Inhibition of the TGF-beta receptor I kinase promotes hematopoiesis in MDS.

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells, providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-beta-mediated gene activation in BM stromal cells, and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.

p38alpha-selective MAP kinase inhibitor reduces tumor growth in mouse xenograft models of multiple myeloma.

Multiple myeloma (MM) is a clonal plasma cell malignancy, which is currently incurable. Therefore, new mono- or combined therapy treatment regimens in the early and advanced phases of MM are urgently needed to combat this disease. Recently, p38 mitogen-activated protein kinase (MAPK) has been implicated as playing an important role in MM. Therefore, the effect of a p38alpha-selective MAPK inhibitor, SCIO-469 (indole-5-carboxamide, ATP-competitive inhibitor), or its structural analog, SD-282 (indole-5-carboxamide, ATP-competitive inhibitor) was examined in mouse xenograft models of MM using human RPMI-8226 or H-929 plasmacytoma inocula. Oral treatment with SCIO-469 (10, 30, 90 mg/kg) twice daily was initiated in mice with palpable tumors of RPMI-8226 origin, a condition that mimics early human myeloma disease. In mice with palpable tumors, 14 days of SCIO-469 treatment significantly reduced RPMI-8226 tumor growth in a dose-dependent manner. A significant dose-dependent reduction in RPMI-8226 tumor growth was also observed when SCIO-469 oral treatment at doses of 10, 30 and 90 mg/kg twice daily was initiated in mice with tumors of pronounced size, a condition that mimics advanced human myeloma disease. In a similar set of studies employing the SCIO-469 analogue SD-282 at 90 mg/kg/bid orally, histological assessment at the end of the study demonstrated a significant reduction in RPMI-8226 tumor growth and angiogenesis. SD-282 treatment was additionally shown to significantly reduced expression of heat-shock protein-27 (HSP-27) and phospho-p38 in the tumor cells. Furthermore, co-administration of SCIO-469 with dexamethasone elicited antitumor properties in dexamethasone-sensitive H-929 tumors at much lower than the typically effective doses of dexamethasone, suggesting its potential for combined therapy. In conclusion, p38 inhibitors reduced human myeloma cell growth in vivo both at early and advanced phases of the disease. The current study also provides evidence of potential for co-therapy with dexamethasone.

Preventive and therapeutic potential of p38 alpha-selective mitogen-activated protein kinase inhibitor in nonobese diabetic mice with type 1 diabetes.

Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that function within T cells in both normal and stress-related pathophysiological states, including type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops T cell-mediated autoimmune pancreatic beta cell destruction that is similar to type 1 diabetes in humans. Because p38 MAPKs have been shown to modulate T cell function, we studied the effects of a p38alpha MAPK-selective inhibitor, indole-5-carboxamide (SD-169), on the development and progression of type 1 diabetes in the NOD mouse. In preventive treatment studies, SD-169 significantly reduced p38 and HSP60 expression in T cells of the pancreatic beta islets. Following treatment, the incidence of diabetes as determined by blood glucose levels was significantly lower, and immuno-histochemistry of pancreatic beta islet tissue demonstrated significant reduction in CD5+ T cell infiltration in the SD-169 treatment group as compared with untreated NOD mice. In therapeutic studies using mildly and moderately hyperglycemic NOD mice, SD-169 treatment lowered blood glucose and improved glucose homeostasis. Furthermore, following cessation of SD-169 treatment, NOD mice showed significant arrest of diabetes. In conclusion, we report that this p38alpha-selective inhibitor prevents the development and progression of diabetes in NOD mice by inhibiting T cell infiltration and activation, thereby preserving beta cell mass via inhibition of the p38 MAPK signaling pathway. These results have bearing on current prophylactic and therapeutic protocols using p38alpha-selective inhibitors in the prediabetic period for children at high risk of type 1 diabetes, in the honeymoon period, and for adults with latent autoimmune diabetes.

Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors.

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 p38alpha 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 p38alpha 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.

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation.

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.

APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA.

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-gamma. The induction of cytokine production by NK cells was preceded by activation of NF-kappaB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.