TGF-β Receptor Kinase Inhibitor LY2109761 Reverses the Anti-Apoptotic Effects of TGF-β1 in Leukemic Cells Co-Cultured with Stromal Cells.

Transforming growth factor β1 (TGF-β1) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. TGF-β1 is also known to affect cell-to-cell interactions between tumor and stromal cells through production of the extracellular matrix and stimulation of integrin receptors. We investigated the role of TGF-β1 in the survival of human leukemic cells growing in the context of bone marrow (BM) microenvironment, and the anti-leukemia effects of the novel TGF-β receptor inhibitor LY2109761. BM-derived mesenchymal stem cells (MSC) produced TGF-β in an autocrine fashion. Treatment with rhTGF-β1 (2ng/mL) inhibited the spontaneous and Ara-C-induced apoptosis in U937 cells (% AnnexinV(+), control 34.5±8.4; TGF-β1 18.4±4.5; Ara-C 88.6±3.0; Ara-C/TGF-β1 60.4±8.0, p=0.04). These effects were more prominent in U937 cells co-cultured with MSC (% AnnexinV(+), control 19.4±2.8; TGF-β1 3.5±1.0; Ara-C 69.0±3.6; Ara-C+TGF-β1 24.9±3.3; p=0.01). In U937 cells co-cultured with MSC, rhTGF-β1 conferred higher cell protective effects on leukemia cells attached to MSC than on floating cells. Conversely, the pro-survival effects of TGF-β1 were inhibited by 5mM LY2109761 (%AnnexinV(+); MSC(−), control 31.8±2.3, TGF-β1 19.5±3.0, LY 28.4±4.4, TGF-β1+LY 37.7±2.0; MSC(+), control 22.1±1.7, TGF-β1 7.8±0.9, LY16.1±2.6, TGF-β1+ LY 18.0±1.1, p<0.01). Similar results were obtained using TGF-β1 neutralizing antibody. TGF-β1 induced pro-survival phosphorylation of Akt in U937 cells cultured alone or co-cultured with MSC, which was abrogated by LY2109761. Further, rhTGF-β1 induced a moderate increase in C/EBPβ gene and LAP isoform (cell cycle arrest inducing) of C/EBPβ protein in U937 cells cultured without MSCs, while markedly upregulating C/EBPβ gene and protein, both LIP (cell proliferation inducing) and LAP isoforms, under MSCs co-culture condition, suggesting the novel role of C/EBPβ in TGF-β1-mediated U937 cell survival. In summary, these results indicate that TGF-β1-secreting BM stromal cells promote the survival of U937 leukemia cells via direct cell-to-cell interaction and promote chemoresistance of leukemia cells through the activation of Akt signaling and upregulation of C/EBPβ. The blockade of TGF-β signaling by LY2109761, which effectively inhibited the pro-survival signaling, may enhance the efficacy of chemotherapy against leukemia cells in the BM microenvironment.