CD97 Expression in Acute Myeloid Leukemia Is Associated with FLT3-ITD Mutation Status

Introduction FLT3 mutations consisting of internal tandem duplications (ITDs) occur in about 30% of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. FLT3-ITD mediates proliferation and survival, blocks myeloid differentiation, and induces leukemic transformation in hematopoietic stem and progenitor cells (HSPCs).

CD97 belongs to a subgroup of adhesion GPCRs which are known to be expressed in lymphoid and myeloid cells as well as on solid tumors but nothing is described so far about the expression and regulation in human normal and malignant HSPCs. Therefore, we investigated a possible implication of CD97 in AML and an association to clinically important mutations.

Methods We studied 292 samples from patients with de novo acute leukemia, comprising AML M0-2 (n=195), AML M3 (n=16), AML M4/5 (n=63), AML M6/7 (n=4) and c-ALL (n=14). A 4-color immunophenotypic measurement was performed using the following antibodies: CD34 PerCp5.5, CD117 FITC, CD97 APC, CD45 V500. FLT3-ITD and NPM1 mutations were detected as reported in detail previously (Thiede C et al. Blood 2002; Thiede C et al. Blood 2006).

In three human AML cell lines, MV4-11, EOL-1 and OCI-AML3, which display a specific FLT3 and NMP1 mutation status, we investigated the mRNA and protein expression levels of CD97 and a possible influence of the protein kinase inhibitors PKC412 (Midostaurin) and SU5614. CD97 knock-down in MV4-11 cells was achieved by lentiviral transduction of plko1.6/shRNACD97. CD34+ HSPCs were isolated from apheresis of healthy donors using MACS microbeads and were transduced with MFGS-FLT3-ITD-IRES-GFP (Jacobi A et al. Exp Hematol 2010).

Results Compared to bone marrow blasts from, healthy donors (n=10), we detected significantly higher CD97 expression levels (mean fluorescence intensity, MFI) in 144 AML samples (49%). In detail, the CD97 expression could be observed in 40% out of cases with M0-2, 100% of cases with M3, 59% of patients with M4/5, 75% of M6/7 and 71% of the c-ALL cases, respectively. Of note, higher CD97 expression was accompanied by a significantly higher BM blast count (75% vs. 53%, p<0.001) and a lower Hb (5.9 vs. 6.5, p=0.02). Interestingly, elevated CD97 expression was associated with mutations in NPM1 (44% vs. 19%, p=0.002) and FLT3 genes (43% vs. 10%, p<0.0001) as well as lower CD34 (52% vs. 83%, p<0.0001) and HLA-DR expression (79% vs. 96%, p<0.0001).

Quantitative real-time PCR analysis of AML cell lines with or without FLT3-ITD or NPM1 mutation revealed higher CD97 mRNA levels in cells carrying this mutation, as MV4-11 and OCI-AML3, compared to normal CD34+ HSPCs or EOL-1 cells, respectively. This result was confirmed at the protein level by flow cytometry.

Treatment of MV4-11 cells with 0.5µM of the small molecule inhibitor PKC412 significantly decreased the CD97 expression. Another more specific FLT3 inhibitor SU5614 resulted in an even more decreased CD97 expression. In contrast, the low CD97 expression levels in EOL-1 cells and normal HSPCs were not affected by these inhibitors.

Interestingly, induction of a leukemic phenotype in normal HSPCs by lentiviral transduction of FLT3-ITD also activated the CD97 expression confirming an association of the molecules.

Knock-down of CD97 expression in MV4-11 cells by lentiviral transduction of shRNA constructs had no effect on the viability and proliferation but resulted in decreased spontaneous migration potential as well as inhibited adhesion to a mesenchymal stromal cell layer. This suggests a modulation of the bone marrow microenvironment by leukemic cells probably by interactions of CD97 with integrins expressed by stromal cells.

Conclusions Our data provide evidence that the expression of CD97 is linked to the malignant phenotype of AML cells and may therefore serve as diagnostic and therapeutic target.