7D and E). Four hours after treatment with mAbCD99 (5 μg/mL) we found an approximately 70% decrease in cell migration in both THP-1 and MOLM-13 cells (P=0.01 and P=0.004, respectively) (Figure 7F and G). Treatment of THP-1 cells with mAbCD99 triggered an increase in CD11b+ popula- tion, indicative of myeloid differentiation (2.79-fold, P=0.04) (Figure 7H). Consistently, Wright-Giemsa stain revealed that mAbCD99 (2.5 μg/mL) induced differentia- tion with morphology resembling more mature cell fates (Figure 7I).
CD99 modulates ERK and SRC signaling pathways in acute myeloid leukemia cells
Because the initial enhanced proliferation of CD99-L expressing cells was serum stimulated and transient, we speculated that growth factor-induced signaling pathways are affected by CD99. In EWS and osteosarcoma, CD99 was found to modulate ERK pathways.35,36 Thus, we examined the effect of ectopic expression of CD99 iso- forms on AKT and ERK kinase activity. We also assessed changes in SRC signaling, previously shown to be affected by CD99.17,37 CD99-L induces transient upregulation of P- ERK, P-AKT and P-SRC compared with CD99-S and EV expressing cells (Figure 8A and Online Supplementary Figure S13) measured 72 h post viral transduction. CD99 knock- down (against both S and L) decreased P-ERK but increased P-AKT and P-SRC (THP-1 cells express mainly CD99-S isoform) (Figure 8B). In EWS, treatment with CD99-antibody induced cell death via rapid decrease of MDM2 and activation of IGF-1R and ERK signaling.35,36 Thus, we asked whether MDM2 is affected by CD99 expression. CD99-L cells exhibit lower MDM2 protein levels compared with CD99-S and EV cells (Figure 8C). In addition, MDM2 is reduced in cells treated with CD99mAb 2-9 h post treatment (Figure 8D), yet P-ERK was not changed (Figure 8E and J). Because MDM2 is known to ubiquitinate IGF-1R, we speculated that CD99- induced reduction in MDM2 may up-regulate IGF-1R downstream target genes such as Cyclin D1.38 CCND1 mRNA levels in CD99-L cells were higher than that in CD99-S (1.75-fold, P=0.02) or EV (2.8-fold, P<0.0001) (Figure 8F). CD99 knockdown also decreased CCND1 mRNA (P<0.0001) (Figure 8G). We also examined P-ERK and P-SRC in stable cells maintained in culture for >2 weeks (Figure 8H). Contrary to the early effect, we found a dramatic decrease in P-SRC in CD99-L cells. Similarly, cells treated with anti-CD99mAb showed a rapid increase in P-SRC observed within 1-2 h followed by a decrease in P-SRC observed 3-9 h post treatment (Figure 8I and J).
Discussion
CD99 upregulation has been implicated in several malignancies and is mostly known for its role in EWS.6,39 Chung et al. have recently shown that CD99+ cells expressed an antigenic profile that enriches leukemia stem cells in the majority of human AML. They also demon-
strated anti-leukemia activity of the CD99-antibody both in cellular and murine models.17 Because AML is a hetero- geneous disease, it is essential to identify patients with high CD99 expression that may benefit from treatments aimed to target this gene. Our analysis revealed an associ- ation between CD99 overexpression and the presence of FLT3-ITD. A previous study has shown that CD123/CD99/CD25(+) cells in a CD34+ cell fraction pre- dict FLT3-ITD mutations.40 Patients with FLT3-ITD have a dismal clinical outcome. Whether targeting CD99 in this patient population provides a therapeutic advantage remains to be investigated. Despite its upregulation in AML, high CD99 was associated with better outcome. Yet this association was only observed in CA-AML and was not significant in multivariate survival analysis. The inverse correlation between high CD99 and P53 muta- tions is likely driving its association with better clinical outcome as P53 mutations are most common in complex karyotype patients and associated with inferior outcome.41
CD99 is expressed as short and long isoforms with tis- sue-specific differential expression. However, the different roles of CD99 isoforms in normal and malignant tissues is only supported by limited research. Considering the increased interest in CD99 as a therapeutic target in AML, investigating the roles of its isoforms in preclinical studies is essential.18 RNA sequencing data of patients’ samples revealed that transcripts coding for CD99-S and L iso- forms are both expressed in AML. We further observed varying levels of CD99 isoform protein expression in HD PBMC and AML cell lines. However, the ability of western blot to compare isoform levels is limited by the antibodies that are generated against different epitopes. Furthermore, CD99 is highly glycosylated, which also affects the size of the protein band. Due to the limited number of cells in our experiments, we were unable to distinguish CD99 iso- form protein expression in primary AML blasts.
The two isoforms have distinct, and maybe opposite, functions in malignancies. Studies of the ectopic expres- sion of CD99-L isoform supported an oncosuppressor function in osteosarcoma.42 Overexpression of CD99-S isoform, however, resulted in decreased cell aggregation and increased cell migration in osteosarcoma and prostate cancer cells.42 In EWS, CD99-S inhibited cell differentia- tion and contributed to the maintenance of stemness.43 Downregulation of CD99-L transformed B lymphocytes to Hodgkin and Reed-Sternberg phenotype.44 Our data suggest that CD99-L cells are more responsive to serum stimuli with increased DNA synthesis and cell growth; however, over time, these cells accumulate higher ROS and DNA damage, resulting in apoptosis. We speculate that CD99 homotypic interaction is likely driving the later reduction of cell viability in CD99-L cells. While this phe- nomenon resembles oncogene-induced senescence,45 only a slight increase in P16 was observed in CD99-L cells (data not shown).
CD99-L consistently delayed disease progression in AML murine models. Whether CD99 interaction with the murine microenvironment inhibits cell homing to the BM is unclear. Yet we observed no change in CXCR4 and CD49d (VLA-4a) on the surface of CD99-L cells (data not shown). Mechanistically, CD99-L induced P-SRC and P- ERK is likely driving the initial increase in cell growth. A CD99-derived agonist peptide that specifically interacts with conserved motifs in the CD99 extracellular domain was previously found to inhibit fibronectin-mediated β1
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