RSK2 kinases [4][5][6][7] . In response to inflammatory cytokines, IKK-α phosphorylates H3S10 at NFkB-responsive promoters 8,9 .In this study, we analyzed the function of PIM1, a constitutively active serine/threonine kinase. To understand the function of PIM1, we investigated the molecular connection between PIM1and MYC. We propose that the recruitment of PIM1 to the chromatin by MYC contributes to transcriptional activation of MYC-target genes by phosphorylating H3S10 at the E box element and suggest that this cooperation is relevant for MYC-dependent tumour formation. RESULTS PIM1 phosphorylates the nucleosome at H3S10.PIM1 mRNA is induced with fast kinetics in HUVEC treated with VEGF-A 11 . Upon VEGF-A treatment PIM1 is induced and localizes in HUVEC nuclei within 40 minutes (see Supplementary Information Fig. S1). As PIM1 is a serine/threonine kinase with putative consensus sites present on histones, its nuclear localization could account for the increase of H3 phosphorylation observed in these cells between 60 and 90 minutes after VEGF-A treatment (see Supplementary Information Fig. S1). We found that PIM1 could directly phosphorylate nucleosomes in vitro by incubating the recombinant GST-PIM1 protein with chromatin fractions obtained from 293 cells in the presence of [γ 32 P] ATP. This analysis revealed that a single protein, with molecular weight corresponding to H3, was phosphorylated by wild type GST-PIM1, but not by the kinase inactive mutant GST-PIM1-K67M (Fig. 1a, lanes 1-3). Specific inhibition of nucleosome phosphorylation was observed in the presence of a peptide derived from the H3 N-terminus but not with a peptide derived from H2B Nterminus or with an unrelated peptide (Fig. 1a, lanes 4-12). PIM1 could phosphorylate PIM1 forms a complex with MYC and MAX.VEGF-A treatment induces MYC and PIM1 co-localization in the cell nuclei at 60 minutes, decreasing thereafter (Fig. 2a). We tested whether PIM1 coimmunoprecipitaes with MYC.Time course analysis in HUVEC treated with VEGF-A showed that upon induction, both MYC and PIM1 were induced with fast kinetics. Immunoprecipitations with anti-MAX antibodies at various time points showed the formation of the MAX/MYC complex from 60 minutes after growth factor stimulation thus corresponding to the appearance of MYC in the nucleus (Fig. 2b, right panel). Importantly, PIM1 co-immunoprecipitated with MYC and MAX suggesting that a complex containing PIM1 is formed. Endogenous PIM1 was also found to co-immunoprecipitate with endogenous MYC and MAX from 293 cells after serum treatment for 120 minutes (Fig. 2c), thus demonstrating that a MAX/MYC/PIM1 endogenous complex is also formed in these cells.As MYC-dependent cell transformation requires MYC boxII (MBII) domain to bind cofactors either in a TRRAP-dependent or -independent manner 24 , we tested whether PIM1 interacts (Fig. 2d). Endogenous TRRAP associated with MYC but not with the FLAG-MYC MBII as previously described 25 . In contrast, PIM1did not co-immunoprecipitate the endogenous TRRAP (lanes 5 and 10) ...

No abstract

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than one year. Although canonical WNT pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-CATENIN or AXIN proteins), an increasing number of studies suggest that elevated WNT signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of WNT signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to WNT inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical WNT signaling and associated proliferative responses in GBM cells. Here we show that the small molecule SEN461 inhibits the canonical WNT signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced AXIN stabilization, increased β-CATENIN phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized WNT signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small molecule-mediated inhibition of WNT signaling may be a potential approach for GBM therapeutics.

No abstract

Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas.

A.Z. planned and performed the experiments and analysed the data. A.D. generated stable clones and perfomed immunoprecipitation experiments. R.S. performed the transformation assays. S.O. planned the experimental design, analysed the data and wrote the manuscript.

<div>Abstract<p>Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels <i>in vitro</i> and <i>in vivo</i>. These include SEN461-induced Axin stabilization, increased β-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells <i>in vitro</i>. Moreover, <i>in vivo</i> administration of SEN461 antagonized Wnt signaling in <i>Xenopus</i> embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule–mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics. <i>Mol Cancer Ther; 12(7); 1180–9. ©2013 AACR</i>.</p></div>

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