Page 162 - Haematologica April 2020

V.P. Vaikari et al.
We then examined the effect of CD99 knockdown on cell viability. Transducing cells with lentiviral-CD99- shRNA significantly reduced primary AML blast viability (n=4) (Figure 3C and Online Supplementary Figure S7C) and AML cell lines (THP-1, MOLM-13, and U937) (40-60%; P<0.05) (Figure 3C). Knockdown was confirmed by qPCR (Figure 3D) and western blot (Online Supplementary Figure S7D). THP-1 and MV4-11 cells had an approximately 35- 50% decrease in cell viability when transiently transfected by elecroporation with CD99-siRNA compared with neg- ative-control-siRNA (P=0.001 and P=0.001, respectively) (Online Supplementary Figure S7E-G).
We also established a gain-of-function approach to study CD99-L and CD99-S isoform functions. We per- formed lentiviral transduction to over-express CD99-L and CD99-S in THP-1, U937 and MOLM-13 cells expressing variable endogenous levels of CD99 isoforms (Figure 3E and Online Supplementary Figure S7H and I). CD99-L trans- duced cells had increased cell proliferation at 72 hours (h) compared with their respective empty vector (EV) controls and CD99-S transduced cells, respectively, counted by try- pan-blue in THP-1 (1.78-fold, P<0.001; 1.61-fold, P<0.01), U937 (1.47-fold, P<0.001; 2.59-fold, P<0.0001), and MOLM-13 cells (2-fold, P<0.0001; 1.45-fold, P<0.0001) (Figure 3F). This was also confirmed by alamar-blue assay (Online Supplementary Figure S8A) and by BrdU staining (1.6-fold, P=0.006) (Online Supplementary Figure S8B) sug- gesting enhanced metabolic activity and DNA synthesis in these cells, respectively.
We also ectopically over-expressed CD99-L in AML blasts (n=7) (Online Supplementary Table S6) using lentiviral transduction. Overexpression of CD99-L was confirmed using western blot and fluorescence microscopy for green fluorescent protein (GFP) (Online Supplementary Figure S8C and D). Higher cell number was observed in lenti-CD99 blasts compared with lenti-EV transduced blasts 96 h after viral transduction (2.8-fold, P<0.0001) (Figure 3G). We also observed a modest increase in the number of colonies on day 14 in 3 of 6 patient samples over-expressing CD99-L compared with their respective controls: AML-3 (6 vs. 15, P=0.02), AML-4 (6 vs. 14, P=0.02), and AML-5 (42 vs. 128, P=0.001) (Online Supplementary Figure S8E). Furthermore, we ectopically expressed CD99-L and CD99-S in three additional AML blast samples (Online Supplementary Figure S8F). Cell viability was measured 96 h after transduction using trypan-blue and alamar-blue. A higher number of live cells was observed in CD99-L transduced blasts com- pared with lenti-EV (1.5-fold, P<0.0001) and CD99-S transduced blasts (1.3-fold, P<0.0001) (Figure 3H). More colonies were observed in one sample over-expressing CD99-L compared with their respective controls; AML-10 (49.5 vs. 101.5, P=0.04) (Online Supplementary Figure S8G).
However, long-term culture of CD99-L transduced cells showed a subsequent drop in cell viability, and cells could not be maintained in culture for more than 4-6 weeks. To validate this, we performed a long-term culture assay for ten days starting approximately two weeks post viral transduction. Proliferation of CD99-L cells started to decline by day 5 of initial serum stimulation compared with EV and CD99-S cells, even when cell density and nutrients were accounted for (Figure 3I). In CD34+ cells, we observed no significant change in the number of viable cells between cells transduced with CD99-L, -S isoform or EV (Online Supplementary Figure S8H) at 96 h post trans- duction. Transducing cells with lentiviral-CD99-shRNA
resulted in a slight decrease in cell viability (Online Supplementary Figure S8I).
Ectopic expression of CD99 long isoform enhances reactive oxygen species levels, DNA damage, and induces cell apoptosis
Because the initial enhanced proliferation of CD99-L cells was serum induced and reversed with further expan- sion in vitro, we speculated that the serum-induced cell growth would stimulate higher production of reactive oxygen species (ROS) in these cells. Indeed, THP-1 cells transduced with CD99-L exhibit a 2.5- and 1.6-fold increase in ROS levels compared with CD99-S and EV cells, respectively (Figure 4A and B). Because of their high- er ROS levels, we asked whether DNA damage is increased in these cells. Western blot analysis showed that CD99-L cells exhibit a higher level of the DNA damage marker H2Axγ compared with CD99-S and EV cells (Figure 4C). Furthermore, apoptosis was also enhanced in CD99-L transduced cells measured by annexin-V staining in THP-1 (CD99-L vs. EV: 3.48-fold, P=0.001; CD99-L vs. CD99-S: 6.32-fold, P=0.0027), U937(CD99-L vs. EV: 3.26- fold, P=0.07; CD99-L vs. CD99-S: 3.67-fold, P=0.10), and MOLM-13 cells (CD99-L vs. EV: 4.88-fold, P=0.0032; CD99-L vs. CD99-S: 4.2-fold, P=0.0042) (Figure 4D and E). The enhanced apoptosis was also confirmed by the increase of cleaved caspase-3 (Figure 4F).
Ectopic expression of CD99-L isoform induces myeloid differentiation and reduces cell migration
Previous studies have demonstrated that CD99 homo- typic interaction in CD99 expressing cells32-34 plays a role in monocyte trans-endothelial migration.4 Thus, we asked which isoform is responsible for cell homotypic interac- tion, and whether they affect cell migration and myeloid differentiation differently. Cells were seeded at 1x105cells/mL per well in a 6-well-plate and images were taken 6 h later; CD99-L THP-1 cells displayed higher cell aggregation compared with EV and CD99-S cells (Figure 5A). CD99-L cells exhibit decreased migration towards SDF-1α in a transwell chamber compared with EV and CD99-S cells; THP-1 (70%, P<0.0001; 66%, P<0.0001), U937 (80%, P<0.0001; 83%, P<0.0001), and MOLM-13 (80%, P<0.0001; P=0.0032) (Figure 5B and C).
CD99-L expressing cells showed an increase in CD11b surface marker measured by flow cytometry 24 h after cells were seeded in THP-1 (CD99-L vs. EV: 2-fold, P=0.0027; CD99-L vs. CD99-S: 1.63-fold, P=0.043), U937 (CD99-L vs. EV: 1.56-fold, P=0.01; CD99-L vs. CD99-S: 1.29-fold, P=0.11), and MOLM-13 (CD99-L vs. EV: 1.89- fold, P<0.0001; CD99-L vs. CD99-S: 1.68-fold, P<0.079) (Figure 5D).
Ectopic expression of CD99 long isoform delayed leukemia engraftment in acute myeloid leukemia murine models
Next, we investigated the effect of ectopic expression of CD99 isoforms in murine leukemia models. THP-1 cells stably over-expressing CD99-L (n=6), CD99-S (n=3), or EV (n=6) were injected into NSG mice. Mice were sacrificed on day 30-32 post implantation. Mice engrafted with CD99-L cells had smaller spleens compared with EV mice (0.04g vs. 0.07g, P=0.010) and CD99-S mice (0.04g vs. 0.06g, P=0.0003) (Figure 6A and B). hCD45 flow analysis revealed that CD99-L mice had significantly less BM engraftment
1004
haematologica | 2020; 105(4)