High levels of BMP2 and BMP4 produced by the CML microenvironment contributes to BMPRIb-induced expression. (A) Enzyme-linked immunosorbent assay quantification of BMP4 and BMP2 in BM plasma obtained from healthy donors and CP-CML samples at diagnosis. Data are categorized according to circulating platelet number measured for CML patients as either normal (≤400 G/L) or abnormally elevated (>400 G/L). Results expressed in picograms per milliliter represent the mean value ± SEM of the indicated number of analyzed samples. *P < .005 and ***P < .0001 indicate differences between the levels of BMP2 and BMP4 in CP-CML samples at diagnosis compared with healthy samples. Comparative expression of BMP2 and BMP4 genes in (B) CD34⁺ and CD34⁻ cells obtained from healthy (n = 19; open bars) and CML (n = 41; closed bars) blood samples, *P < .0001 indicates differences between the expression of BMP2 and BMP4 genes in CML patient blood samples in CP at diagnosis compared with healthy samples and in (C) stromal cells derived from 3–4 weeks of culture of unmanipulated BM samples from healthy (n = 4; open bars) and at diagnosis CP-CML (n = 6; closed bars) samples. (D) In situ staining of BMP2 and BMP4 and their control antibody performed on sections of BM biopsies from nonhematologic malignancy patients (n = 2; upper panels) and CP-CML patients at diagnosis (n = 3; lower panels). Pictures were captured on a DMR microscope using PL Fluotar objective (Leica) at a magnification of ×40/1.00–0.50 oil. The following cells are indicated in the picture: (1) polynuclear neutrophils, (2) immature granular neutrophils (promyelocytes/myelocytes), (3) megakaryocytes, (4) erythroblasts, and (5) endothelial cells of the sinusoid. (E) Flow cytometry analysis of BMPRIb receptor cell surface expression on parental TF1 cells either transduced with an empty vector (non–BCR-ABL; open bars) or transduced with a vector containing BCR-ABL expressing sequence (closed bars) after chronic exposure for 4 weeks to either BMP2 or BMP4 (50 ng/mL). Results represent the mean value ± SEM of 8 experiments. *P < .05 indicates differences between parental TF1 cells transduced with an empty vector and BCR-ABL–transduced TF1 cells. (F) Selected CD34⁺ cells isolated from CP-CML samples were incubated in serum-free medium (6 × 10⁵/mL) for 7 days in the presence of BMP4 or BMP2 (50 ng/mL), with or without coculture on a stroma composed of HS27A cells seeded at 2 × 10⁴ cells/mL 1 day before the experiment. Comparative expression of the BMPRIb gene was then performed on CP-CML CD34⁺cells. Results represent the mean value ± SEM of 3 experiments.