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. 2017 Jan 25;9(374):eaaj2025.
doi: 10.1126/scitranslmed.aaj2025.

CD99 Is a Therapeutic Target on Disease Stem Cells in Myeloid Malignancies

Affiliations
Free PMC article

CD99 Is a Therapeutic Target on Disease Stem Cells in Myeloid Malignancies

Stephen S Chung et al. Sci Transl Med. .
Free PMC article

Abstract

Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are initiated and sustained by self-renewing malignant stem cells; thus, eradication of AML and MDS stem cells is required for cure. We identified CD99 as a cell surface protein frequently overexpressed on AML and MDS stem cells. Expression of CD99 allows for prospective separation of leukemic stem cells (LSCs) from functionally normal hematopoietic stem cells in AML, and high CD99 expression on AML blasts enriches for functional LSCs as demonstrated by limiting dilution xenotransplant studies. Monoclonal antibodies (mAbs) targeting CD99 induce the death of AML and MDS cells in a SARC family kinase-dependent manner in the absence of immune effector cells or complement, and anti-CD99 mAbs exhibit antileukemic activity in AML xenografts. These data establish CD99 as a marker of AML and MDS stem cells, as well as a promising therapeutic target in these disorders.

Conflict of interest statement

Current Affiliation, Department of Pathology, New York University School of Medicine, New York, NY 10016.

Competing interests: None of the authors have any financial conflicts of interest to report.

Figures

Fig. 1
Fig. 1. Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) stem cells express high levels of CD99
(A) Hematopoietic stem cells (HSCs; lineage negative [LN] CD34+CD38−CD90+CD45RA−) from patients with MDS (n=24) and normal cord blood (CB) controls (n=24) were analyzed by flow cytometry (FC) for CD99 expression, (B) shown as fold-change CD99 mean fluorescence intensity (MFI) on MDS HSCs compared with CB HSCs. Unfractionated blasts (CD45[low]SSC[low]) from diagnostic (n=39) or relapse (n=40) AML specimens and normal CB HSCs (n=18) were analyzed by FC for CD99 expression, shown as fold-change CD99 MFI on AML blasts compared with CB HSCs. Error bars represent ±SEM. *P<0.0001 (paired t-test). (C) CD99 expression was evaluated by FC on CD3-CD19-CD34+CD38− cells from AML specimen MSK AML-003. By immunophenotype, CD99 negative cells include HSCs and multipotent progenitors (MPPs; LN CD34+CD38−CD90−CD45RA−), while CD99 positive cells are almost entirely composed of lymphoid-primed multipotent progenitor-like cells (LMPP-like; LN CD34+CD38−CD90−CD45RA+). (D) When these populations were plated in methylcellulose (750 cells in triplicate), normal myeloid/erythroid colonies formed only from the CD99 negative fraction. Error bars represent ±SEM of triplicates. (E) All CD99 negative derived methylcellulose colonies lacked the heterozygous FLT3-ITD and NPM1 abnormalities present in MSK AML-003, while 14 out of 33 sequenced colonies harbored a heterozygous DNMT3A R882P abnormality. CD99 negative sorted cells and xenografts lacked DNMT3A, NPM1, and FLT3 mutations, while all three abnormalities were present in CD99 positive sorted cells and xenografts. (F) Summary of the percentage of CD99 negative cell derived colonies from ten AML specimens that were positive for disease associated mutations in the indicated alleles (which were confirmed to be present in the bulk fraction of each corresponding AML). Each data point represents a summary of results for each AML harboring a particular mutation (shown in detail in Supp. Fig. 4). (G) CD99 negative cells (2,500 cells) were transplanted into sublethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (n=3), with mice demonstrating human lymphomyeloid engraftment in the BM 10 months after transplantation. Representative FACS-plots are shown from two CD99 low xenografts. (H) Sanger sequencing traces reveal the absence of DNMT3A R882P and NPM1 W288fs mutations in CD99 negative cell derived xenografts, and the presence of both of these abnormalities in CD99 positive cell derived xenografts.
Fig. 2
Fig. 2. CD99 expression enriches for functional leukemic stem cells (LSCs)
(A) In AML specimens with an identifiable LMPP-like (LN CD34+CD38−CD90−CD45RA+) LSC-enriched population (n=69), CD99 expression was higher in LMPP-like blasts compared with bulk unfractionated blasts. **P<0.0001 (paired t-test). (B) CD99 expression was higher on bulk unfractionated AML blasts from relapse samples (n=40) as compared with diagnostic samples (n=39). Error bars represent ±SEM. *P=0.01 (paired t-test). (C) Gating strategy for purifying LSC-enriched LMPP-like cells from AML specimen UP31. From this fraction, the highest and lowest CD99 expressing blasts (top and bottom 10%, respectively) were FACS-purified to >95% purity and transplanted at limiting dilution into sublethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Sort gates were drawn based on normal HSC, MPP, and LMPP populations in CB as depicted in Fig. 1A. (D) Leukemic engraftment (defined as >0.1% detectable human cells in the bone marrow >12 weeks after transplantation) was only observed in mice transplanted with “top 10%” CD99 expressing LMPP-like blasts. (E) The highest and lowest CD99 expressing blasts (top and bottom 15%, respectively) from the CD34 negative AML specimen UP32 were FACS-purified to >95% purity and transplanted at limiting dilution into NSG mice. Leukemic engraftment was only observed in mice transplanted with “top 15%” CD99 expressing blasts. (F) The highest and lowest CD99 expressing blasts (top and bottom 10%, respectively) from the bulk (CD45[low] SSC[low] CD45RA+) fraction of AML specimen MSK AML-003 were FACS-purified to >95% purity and transplanted at limiting dilution into NSG mice. Leukemia-initiating cell activity was estimated to be 10-fold higher in the “top 10%” CD99 expressing blasts. (G–I) The top 10% and bottom 10% of CD99 expressing blasts within the LMPP-like fraction of eight independent primary AML specimens, as well as the bulk fraction from six of these AML specimens, were FACS-purified and RNA-sequencing was performed. Gene set enrichment analysis revealed in “top 10%” CD99 expressing blasts enrichment for LSC (–29) and HSC gene signatures (28, 30), as well depletion of ribosomal gene transcripts (KEGG)(54) and gene signatures associated with translation (REACTOME)(55, 56).
Fig. 3
Fig. 3. An anti-CD99 monoclonal antibody (mAb) is cytotoxic to MDS and AML cells
(A) Incubation of purified CD34+ cells from MSK MDS-001 and MDS-002 with anti-CD99 mAb (clone H036-1.1) for 48 hours led to a significant decrease in cell number. Similar results were obtained with (B) CD34+CD38−CD90−CD45RA+ LMPP-like cells from AML specimens MSK AML-001 and UP31, as well as (C) CD34+ cells from MSK AML-004. CD34+ cells from the BCR-ABL positive AML MSK AML-005 were incubated with anti-CD99 mAb (H036-1.1) for 48 hours. The IC50 was not reached using mAb concentrations up to 35 μg/ml. (D) Incubation of bulk blasts (CD45[low]SSC[low]) purified from UP32, UA8, UP4, UA16, UP34, and MSK AML-003 with anti-CD99 mAb (clone H036-1.1) for 48 hours led to a significant decrease in cell number. (E) At the end of the incubation of MSK MDS-001 and MSK MDS-002 with anti-CD99 mAb, CD34 and CD38 expression was measured on remaining viable cells, demonstrating selective depletion of CD34+CD38− cells. Representative FACS-plots are shown. *P<0.05, **P<0.01 (unpaired t-test). (F) At the end of incubation of MSK AML-004, UP4, UA16, UP34, and MSK AML-003 with anti-CD99 mAb, CD34 and CD38 expression was measured on remaining viable cells, demonstrating selective depletion of CD34+CD38− cells (or CD34+ cells in UP4). Representative FACS-plots are shown here and in Supp. Fig. 12. *P<0.05, **P<0.01 (unpaired t-test). (G) MOLM13 cells were incubated with anti-CD99 mAb (H036-1.1) for 48 hours, leading to a marked decrease in cell number. (H) Incubation of MOLM13 cells with anti-CD99 mAb (H036-1.1, 20 μg/ml) led to induction of apoptosis over the course of 36 hours. **P<0.01, ***P<0.001, ****P<0.0001 (unpaired t-test). (I) 700 HSCs (LN CD34+CD38−CD90+CD45RA−) purified from CB were incubated with anti-CD99 mAb (H036-1.1) for 48 hours. The IC50 was not reached using mAb concentrations up to 35 μg/ml. Juxtaposed for comparison is the sensitivity of MSK MDS-001 and MSK AML-001 to anti-CD99 mAb as shown in panels A and B. (J) Similar results were obtained when human umbilical vein endothelial cells (HUVECs) were incubated with anti-CD99 mAb (H036-1.1) for 48 hours. For panels A–J, error bars represent ±SEM of biological triplicates.
Fig. 4
Fig. 4
(A) Schematic for combined ex vivo and in vivo anti-CD99 mAb (H036-1.1) treatment of AML specimen UP32. (B) Summary of engraftment in UP32 xenografts five months after transplantation (>0.1% threshold for human engraftment demarcated with dotted gray line). Error bars represent ±SD. (C) Representative FACS plots of human engraftment levels in anti-CD99 mAb and isotype control treated animals. (D) Schematic for combined ex vivo and in vivo anti-CD99 mAb (H036-1.1) treatment of normal CB HSCs (LN CD34+CD38−CD90+CD45RA−). (E) Summary of engraftment based on assessment of human CD45+ cells two months after transplantation and one month after antibody treatment. (F) Schematic for engraftment of primary AML specimens and in vivo treatment with anti-CD99 mAbs (H036-1.1 or 10D6). (G) Human leukemic chimerism in xenografts relative to pre-treatment chimerism after four weeks of antibody treatment with the indicated anti-CD99 mAb clone or isotype. **P<0.01, ***P<0.001, ****P<0.0001 (Mann-Whitney U test). Representative FACS plots of human engraftment levels in the BM and PB before and after anti-CD99 mAb treatment. (H) Schematic for in vivo treatment of mice engrafted with normal CB HSCs with anti-CD99 mAb (H036-1.1 or 10D6). (I) Summary of CB HSC derived human engraftment in the BM and PB after four weeks of antibody treatment with the indicated anti-CD99 mAb clone or isotype. *P<0.05 (Mann-Whitney U test).
Fig. 5
Fig. 5. Anti-CD99 mAbs induce apoptosis by promoting SRC-family kinase (SFK) activation
(A) MOLM13 cells were transduced with an shRNA targeting CD99. Western blot confirmed knockdown of CD99, which was accompanied by an increase in SFK activation, as measured by pSRC (Y416). (B) MOLM13 cells were transduced to overexpress CD99 under a doxycycline inducible promoter, and 1 μg/ml doxycycline was added to the media. Western blot confirmed overexpression of CD99 24 hours after addition of doxycycline, which was accompanied by a decrease in pSRC (Y416). (C) Incubation of MOLM13 cells with anti-CD99 mAb (clone H036-1.1, 20 μg/ml) induced rapid SFK activation. (D) Incubation of the indicated primary AML specimens with anti-CD99 mAb (clone H036-1.1, 36 μg/ml) induced rapid SFK activation. (E) MOLM13 cells were incubated with dasatinib (1 μM) or DMSO for eight hours prior to incubation with anti-CD99 mAb (H036-1.1, 5 μg/ml) for 48 hours. Anti-CD99 mAb induced a significant reduction in cell number that was partially rescued by dasatinib treatment. *P<0.05, **P<0.01, ***P<0.001 (unpaired t-test). Error bars represent ±SEM of biological triplicates. (F) Primary AML blasts were incubated with PP2 (20 μM) or DMSO immediately prior to incubation with anti-CD99 mAb (H036-1.1, 36 μg/ml) for 48 hours. Anti-CD99 mAb induced a significant reduction in cell number that was partially rescued by dasatinib treatment. *P<0.05, **P<0.01, ***P<0.001 (unpaired t-test). Error bars represent ±SEM of biological triplicates. (G) Incubation of MOLM13 cells with anti-CD99 mAb (H036-1.1, 20 μg/ml) for 36 hours leads to a marked redistribution of cells from the G1 to the G0 or S/G2/M phases of the cell cycle. *P<0.05, **P<0.01, ***P<0.001 (unpaired t-test). (I) MOLM13 cells were incubated with anti-CD99 mAb (H036-1.1, 36 μg/ml) and after 24 hours live cells (propidium iodide negative) were FACS-purified and RNA-sequencing was performed. Gene set enrichment analysis revealed enrichment for gene signatures associated with DNA-damage response(57), cell cycle arrest, replication stress, and the unfolded protein response(55, 58). (J) K562 cells were incubated with anti-CD99 mAb (H036-1.1) for 48 hours. The IC50 was not reached using mAb concentrations up to 17.4 μg/ml. The sensitivity of MOLM13 cells to anti-CD99 mAb, as shown in Fig. 3G, is juxtaposed for comparison. Error bars represent ±SEM of biological triplicates. (K) A constitutively active SRC (Y530F) mutant was generated by site directed mutagenesis. (L) MOLM13 and K562 cells were transduced to overexpress wild-type SRC or mutant SRC (Y530F) using a doxycycline-inducible system; SRC (Y530F) expression induced a greater decrease in cell growth in MOLM13 as compared with K562 cells. Error bars represent ±SEM of biological triplicates. **P<0.01, ***P<0.001 (unpaired t-test). (M) SRC (Y530F) expression induced apoptosis, as measured by activated caspase 3, in MOLM13 but not K562 cells. Error bars represent ±SEM of biological triplicates. ***P<0.001 (unpaired t-test).

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