Embryonic stem cells (ESCs) represent an important research tool and a potential resource for regenerative medicine. Generally, ESCs are cocultured with a supportive feeder cell layer of murine embryonic fibroblasts, which maintain the ESCs' capacity for selfrenewal and block spontaneous differentiation. These cumbersome conditions, as well as the risk of xenobiotic contamination of human ESCs grown on murine embryonic fibroblasts, make it a priority to develop chemically defined methods that can be safely used for the expansion of ESCs. Using a high-throughput, cellbased assay, we identified the small molecule IQ-1 that allows for the Wnt/␤-catenin-driven long-term expansion of mouse ESCs and prevents spontaneous differentiation. We demonstrate that IQ-1, by targeting the PR72/130 subunit of the serine/threonine phosphatase PP2A, prevents ␤-catenin from switching coactivator usage from CBP to p300. The increase in ␤-catenin/CBP-mediated transcription at the expense of ␤-catenin/p300-mediated transcription is critical for the maintenance of murine stem cell pluripotency. p300 ͉ PP2A ͉ Wnt signaling ͉ Nkd ͉ small molecule

In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly upregulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-b1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-b was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/b-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 micro-RNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia, however little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CBP) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300, leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using ChIP assay, we demonstrate occupancy by CBP of the survivin promoter, which is decreased by ICG-001 in primary ALL. CBP-mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

No abstract