Distinct protein signatures of acute myeloid leukemia bone marrow-derived stromal cells are prognostic for patient survival

Abstract

Mesenchymal stromal cells (MSC) support acute myeloid leukemia (AML) cell survival in the bone marrow (BM) microenvironment. Protein expression profiles of AML-derived MSC are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from AML-MSC (n=106) with MSC from healthy donors (n=71). Protein expression differed significantly between the two groups with 19 proteins over-expressed in leukemia stromal cells and 9 over-expressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these 28 proteins revealed three protein constellations whose variation in expression defined four MSC protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cell populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and remission duration compared to other groups. Comparison of leukemia MSC at first diagnosis with those obtained at salvage (i.e. relapse/refractory) showed differential expression of 9 proteins reflecting a shift toward osteogenic differentiation. Leukemia MSC are more senescent compared to their normal counterparts, possibly due to the overexpressed p53/p21 axis as confirmed by high β-galactosidase staining. In addition, overexpression of BCL-X_L in leukemia MSC might give survival advantage under conditions of senescence or stress and overexpressed galectin-3 exerts profound immunosuppression. Together, our findings suggest that the identification of specific populations of MSC in AML patients may be an important determinant of therapeutic response.

Figure 1.

Mesenchymal stromal cell (MSC) protein expression signatures. Protein expression is distinct between normal MSC and acute myeloid leukemia (AML)- MSC. (A) Principal component analysis (PCA) of 151 proteins examined in Class 1 (yellow), Class 2 (light blue), Class 3 (orange) and Class 4 (dark blue). (B) Unbiased hierarchical clustering identifies 3 protein signature groups: Group 1 (11 members), Group 2 (8 members) and Group 3 (9 members) in Class 1 (yellow), Class 2 (light blue), Class 3 (orange) and Class 4 (dark blue) groups, identified in top row as “MSC protein type”. MSC derived from normal donor (light blue) or AML patient (dark blue) is shown in the second row marked “cell type”.

Figure 2.

Survival and remission duration differ in patients based on mesenchymal stromal cell (MSC) population. Kaplan-Meir curves showing overall survival (A) and remission duration (B). N: number.

Figure 3.

Proteins differentially expressed in acute myeloid leukemia (AML) and normal mesenchymal stromal cell (MSC) are highly interactive. (A) String analysis was performed by String 10.0 using interactions based on “action”; available from: http://string-db.org. (B) Model of involvement of Group 1 and 2 proteins in AKT signaling. Red: proteins are members of Group 1 or 2. Yellow; proteins are non-members but possible links.

Figure 4.

Acute myeloid leukemia (AML) mesenchymal stromal cell (AML-MSC) are more senescent than normal MSC (NL-MSC). (A) Representative microscopy of an AML-MSC and a normal MSC with two different slide areas. (B). Level of β-galactosidase was assessed by enzymatic assay in normal MSC (n=5) and AML-MSC (n=4). Statistical significance determined by Student t-test; ^*P=0.027.

Figure 5.

A distinct set of proteins is associated with acute myeloid leukemia (AML) patient salvage status. (A) Reverse phase protein arrays (RPPA) reveals protein expression in AML mesenchymal stromal cells (MSC) differs between diagnosis and the salvage setting for 9 proteins (P=0.05; false discovery rate=0.68).