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) ...

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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.

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