Bone Marrow Stroma-Produced TGF-beta1 Confers Chemoresistance of Leukemic Cells.

TGF-beta1 is secreted at high levels by bone marrow stromal cells and osteoblasts, however its role in leukemia/stroma interactions is unknown. We investigated the effects of TGF-beta1 on survival and proliferation of human monoblastic U937 cells. First, the production of TGF-beta1 by bone marrow-derived mesenchymal stem cells (MSCs) and by U937 cells was analyzed by ELISA. U937 cells secreted approximately 0.1 ng/ml of TGF-beta, while TGF-beta1 level was over 20-fold higher in MSC conditioned medium (2.1±1.7 ng/ml at 72 hours). When U937 cells were co-cultured with MSC, TGFbeta concentration was 2.2±0.2 ng/ml. Hence, we selected 2 ng/ml concentration of recombinant TGF-beta to examine the effects on survival and proliferation of U937 cells exposed to chemotherapy or serum starvation. TGF-beta1 significantly reduced Ara-C or serum withdrawal-induced apoptosis of U937 cells in particular when U937 cells were co-cultured with MSCs (AnnexinV(+)%;U937 alone, control 34.5±8.4, TGF-beta1 18.4±4.5, Ara-C 88.6±3.0, Ara-C/TGF-beta1 60.4±8.0, p=0.04; U937 co-cultured with MSC, control 19.4±2.8, TGF-beta1 3.5±1.0, Ara-C 69.0±3.6, Ara-C+TGF-beta1 24.9±3.3, p=0.01). Treatment with rhTGF-beta1 also enhanced percentage of cells in G_0/1 cell cycle phase hence promoting quiescence (G₀/G₁ phase; control 50.1±6.7, TGF-beta1 63.8±5.5, p=0.04). Most importantly, inhibition of TGF-beta1 by neutralizing antibody (TGF-NA) significantly diminished the pro-survival effects of rhTGF-beta1 in serum-deprived U937 cells co-cultured with MSC (AnnexinV(+)%; U937 alone, control 27.9±9.0, TGF-beta1 20.1±3.3, TGF-NA 21.8±2.2, TGF-beta1+ TGF-NA 18.9±4.1; U937 co-cultured with MSC, control 22.2±1.9, TGF-beta1 10.9±2.5, TGF-NA 29.6±5.1, TGF-beta1+ TGF-NA 20.2±0.9, p<0.001). Dissection of molecular mechanisms demonstrated that TGF-beta1 induced anti-apoptotic Bcl-2 protein expression in U937 cells. Furthermore, concomitant exposure of U937 cells to rhTGF-beta1 in MSC co-cultures results in activation of AKT signaling and pro-survival phosphorylation of Bcl-2, implicating their role in anti-apoptotic effects of TGF-beta1. Taken together, these findings demonstrate that TGF-beta1 secreted by BM stroma cells promotes survival of U937 cells and confers chemoresistance of leukemic cells within the bone marrow microenvironment. Since small molecule inhibitors of TGF-beta signaling are undergoing final steps of pre-clinical development, this approach may represent a viable strategy to enhance efficacy of chemotherapy in AML.