Inhibition of p38 MAPK by SCIO-469 Suppresses TNF Generation and Promotes CD34+ Cell Survival in an In Vitro MDS Cell Culture Model.

Progress in the development of more effective therapeutics for myelodysplastic syndrome (MDS) has been limited by the lack of targets critical to the pathobiology of the disease. Ineffective hematopoiesis in MDS is characterized by accelerated proliferation and premature apoptotic death of progenitors and their progeny that is potentiated by the local generation of inhibitory molecules, including TNFa, TGFß, FasL, and VEGF. To identify upstream regulatory signals that may coordinate activation of inhibitory molecules, we used an in vitro cell culture model incorporating a CD34+ MDS cell line isolated from a RAEB-t patient, normal bone marrow stromal cells (BMSC), and/ or bone marrow mononuclear cells (BMMNC) to determine effects of cell-cell interactions on secretion of inhibitory hematopoietic cytokines. The role of p38 MAP kinase, a regulatory kinase involved in the convergence of inhibitory cytokine activation and signaling, was evaluated in this interaction. We found that p38 MAPK is induced under basal culture conditions in the MDS cell line and is further activated by TNFa or TGFß. In all cases, p38 activation was reduced by SCIO-469, a potent and specific inhibitor of p38a activity. SCIO-469 does not directly block p38 activation, suggesting a feedback loop is interrupted when p38 kinase activity is inhibited in MDS cells. To determine the effects of cellular interactions, the MDS cell line was co-cultured with either BMSC, BMMNCs or both from normal donors, and TNFa and FasL secretion were measured after 3 days incubation. TNFa and FasL were detected in culture supernatants when the MDS cell line was co-cultured with BMMNC but not when co-cultured with BMSC. TNFa secretion by BMMNCs was dependent on MDS cell contact and was significantly inhibited by SCIO-469. The addition of BMSC to the MDS and BMMNC co-culture prevented TNFa elevation, suggesting BMSCs as a dominant source for anti-inflammatory signal(s). VEGF, FGF-ß, TGFß2, BDNF, TIMP-1, TIMP-2 and IL-6 secretion by BMSC was induced by MDS co-culture, whereas SCIO-469 blocked cytokine induction. To determine the effects of SCIO-469 and MDS clone-induced BM cytokine secretion on normal CD34+ proliferation, we co-cultured BMMNCs and BMSC in transwell inserts in the presence or absence of the MDS cell line with or without SCIO-469. CD34+ proliferation was assessed in cells cultured in outer wells. CD34+ progenitors proliferated in culture at the same rate as those co-cultured with BMSC, BMMNC and MDS for 6 days. At longer intervals, viability of progenitors cultured with the MDS line declined, whereas treatment with SCIO-469 abrogated the decrease in CD34+ viability. These results implicate p38a as a critical target in the induction of pro-apoptotic cytokines in MDS, and that selective inhibition of p38 by SCIO-469 may provide a novel therapeutic strategy for MDS.