Interaction between FLT3 and CD99 in Acute Myeloid Leukemia

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). These results were consistent with Chung et al, recent study that demonstrated the therapeutic potential of targeting CD99 on disease stem cells in myeloid malignancies. However, the functional and mechanistic role of CD99 in AML and how it is upregulated remains unknown.

To investigate the function of CD99 in AML, we transduced blasts obtained from patients with AML (N=3) and AML cell lines (THP-1 and U937) with CD99 overexpressing lentivirus. CD99 -lentiviral transduced primary blasts had a significant increase in cell number compared with control cells (day 5, 3.5-fold, p<0.001). Similarly, CD99 ectopic expression increased cell proliferation in THP-1 and U937 (1.83-fold, p<0.0001 and 1.85-fold, p<0.0001, respectively). Consistently, CD99 monoclonal antibody treatment (2.5ug/ml) induced cell differentiation in THP-1 cells, assessed by Wright-Giemse staining and by flow cytometry measurement of CD11b and enhanced apoptosis as measured by annexin and PI staining.

We previously found that patients with FLT3 -ITD mutation exhibit higher levels of CD99 compared with patients with wild type FLT3 ( FLT3 -WT). Here we hypothesize thatFLT3 -ITD upregulates CD99 expression in AML and that both surface proteins cooperate to maintain leukemia stem cell survival. To test this hypothesis, we treated FLT3 -ITD positive cells (MV4-11 and MOLM-13) with 0.5uM of midostaurin (FLT3 inhibitor), and assessed CD99 expression levels. We found CD99 protein levels were significantly reduced following midostaurin treatment (>80%). On the other hand, CD99 knockdown resulted in increased FLT3- ITD protein levels in MV4-11 cells ( FLT3- ITD homozygous). Furthermore, the combination of CD99 siRNA and midostaurin resulted in increased P- FLT3 and total FLT3 protein levels in MV4-11 cells compared to either treatment alone. We also co-expressed CD99 with FLT3-WT or FLT3-ITD in 293T cells and performed immunoprecipitation using CD99 pull down with flag tag. This assay demonstrated that CD99 bound to both FLT3 -WT and -ITD.

Next, we characterized CD99 expression in patients with AML according to their clinical outcome. In 186 patients from The Cancer Genome Atlas (TCGA) AML dataset, CD99 was overexpressed in the favorable risk group as compared with the intermediate and poor-risk groups (p< 0.01, p<0.0001 respectively). To determine the clinical relevance of CD99 , patients were dichotomized into CD99 high and low based on CD99 median expression. The median overall survival (OS) of the CD99 high group was significantly longer than that of the CD99 low patients (OS, 27.4 vs 11.9 months, p=0.0063). Also, the median event-free survival (ES) of the CD99 high group were significantly longer compared with that of CD99 low patients (ES, 13.4 vs 8.9 months p=0.025). When patients were stratified into normal (CN) vs abnormal cytogenetic (Non-CN) AML, we found that, in CN-AML, the CD99 high group survived significantly longer than CD99 low patients (OS, 45.8 vs 12 months p=0.020). Similarly, there was a significant increase in the event free survival of CD99 high patients (ES, 19.3 vs 8.4 months p=0.026). There was no significant difference in CD99 expression between CN AML and non-CN AML. Furthermore, high CD99 was associated with longer overall (OS, 17.15 vs 7.7 months, p=0.0006) and event free survival (ES, 11.6 vs 5.65 months, p=0.001) in patients who did not receive hematopoietic stem cell transplant, but not in patients who received a transplant. In a multivariable analysis, CD99 high expression association with overall survival was not significant when adjusted by age, Cytogenetic Risk, Transplant status, DNMT3A, Tp53 and CD99 (p=0.364).

In summary, although CD99 is a potential therapeutic target in AML, high CD99 is associated with better clinical outcome. Our study also demonstrates an interplay between CD99 and FLT3 , which may elucidate the upregulation of CD99 in patients with FLT3 -ITD. Further investigations are ongoing to determine the functional relevance of CD99 and FLT3 interaction.