CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes
Authors: Chung Stephen S. , Tavakkoli Montreh , Devlin Sean M. , Park Christopher Y. 
Published: October 2019

Abstract
The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. We performed transcriptomal profiling of purified MDS hematopoietic stem cells (HSCs) and identified CD99 as a highly expressed cell surface transcript. Flow cytometry of MDS patient bone marrow (BM) samples (n=63) confirmed that CD99 is frequently increased on MDS HSCs (85%). Assessment of 78 paired diagnosis/relapse AML specimens revealed that CD99 is also frequently overexpressed in AML at diagnosis (81%) and relapse (83%). CD99 is a potential leukemic stem cell (LSC) marker, as CD99 high cells exhibit a CD34+CD38-CD90-CD45RA+ LMPP-like immunophenotype, previously shown to be enriched for LSC activity, whereas CD99 low cells exhibit a CD34+CD38-CD45RA- phenotype resembling normal HSCs and MPPs.
Providing additional support that CD99 is a LSC marker in AML, CD99 surface expression is significantly higher in the LSC-enriched CD34+CD38- fraction compared to bulk blasts (p=0.003), as well as in relapsed as compared to diagnostic specimens (p=0.007). To determine if CD99 is preferentially expressed on functional LSCs, we purified the top 10% and bottom 10% of CD99 expressing leukemic blasts from the CD34+CD38-CD90-CD45RA+ fraction of a primary AML specimen. Limiting dilution xenotransplantation experiments demonstrated an estimated LSC frequency of 1 in 24,401 in the top 10% of CD99 expressors, whereas transplantation of up to 360,000 of the bottom 10% of CD99 expressors did not lead to any engraftment. Thus, within the LMPP-like LSC-enriched compartment, LSC activity appears to be restricted to high CD99 expressors.
To determine the function of CD99 in AML, we stably transduced MOLM13 AML cells with a CD99 shRNA (8.0-fold knockdown) and xenografted them into NSG mice. Animals transplanted with these cells showed improved survival compared to vector controls (58d vs. 34d, p=0.02). Consistent with its described role in leukocyte trafficking, overexpression of CD99 in AML cell lines promoted transendothelial migration in transwell assays. In primary AML specimens, CD99 expression was higher on PB as compared with BM specimens (p=0.03). Together, these findings suggest that CD99 may promote AML aggressiveness by enhancing transendothelial migration and mobilization.
Unexpectedly, CD99 transcript expression in the ECOG 1900 AML patient cohort (n=308) positively correlated with survival (p=0.001). We propose that CD99 may improve survival in the context of chemotherapy by promoting mobilization and thus chemosensitivity of AML cells, and AML cells with low levels of CD99 may exhibit increased retention in chemoprotective BM niches. In line with this hypothesis, the poor prognosis of low CD99 expression appeared to be mitigated by intensification of chemotherapy.
To determine whether CD99 may be a relevant therapeutic target, we tested the ability of monoclonal antibodies (mAb) against CD99 to induce direct cytotoxicity in vitro. Anti-CD99 mAbs induced significant cell death in 11 AML and two MDS-derived cell lines, as well as in primary AML blasts (n=7) and MDS HSCs/CD34+ cells (n=3). These mAbs induced cytotoxicity in a CDC and ADCC independent manner, with relative sparing of normal cells expressing low levels of CD99 such as primary human HSCs and endothelial cells, suggesting a mechanism of cytotoxicity unique to AML and MDS. Pre-coating of primary human LSCs with an anti-CD99 mAb prior to transplantation into NSG mice led to impaired engraftment at eight weeks (20% vs. 67%, p=0.009) and improved survival (p=0.05). Immunofluorescence imaging demonstrated that mAb ligation promotes clustering of CD99, and this clustering appears to be critical for cytotoxicity. Accordingly, cross-linking of IgG isotype anti-CD99 mAbs with an anti-IgG secondary antibody enhanced cytotoxicity 3.4-fold. Anti-CD99 mAb treatment was also associated with activation of Src-family kinases, and we propose that anti-CD99 mAbs may promote cytotoxicity by inducing oncogenic stress via dysregulated Src-family kinase activation.
Our results establish CD99 as a cell surface marker expressed in AML and MDS stem cells, as a mediator of transendothelial migration, and as a promising therapeutic target for direct targeting by mAbs. Further studies are needed to evaluate the potential of targeting CD99 in AML/MDS in vivo and to further characterize the mechanisms of CD99 mediated cell death.

Disclosures:
No relevant conflicts of interest to declare.