Page 158 - Haematologica April 2020

V.P. Vaikari et al.
CD99 immunoreactivity was found in AML but rarely in myeloproliferative disorders, myelodysplastic syn- dromes, remission, and normal marrow samples.16 A recent study, however, showed that CD99 is a disease stem cell marker, and CD99 antibody proved beneficial in xenograft mice models of myeloid malignancies.17 With growing evidence that CD99 plays a role in cancer, and particularly in AML, and that CD99 isoforms are differen- tially expressed and play different roles in different hematopoietic cells,18 investigating the roles of the two isoforms is crucial for CD99 preclinical development as a therapeutic target. Here we characterize CD99 upregula- tion in patients with AML and its association with clinical and molecular characteristics, and determine the function of CD99 long (L) and short (S) isoforms in preclinical leukemia models.
Methods
Patients’ samples
Diagnostic or relapse blood was obtained from AML patients treated at the Norris Comprehensive Cancer Center at the University of Southern California (USC) after obtaining written informed consent. The use of human materials was approved by the Institutional Review Boards of the USC in accordance with the Declaration of Helsinki.
Patient datasets and gene expression analysis
The Cancer Genome Atlas (TCGA) AML dataset was down- loaded from oncomine.19,20 Patients data from the GSE7186,21 GSE13159,22 GSE1159,23 GSE15434,24 GSE3077,25 GSE425,26 GSE12417,27 and GSE1785528 datasets were downloaded from the GEO database. Details of analysis methods are available in the Online Supplementary Appendix.
Cell lines and primary blasts
Acute myeloid leukemia cell lines were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 100U/mL penicillin. Peripheral blood mononuclear cells (PBMC) from AML patients and from healthy donors were isolated by density gradient centrifugation using Ficoll-Paque. Primary cells were grown in RPMI plus 20% FBS and cytokine cocktails CC100 (Flt3L, SCF, IL-3 and IL-6).
In vivo studies
Animal protocols were approved by the Institution for Animal
Care and Use Committee (IACUC) of the USC. For THP-1 and MOLM-13 xenograft experiments, 2.5x106 cells were injected via tail-vein (IV) into 4-6 week-old NOD-scid /Il2rg-/- (NSG) mice (Jackson). For the primary blasts xenograft experiment, 1x106 cells were IV engrafted into irradiated mice. Details of the methods used, including plasmids, primer sequences, antibodies and in vivo experiments are available in the Online Supplementary Appendix.
Results
CD99 is up-regulated in acute myeloid leukemia
In an effort to identify target genes that were differen- tially over-expressed in AML, we compared gene expres- sion profiles between normal and AML cells from various available public datasets. We found that CD99 was signif- icantly up-regulated in AML compared with normal cells in five datasets with available measurements of CD99
RNA levels in both leukemia and normal cells (oncomine median ranking among up-regulated measured genes 155, P=0.013; TCGA data did not have CD99 levels in normal cells). CD99 was significantly higher in 23 AML samples compared with six normal bone marrow (BM) samples (GSE7186: 3.5-fold, P<0.0001) (Figure 1A). Consistently, CD99 was significantly over-expressed in blasts of 542 patients with AML compared with PBMC from 74 healthy donors (HD) (GSE13159: 2-fold, P<0.0001) (Figure 1B). Similarly, CD99 expression was 1.8-fold higher in blasts from 285 patients with AML compared with HD cells (GSE1159: P=0.003) (Online Supplementary Figure S1A). There was a 2.3-fold increase in CD99 expression (GSE13164: P<0.001) (Online Supplementary Figure S1B) in blasts of 257 AML patients compared with PBMC of 58 HD. In the GSE995 dataset, CD99 expression was 3-fold higher in blasts from nine AML patients compared with that in cells from six HD (P=0.028) (Online Supplementary Figure S1C). We also analyzed the expression of CD99 protein coding transcripts in patients with AML (GSE106291; n=246). ENST00000381192.10 (CD99-L) and ENST00000611428.5 (CD99-S) were the top expressed transcripts (Figure 1C). The combined expression of ENST00000482405.7 and ENST00000611428.5 (both code for CD99-S) was significantly higher (P=0.0073) than the level of ENST00000381192.10 (CD99-L) (Online Supplementary Figure S1D). Transcript Ensembl IDs are described in Online Supplementary Table S1. The transcript expression of the two isoforms were positively correlated (Online Supplementary Figure S1E and F).
Several reports suggested that CD99 is up-regulated on leukemia stem cells.29-31 To validate this, we assessed CD99 expression in the GSE3077 dataset that has gene expres- sion data of leukemia blasts obtained from 23 patients with AML sorted according to their CD34 and CD38 expression levels. We found that CD99 expression was significantly higher (1.34-fold, P<0.001) (Figure 1D) in the CD34+CD38+ and CD34+CD38- combined subpopulation compared with CD34-CD38- and CD34-CD38+ combined.
CD99 overexpression is positively associated with FLT3-ITD and reversely with TP53 mutations
We assessed the association between CD99 upregula- tion and the presence of AML mutations. In the TCGA dataset, CD99 expression was significantly higher (P=0.004) in patients with FLT3-ITD (n=37) compared with patients with FLT3 point mutations (n=16) and FLT3 wild-type (n=133) (1.3 fold, P=0.002) (Figure 1E). Similarly, there was a significant association between high CD99 (above the median) and the presence of FLT3-ITD muta- tion (Fisher's exact test, P=0.04) (Online Supplementary Table S2). A similar association was found in the GSE17855 and GSE15434 datasets with childhood AML and patients with cytogenetically normal karyotype, respectively. CD99 is significantly higher in patients with FLT3-ITD compared with patients with FLT3 wild-type (FLT3-WT) (1.3-fold, P=0.03, Online Supplementary Figure S1G; 1.4-fold, P<0.001, Online Supplementary Figure S1H; respectively). We next examined the association of each CD99 transcript with FLT3-ITD mutation. ENST00000381187.8 and ENST00000624481.4 were sig- nificantly higher in FLT3-ITD compared with FLT3-WT patients (P=0.001 and P=0.002) (Online Supplementary Figure S1I-N). ENST00000611428.5 (CD99-S) was not sig- nificantly associated with FLT3-ITD after adjusting for
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