Abstract
CD99 is a 32-kDa glycoprotein involved in leukocyte migration and homotypic cell aggregation. Since its initial discovery as a marker on acute lymphoblastic leukemia (ALL), few studies have investigated its potential targeting and biological role in this disease. We have shown that CD99 is up-regulated in malignant stem cells in acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS), and that monoclonal antibodies (mAbs) targeting CD99 induce cell death. Given that targeting CD99 holds promise in AML/MDS, we sought to determine whether it is an effective target in other hematologic malignancies.
We began by screening 15 T-, B-, and plasma cell lines as well as normal peripheral blood and umbilical cord CD34+ cells for CD99 expression by flow cytometry. CD99 expression was 7- and 10-fold higher on 1/1 T-ALL and 1/2 anaplastic large cell lymphoma (ALCL) cell lines compared with CD34+ cells, and 2- and 3-fold higher relative to normal peripheral blood T cells, respectively. However, it was minimally expressed in 11/12 B cell lymphomas, plasma cell dyscrasias, and peripheral T cell neoplasms. CD99 expression (degree, localization) was also assessed on 264 lymphoma patient samples by immunohistochemistry (IHC) using the CD99 mAb, 12E7. We found that 11/20 (55%) T-lymphoblastic lymphomas, 7/16 (44%) angioimmunoblastic T-cell lymphomas, 4/13 (31%) ALCLs, 10/63 (16%) peripheral T-cell lymphomas, and 0/3 (0%) of NK/T cell lymphomas express CD99 by IHC, while only 1/70 (1.4%) diffuse large B cell lymphomas, 2/24 (8%) mantle cell lymphomas, 2/17 (12%) follicular lymphomas, 4/22 (18%) chronic lymphocytic leukemias, and 3/16 (19%) marginal zone lymphomas express CD99. Staining was predominately moderate and cytoplasmic. Using a BioGPS dataset from T-ALL patient bone marrow samples, CD99 transcript was found to be up-regulated in T-ALL bone marrow (n=117) relative to normal bone marrow (n=7) (p<0.0001), and was expressed at similar levels at diagnosis (n=14) and relapse (n=14), suggesting it is stably expressed and may be a candidate therapeutic target.
To test whether CD99 mAbs are cytotoxic to T-ALL and ALCL cell lines, cells were incubated with 5µg/ml CD99 mAb in the presence of 7µg/ml anti-IgG antibody, and cell survival was assessed by flow cytometry following 72-hours relative to IgG isotype control. 4/5 T-ALL cell lines (KOPTK1, Loucy, CCRF HSB-2, PF283) were sensitive to the cytotoxicity of CD99 mAb, mediating 30-96% cell death (p≤0.003), with 2/4 cell lines displaying 90-96% cytotoxicity. Remarkably, incubating CD99 mAb with a primary T-ALL patient sample induced 100% cell death within 48 hours of treatment (p<0.0001). 1/2 ALCL cell lines (Karpas-299) were sensitive to cytotoxic CD99 mAb (46% cell death, p=0.02). Furthermore, CD99 mAb treatment induced Annexin V positivity, and cell death occurred independent of complement and within 3 hours of treatment.
To determine whether CD99 mAb cytotoxicity depends on the level of CD99 expression, we stably transduced KOPTK1 cells with an optimized CD99 shRNA (199-fold reduction in CD99 mean fluorescence intensity [MFI]), stably transduced CD99-low Mac2A (ALCL) cells with TetOn CD99 (17-fold increase in CD99 MFI), and analyzed the cells for cytotoxicity following 24-hour incubations with CD99 mAbs. CD99 mAb-induced cell death increased from 4.4% to 88% upon overexpressing CD99, and decreased from 89% to 20% upon knocking down CD99, suggesting that cell death is dependent on the level of CD99 expression.
To elucidate the functional role of CD99 in T-cell neoplasms, we xenografted KOPTK1 cells expressing CD99 shRNA into sublethally irradiated NOD/SCID/IL-2Rgc-null (NSG) mice. Animals transplanted with CD99 knockdown showed no improved survival compared to controls (n=4 and 5 in each group, respectively). We further evaluated the potential oncogenic role of CD99 in vitro, and observed no effect of CD99 knock down in KOPTK1 or overexpression in Mac2A on cell cycle status or proliferation by PI staining and cell counting.
Our data indicate that CD99 is expressed in a subset of T-lineage neoplasms. While there is no evidence for a functional role of CD99 in the growth or survival of T-ALL and ALCL, CD99 can be targeted by CD99 mAbs to induce apoptosis with rapid kinetics and in a manner that is dependent on levels of CD99 expression and independent of complement. Thus, CD99 is a promising target in the treatment of a subset of T-cell neoplasms.
Disclosures
No relevant conflicts of interest to declare.
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