CD99 Identifies Disease Stem Cells in Acute Myeloid Leukemia and the Myelodysplastic Syndromes
Authors: Chung Stephen S. , Pang Wendy W , In Gino K. , Jan Max , Klimek Virginia M. , Melnick Ari , Weissman Irving L. , Majeti Ravi , Park Christopher Y. 
Published: October 2019

Abstract
Abstract 210
The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. Our data indicate that HSCs (Lin-CD34+CD38-CD90+CD45RA-) from MDS patients exhibit widespread gene dysregulation (3,258 differentially expressed genes, FDR<0.1), including 25 cell surface protein transcripts. As these proteins can be easily measured by flow cytometry, we hypothesized that they may represent promising biomarkers.
We used flow cytometry to assess the expression of 25 candidate disease markers on HSCs in bone marrow (BM) samples from patients with therapy related MDS (n=26). Nine surface markers were upregulated on MDS HSCs as compared with cord blood HSCs in at least 50% of samples. The most frequently upregulated surface marker was CD99 (85%). CD99 was also upregulated on HSCs in 27/37 (73%) de novo MDS cases. To determine whether CD99 upregulation was specific to MDS, we analyzed 78 paired diagnosis/relapse AML specimens encompassing all non-M3 subtypes of AML and a variety of cytogenetic subgroups. CD99 was found to be frequently overexpressed both at diagnosis (81%) and at relapse (83%). Notably, the mean intensity of CD99 expression was significantly higher at relapse (p=0.007). This suggests that CD99 is selected for during relapse and may represent a high risk AML marker. To investigate whether CD99 is upregulated in other myeloid disorders, we measured CD99 in CML samples (n=12). CD99 was decreased in chronic phase CML HSCs (7/10) but increased in blast crisis CML HSCs (2/2), suggesting that it may be a marker of leukemic transformation. Nevertheless, its overexpression in low-risk MDS suggests a distinct role in MDS biology.
In CD34+ AMLs, CD99 expression was significantly higher in the stem cell enriched CD34+CD38- fraction compared with bulk blasts (p=0.003). In most samples (91%), CD99 high cells within the CD34+CD38- fraction exhibited a CD90-CD45RA+ “LMPP-like” immunophenotype, previously shown to be enriched for leukemia-initiating cell activity, whereas CD99 low cells exhibited a CD45RA- phenotype resembling normal HSCs and MPPs. Thus, CD99 may be preferentially expressed at higher levels on leukemic stem cells (LSC). We therefore used CD99 expression levels to prospectively purify potential LSCs (high CD99) from residual normal HSCs (low CD99) by sorting CD34+CD38- cells from three primary AML specimens into complete methylcellulose. CD99 low cells generated more myeloid colonies than CD99 high cells (mean 33 and 12, respectively, p=0.02). PCR-based assays revealed that molecular abnormalities associated with the prospectively sorted leukemias were present in CD99 high derived colonies, but absent in CD99 low derived colonies (two Flt3-ITD+ and one AML1-ETO+ samples tested). Transplantation of CD34+CD38-CD99 low cells from a Flt3-ITD+ AML sample into sublethally irradiated NOD/SCID/IL2-R null mice (NSG) (n=2) led to lympho-myeloid engraftment at 12 weeks (2/2 transplanted mice), with Flt3-ITD absent in the engrafted cells. Together, these data indicate that CD99 expression can distinguish LSCs from residual normal HSCs.
To study the function of CD99 in AML, we measured CD99 expression on 14 AML cell lines, identifying 12 with high levels, including HL60 and MOLM13. Using a lentiviral vector to introduce a CD99 shRNA into HL60 cells, we achieved an 8.2-fold reduction in CD99 surface expression. Cells expressing the CD99 shRNA exhibited decreased growth in vitro compared with vector control (p=0.05). When these cells were transplanted into NSG mice, animals transplanted with CD99 shRNA expressing cells demonstrated a significant improvement in survival compared with vector controls (median OS 26d vs. 22d, p=0.04, log-rank). Conversely, NSG mice transplanted with MOLM13 cells overexpressing CD99 (4.7-fold increased surface expression) demonstrated a significantly impaired survival as compared with vector controls (median OS 11d vs. 28d, p=0.05, log-rank).
In summary, our results establish CD99 as a cell surface protein that is highly expressed in MDS HSCs, AML blasts, and LSCs. Using CD99 expression to prospectively isolate residual normal HSCs from LSCs may provide a useful tool for the performance of purged autologous stem cell transplants. Furthermore, CD99 expression appears to promote leukemic cell growth, particularly in vivo. Further studies are needed to fully establish the role of CD99 in the biology of MDS and AML.

Disclosures:
No relevant conflicts of interest to declare.