CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia

Abstract

Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34(+)CD38(-) AML cells in many cases (74.0 +/- 25.3% in 19 of 29 cases), whereas only a few (4.9 +/- 1.6%) cells in the normal HSC-enriched population (Lin(-)CD34(+)CD38(-)CD90(+)) expressed CD96 weakly. To examine whether CD96(+) AML cells are enriched for LSC activity, we separated AML cells into CD96(+) and CD96(-) fractions and transplanted them into irradiated newborn Rag2(-/-) gamma(c)(-/-) mice. In four of five samples, only CD96(+) cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.

Fig. 1.

SST screening of CD34⁺CD38⁻ AML-LSCs. (A) The scheme of the SST screening of CD34⁺CD38⁻ AML-LSCs. Full-length cDNA, either digested into pieces (experiment 1, Left) or undigested (experiment 2, Right), was ligated with a BstXI linker and subcloned into pMX-SST vector. TPOR, thrombopoietin receptor (a constitutively active mutant); TM, transmembrane. (B) CD96 mRNA expression level in CD34⁺CD38⁻ cells from three different AML (M2) samples and normal CD34⁺CD38⁻Lin⁻ cells. The expression levels are shown relative to those in normal CD34⁺CD38⁻Lin⁻ BM cells.

Fig. 2.

CD96 expression in normal BM cells. (A) Lin⁻ BM cells were separated into subpopulations according to the expression of CD34 and CD38 and then analyzed for CD90 and CD96 expression, with the anti-CD96 clones, G8.5 or TH-111. Numbers represent the percentages of cells in the gated populations. Representative data from three different normal BM samples are shown. (B) Summary of the analysis of CD96 expression in normal Lin⁻ BM cells (n = 3). Error bars show the SD.

Fig. 3.

CD96 expression in AML. (A and B) Expression profiles of CD34, CD38, CD90, and CD96 in AML samples. Cells were separated into subpopulations according to the expression of CD34 and CD38 and then analyzed for CD90 and CD96 expression. Representative data from AML samples that contain high (A) and intermediate (B) percentages of CD96⁺ cells in the CD34⁺CD38⁻ population are shown. (C) Percentages of CD96⁺ cells in the AML CD34⁺CD38⁻ population. Each bar represents a single AML sample. Pts 10, 15, 16, and 24 are not included in this graph because of the lack of information about FAB classification. The frequencies of CD96⁺ cells in normal BM CD34⁺CD38⁻ cells are shown for comparison. The dotted line represents the average in normal BM CD34⁺CD38⁻ cells. (D) Expression of lineage markers and CD96 in CD34⁺CD38⁻ AML blasts. Numbers represent the percentage of CD96⁺Lin⁻ cells in CD34⁺CD38⁻ AML blasts.

Fig. 4.

CD96⁺ AML cells are enriched in LSC activity. (A) Representative results of transplantation of CD34⁺CD38⁻CD96⁺ or CD96⁻ AML cells from Pt 3. (Left) Expression profiles of CD34, CD38, and CD96 and the sorting gates. CD96⁺ or CD96⁻ AML cells were transplanted into sublethally irradiated newborn Rag2^−/− γ_c ^−/− mice. (Right) Analyses of human (hCD45⁺) versus mouse (mouse CD45²⁺) chimerism in the bone marrow (BM) at 6 weeks after transplant. (B) Wright–Giemsa stain of FACS-sorted hCD45⁺ BM cells from an engrafted mouse transplanted with CD96⁺ AML cells from Pt 3 (X630), showing that engrafted hCD45⁺ cells have myeloblastic morphology. (C) CD96 expression on engrafted hCD45⁺ cells in the BM of mice transplanted with CD96⁺ AML cells from Pt 3. (D) (Upper) Expression profiles of CD34 and CD96 and the sorting gates for CD96⁺ or CD96⁻ populations for each AML sample. Note that the CD34⁺CD38⁻ population is pregated in the case of Pt 3. CD96⁺ or CD96⁻ AML cells were transplanted into irradiated newborn Rag2^−/− γ_c ^−/− mice. Analyses of human engraftment are shown as the percentage of hCD45⁺cells in the BM at 6 to 10 weeks after transplantation. Each dot corresponds to an individual mouse recipient.