The Wnt canonical ligands elicit the activation of β-catenin transcriptional activity, a response dependent on, but not limited to, β-catenin stabilization through the inhibition of GSK3 activity. Two mechanisms have been proposed for this inhibition, one dependent on the binding and subsequent block of GSK3 to LRP5/6 Wnt coreceptor and another one on its sequestration into multivesicular bodies (MVBs). Here we report that internalization of the GSK3-containing Wnt-signalosome complex into MVBs is dependent on the dissociation of p120-catenin/cadherin from this complex. Disruption of cadherin-LRP5/6 interaction is controlled by cadherin phosphorylation and requires the previous separation of p120-catenin; thus, p120-catenin and cadherin mutants unable to dissociate from the complex block GSK3 sequestration into MVBs. These mutants substantially inhibit, but do not completely prevent, the β-catenin upregulation caused by Wnt3a. These results, besides elucidating how GSK3 is sequestered into MVBs, support this mechanism as cause of β-catenin stabilization by Wnt.

p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. We describe here that p120-catenin is required for Wnt pathway signaling. p120-catenin binds and is phosphorylated by CK1ε in response to Wnt3a. p120-catenin also associates to the Wnt co-receptor LRP5/6, an interaction mediated by E-cadherin, showing an unexpected physical link between adherens junctions and a Wnt receptor. Depletion of p120-catenin abolishes CK1ε binding to LRP5/6 and prevents CK1ε activation upon Wnt3a stimulation. Elimination of p120-catenin also inhibits early responses to Wnt, such as LRP5/6 and Dvl-2 phosphorylation and axin recruitment to the signalosome, as well as later effects, such as β-catenin stabilization. Moreover, since CK1ε is also required for E-cadherin phosphorylation, a modification that decreases the affinity for β-catenin, p120-catenin depletion prevents the increase in β-catenin transcriptional activity even in the absence of β-catenin degradation. Therefore, these results demonstrate a novel and crucial function of p120-catenin in Wnt signaling and unveil additional points of regulation by this factor of β-catenin transcriptional activity different of β-catenin stability.

Activation of the Wnt pathway promotes the progressive phosphorylation of coreceptor LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6), creating a phosphorylated motif that inhibits glycogen synthase kinase 3␤ (GSK-3␤), which in turn stabilizes ␤-catenin, increasing the transcription of ␤-catenin target genes. Casein kinase 1 (CK1) kinase family members play a complex role in this pathway, either as inhibitors or as activators. In this report, we have dissected the roles of CK1 isoforms in the early steps of Wnt signaling. CK1 is constitutively bound to LRP5/6 through its interaction with p120-catenin and E-cadherin or N-cadherin and is activated upon Wnt3a stimulation. CK1␣ also associates with the LRP5/6/p120-catenin complex but, differently from CK1, only after Wnt3a addition. Binding of CK1␣ is dependent on CK1 and occurs in a complex with axin. The two protein kinases function sequentially: whereas CK1 is required for early responses to Wnt3a stimulation, such as recruitment of Dishevelled 2 (Dvl-2), CK1␣ participates in the release of p120-catenin from the complex, which activates p120-catenin for further actions on this pathway. Another CK1, CK1␥, acts at an intermediate level, since it is not necessary for Dvl-2 recruitment but for LRP5/6 phosphorylation at Thr1479 and axin binding. Therefore, our results indicate that CK1 isoforms work coordinately to promote the full response to Wnt stimulus.The Wnt pathway plays diverse roles in embryonic development and has been implicated in human diseases, including cancer (9). A key element in this pathway is the Ecadherin-associated protein ␤-catenin. When released from the junctional complex, ␤-catenin translocates to the nucleus, where it interacts with the Tcf family of transcriptional factors and regulates the expression of a variety of genes. The translocation of ␤-catenin to the nucleus is tightly controlled by the activity of a complex involved in ␤-catenin degradation. This complex includes the product of the tumor suppressor adenomatous polyposis coli (APC) gene, axin, and the associated Thr/Ser protein kinases, CK1␣ and glycogen synthase kinase 3␤ (GSK-3␤) (12). As a result of the activity of this complex, ␤-catenin is phosphorylated and degraded by the proteasome. The activity of the degradation complex is blocked by canonical Wnt factors, which activate a signaling pathway leading to the stabilization of cytosolic ␤-catenin (12, 13).Wnt ligands form a complex with low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and Frizzled (Fz) receptors (7). Upon Wnt ligand binding, the LRP5/6 cytosolic domain gets phosphorylated in different residues by the action of several protein kinases (15). For, instance Thr1479 is phosphorylated by casein kinase 1␥ (CK1␥), a modification that is also dependent on the Fz-associated protein Dishevelled (Dvl) (1,4,28). Other members of this family, such as CK1ε and CK1␣, also contribute to the phosphorylation of LRP5/6 and Dvl (17,21,27). Phosphorylation of LRP5/6 promotes the recruitment of ...

There was an error published in J. Cell Sci. 125, 5288-5301.In Fig. 2B, panels labelled as the actin input and p120-catenin IP:p120-catenin panels were inadvertently assembled to show the same blots. Concerns were also raised about possible duplications and/or splices in Figs 4A, 4E, 5D, 6A and 6C. The authors were unable to obtain the original data in order to address these concerns and therefore repeated the experiments. The new data are shown in the figures below and have been verified by the corresponding author's institute as supporting the original conclusions of the study. There are no changes to the figure legends, which are accurate.The authors acknowledge the increased contribution of Beatriz Del Valle-Pérez in repeating the experiments and apologise to the readers for any confusion that these errors might have caused. Fig. 2. Wnt-induced Rac1 activation is dependent on Vav2 interaction with p120-catenin. Vav2 (A) and p120-catenin (B) were immunoprecipitated from 500 µg SW-480 whole-cell extracts treated with control or Wnt3a-conditioned medium for the times indicated. Protein complexes were analyzed by western blot (WB) with anti-p120-catenin, anti-Vav2, anti-E-cadherin and anti-Rac1. 10 µg of SW-480 whole-cell extracts were included as internal reference (input). The graphs on the right are autoradiograms from four different experiments that were quantified and the mean ± s.d. obtained after 2 hours of incubation with Wnt3a medium. Each value is presented relative to that obtained in cells treated with control medium and normalized with respect to the amount of immunoprecipitated Vav2 or p120-catenin. (C) SW-480 cells stably expressing scrambled or shRNA specific for Vav2 were treated with control or Wnt3a-conditioned medium for 2 hours. GST-PAK pull-down assays were performed and active Rac1 was determined by WB. Autoradiograms from five different experiments performed in triplicate were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nondepleted cells treated with control medium. (D) Cytosolic and nuclear lysates were obtained from control and Vav2-depleted SW-480 cells treated with control or Wnt3a-conditioned medium for 15 hours. β-catenin distribution between the two cell compartments was analyzed by WB. 2120© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016Science ( ) 129, 2120Science ( -2123Science ( doi:10.1242 Journal of Cell Science 480 (E,F) cells overexpressing either GFP-p120-catenin wt isoform 1, GFP-p120-catenin point mutants Y112E or Y217E or the empty vector phrGFP, treated when indicated with Wnt3a-conditioned medium for 15 hours. The nuclear fraction was separated from the cytosolic and membrane-associated fraction as detailed in Materials and Methods. β-catenin levels in each cellular fraction were analyzed by WB. Lamin-β1 was used as a nuclear marker and pyruvate kinase as a marker for the cytosolic-plus-membrane fraction. In the right panel of C, p120-catenin wt, Y112E and Y217E...

B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, and high-hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD is an initiating oncogenic event affiliated to childhood B-ALL, the mitotic and chromosomal defects associated to HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated to chromosome alignment defects at the metaphase plate leading to robust chromosome segregation defects and non-modal karyotypes. Mechanistically, biochemical, functional and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness and mis-localization of the chromosome passenger complex proteins Aurora B Kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and impaired spindle assembly checkpoint (SAC) thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated to defective condensin complex, AURKB and SAC.

BackgroundAlthough adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells (CD19-CAR T-cells) achieves high rates of complete response in patients with B-cell acute lymphoblastic leukemia (B-ALL), relapse is common. Bone marrow (BM) mesenchymal stem/stromal cells (BM-MSC) are key components of the hematopoietic niche and are implicated in B-ALL pathogenesis and therapy resistance. MSC exert an immunosuppressive effect on T-cells; however, their impact on CD19-CAR T-cell activity is understudied.MethodsWe performed a detailed characterization of BM-MSC from pediatric patients with B-ALL (B-ALL BM-MSC), evaluated their immunomodulatory properties and their impact on CD19-CAR T-cell activity in vitro using microscopy, qRT-PCR, ELISA, flow cytometry analysis and in vivo using a preclinical model of severe colitis and a B-ALL xenograft model.ResultsWhile B-ALL BM-MSC were less proliferative than those from age-matched healthy donors (HD), the morphology, immunophenotype, differentiation potential and chemoprotection was very similar. Likewise, both BM-MSC populations were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, BM-MSC failed to impair CD19-CAR T-cell cytotoxicity or cytokine production in vitro using B-ALL cell lines and primary B-ALL cells. Finally, the growth of NALM6 cells was controlled in vivo by CD19-CAR T-cells irrespective of the absence/presence of BM-MSC.ConclusionsCollectively, our data demonstrate that pediatric B-ALL and HD BM-MSC equally immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicated by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged (MLLr) and non-MLLr iB-ALL leukemias uniformly treated according to Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression and gene co-expression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer-cell vulnerabilities, revealed a robust iB-ALL-specific gene expression-correlating dmCpG signature and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient-derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL.

Canonical Wnt signaling induces the stabilization of β-catenin, its translocation to the nucleus and the activation of target promoters. This pathway is initiated by the binding of Wnt ligands to the Frizzled receptor, the association of the LRP5/6 coreceptor and the formation of a complex comprising Dvl-2, Axin and protein kinases CK1α, ε, γ and GSK3. Among these, activation of CK1ε, constitutively bound to LRP5/6 through p120-catenin, is required for the association of the rest of the components. We describe here that CK1ε is activated by the PP2A/PR61ε phosphatase. Binding of Wnt ligands promotes the interaction of LRP5/6-associated CK1ε with Frizzled-bound PR61ε regulatory subunit, facilitating the access of PP2A catalytic subunit to CK1ε and its activation, what enables the recruitment of Dvl-2 to the receptor complex and the initiation of the Wnt pathway. Our results uncover the mechanism of activation of the canonical Wnt pathway by its ligands.