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The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38α by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38α isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS.

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

Despite recent advances in the treatment of multiple myeloma (MM), this disease remains incurable. Accumulating evidence suggest that the bone marrow (BM) microenvironment of MM plays a critical role in tumor growth, survival, and drug resistance. A key aspect of this tumor-supportive environment is elevated levels of cytokines and other soluble factors. Most prominent among these is IL-6, which acts as a survival factor for MM cells and promotes their proliferation, migration, and drug resistance. Other mediators also implicated in the disease are VEGF and TNFa. The p38 MAPK is activated by a multitude of signals, including pro-inflammatory cytokines (e.g., TNFa and IL-1ß) and environmental stress. Furthermore, p38 activation has been shown to be important for the synthesis and secretion of IL-6, VEGF, and TNFa. Consequently, inhibition of p38 is postulated to reduce the production of these factors implicated in MM and to have therapeutic benefit by suppressing the tumor-supportive state of the BM microenvironment. Here, we demonstrate that SCIO-469, a specific and potent inhibitor of p38a MAPK, strongly inhibits MM cell proliferation by affecting MM cells directly as well as the BM microenvironment. SCIO-469 directly inhibits MM cell proliferation in long term culture. Importantly, SCIO-469 potently inhibits IL-6 and VEGF secretion from BM stromal cells (BMSC). To examine the effect of inhibiting BMSC-derived factors important in MM, we measured MM cell proliferation using transwell plates that separate BMSC from MM cells via a porous membrane. In transwell plates containing only MM cells, MM cell proliferation was modest and was inhibited by SCIO-469. In contrast, the presence of BMSC in transwell inserts dramatically increased the proliferation of MM cells over the course of the study. This result suggests that factors (e.g., IL-6) secreted by BMSC greatly stimulate MM cell proliferation. When SCIO-469 was added to these transwell cultures containing BMSC, MM cell proliferation was inhibited significantly. Consistent with these results, we show that levels of IL-6 under these conditions mirror exactly the proliferation of MM cells; IL-6 level is high in vehicle-treated cultures and is suppressed in SCIO-469-treated cultures. Finally, in a mouse xenograft plasmacytoma model of MM, we show that p38 inhibition significantly inhibited the increase in MM tumor volume. Collectively, our data indicate that SCIO-469 is a suppressor of the BM microenvironment and an effective inhibitor of MM cell proliferation in vitro and in vivo. Since SCIO-469 also inhibits secretion of osteoclast-stimulating factors (RANKL, IL-11, and MIP1a) in the microenvironment, SCIO-469 may not only inhibit MM cell survival but may also alleviate bone-related pathologies (bone destruction and osteolytic lesions) commonly associated with MM. Therefore, SCIO-469 may offer great promise for an improved outcome for patients with MM.

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.