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1.  Wnt Inhibition Leads to Improved Chemosensitivity in Pediatric Acute Lymphoblastic Leukemia 
British journal of haematology  2014;167(1):87-99.
While childhood acute lymphoblastic leukemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis - relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analyzed the expression of Activated β-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β-catenin at relapse in 6 /10 patients. Furthermore, treatment of leukemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach.
PMCID: PMC4207443  PMID: 24995804
acute lymphoblastic leukemia; phosphoflow cytometry; Wnt inhibition; chemosensitivity; relapse
2.  Antagonistic Effect of Small Molecule Inhibitors of Wnt/β-catenin in Multiple Myeloma 
Anticancer research  2012;32(11):4697-4707.
Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment, and within the BM, a number of factors are secreted, including the Wnt ligands. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate Wnt signaling in multiple myeloma. The canonical Wnt pathway which is, mediated through the transcriptional effector β-catenin (β-cat) is commonly deregulated in many cancers. Cells with active β-cat-regulated transcription (CRT) are protected against apoptosis; conversely inhibition of CRT may prevent cell proliferation.
Materials and Methods
In this study, we tested the efficacy of recently described inhibitors of CRT (iCRTs; oxazole and thiazole) for their selective antagonistic effect on Wnt-β-cat response in MM cells MM1, U266, BMSC and primary BMMC obtained from patient samples (n=16).
We demonstrate that iCRTs we used block Wnt/β-cat reporter activity, down regulate β-cat expression and inhibit cell proliferation in a dose dependent manner with an optimal dose closer to 15 µM. Our data further indicate that iCRTs do not influence the expression of the upstream components of the Wnt pathway DKK1 at the optimal dose, suggesting that iCRTs may specifically target β-cat in MM cells. Additionally, iCRT-treatment of MM cells co-cultured with BMSC showed an inhibitory effect on VEGF and cell migration.
This study provides the first in vitro data evaluation of newly described iCRTs as potential Wnt-β-cat/VEGF pathway antagonists in multiple myeloma.
PMCID: PMC3983472  PMID: 23155232
Multiple myeloma; Wnt; β-catenin; transcription; VEGF and DKK1
3.  HIV’s Nef Interacts with β-Catenin of the Wnt Signaling Pathway in HEK293 Cells 
PLoS ONE  2013;8(10):e77865.
The Wnt signaling pathway is implicated in major physiologic cellular functions, such as proliferation, migration, cell fate specification, maintenance of pluripotency and induction of tumorigenicity. Proliferation and migration are important responses of T-cells, which are major cellular targets of HIV infection. Using an informatics screen, we identified a previously unsuspected interaction between HIV’s Nef protein and β-catenin, a key component of the Wnt pathway. A segment in Nef contains identical amino acids at key positions and structurally mimics the β-catenin binding sites on endogenous β-catenin ligands. The interaction between Nef and β-catenin was confirmed in vitro and in a co-immunoprecipitation from HEK293 cells. Moreover, the introduction of Nef into HEK293 cells specifically inhibited a Wnt pathway reporter.
PMCID: PMC3795062  PMID: 24130899
4.  Regulation of Pluripotency and Cellular Reprogramming by the Ubiquitin Proteasome System 
Cell stem cell  2012;11(6):783-798.
While transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for post-translational modifications in these processes. A key mechanism of post-translational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here we map, using shotgun proteomics, the ubiquitinated protein landscape during ES cell differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify novel regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme, Psmd14, and the E3 ligase, Fbxw7, and characterize their importance in ES cell pluripotency and cellular reprogramming. This is the first global characterization of the UPS as a key regulator of stem cell pluripotency, opening the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.
PMCID: PMC3549668  PMID: 23103054
5.  A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells 
Development (Cambridge, England)  2013;140(6):1171-1183.
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.
PMCID: PMC3585656  PMID: 23444350
Oct4; Wnt signaling; Mouse embryonic stem cells; Pluripotency; β-Catenin
6.  PAPTi: A Peptide Aptamer Interference Toolkit for Perturbation of Protein-Protein Interaction Networks 
Scientific Reports  2013;3:1156.
Signaling proteins often form dynamic protein-protein interaction (PPI) complexes to achieve multi-functionality. Methods to abrogate a subset of PPI interfaces without depleting the full-length protein will be valuable for structure-function relationship annotations. Here, we describe the use of Peptide Aptamer Interference (PAPTi) approach for structure-function network studies. We identified peptide aptamers against Dishevelled (Dsh) and β-catenin (β-cat) to target the Wnt signaling pathway and demonstrate that these FN3-based MONOBODYs (FNDYs) can be used to perturb protein activities both in vitro and in vivo. Further, to investigate the crosstalk between the Wnt and Notch pathways, we isolated FNDYs against the Notch Ankyrin (ANK) region and demonstrate that perturbing the ANK domain of Notch increases the inhibitory activity of Notch towards Wnt signaling. Altogether, these studies demonstrate the power of the PAPTi approach to dissect specific PPI interactions within signaling networks.
PMCID: PMC3557448  PMID: 23362456
7.  A Systematic Screen for Micro-RNAs Regulating the Canonical Wnt Pathway 
PLoS ONE  2011;6(10):e26257.
MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of â-catenin (β-cat), miR-1 and miR-613 act upstream of β-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive β-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer.
PMCID: PMC3197157  PMID: 22043311
8.  Bili Inhibits Wnt/β-Catenin Signaling by Regulating the Recruitment of Axin to LRP6 
PLoS ONE  2009;4(7):e6129.
Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ß-catenin pathway.
Methodology/Principal Findings
In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a β-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/β-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/β-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation.
These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/β-catenin pathway.
PMCID: PMC2701632  PMID: 19572019
9.  Notch modulates Wnt signalling by associating with Armadillo/ß-catenin and regulating its transcriptional activity 
Development (Cambridge, England)  2005;132(8):1819-1830.
The establishment and stability of cell fates during development depend on the integration of multiple signals, which ultimately modulate specific patterns of gene expression. While there is ample evidence for this integration at the level of gene regulatory sequences, little is known about its operation at other levels of cellular activity. Wnt and Notch signalling are important elements of the circuitry that regulates gene expression in development and disease. Genetic analysis has suggested that in addition to convergence on the transcription of specific genes, there are modulatory cross regulatory interactions between these signalling pathways. Here we report that the nodal point of these interactions is an activity of Notch which regulates the activity and the amount of the active/oncogenic form of Armadillo/ß-catenin. This activity of Notch is independent of that induced upon cleavage of its intracellular domain and which mediates transcription through Su(H)/CBF1. The modulatory function of Notch described here, contributes to the establishment of a robust threshold for Wnt signalling which is likely to play important roles in both normal and pathological situations
PMCID: PMC2500123  PMID: 15772135
10.  Links between signal transduction, transcription and adhesion in epithelial bud development 
Nature  2003;422(6929):317-322.
The morphogenesis of organs as diverse as lungs, teeth and hair follicles is initiated by a downgrowth from a layer of epithelial stem cells1,2. During follicular morphogenesis, stem cells form this bud structure by changing their polarity and cell–cell contacts. Here we show that this process is achieved through simultaneous receipt of two external signals: a Wnt protein to stabilize β-catenin, and a bone morphogenetic protein (BMP) inhibitor to produce Lef1. β-Catenin then binds to, and activates, Lef1 transcription complexes that appear to act uncharacteristically by downregulating the gene encoding E-cadherin, an important component of polarity and intercellular adhesion. When either signal is missing, functional Lef1 complexes are not made, and E-cadherin downregulation and follicle morphogenesis are impaired. In Drosophila, E-cadherin can influence the plane of cell division and cytoskeletal dynamics3. Consistent with this notion, we show that forced elevation of E-cadherin levels block invagination and follicle production. Our findings reveal an intricate molecular programme that links two extracellular signalling pathways to the formation of a nuclear transcription factor that acts on target genes to remodel cellular junctions and permit follicle formation.
PMCID: PMC2424170  PMID: 12646922
11.  A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila 
Genome Biology  2007;8(9):R203.
A second generation dsRNA library was used to re-assess factors that influence the outcome of transcriptional reporter-based whole-genome RNAi screens for the Wnt/Wingless (wg) and Hedgehog (hh)-signaling pathways.
Off-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.
PMCID: PMC2375041  PMID: 17903264
12.  A developmental conundrum 
The Journal of Cell Biology  2002;158(2):331-344.
Wnt signaling orchestrates morphogenetic processes in which changes in gene expression are associated with dramatic changes in cell organization within developing tissue/organss. Upon signaling, excess β-catenin not utilized at cell–cell junctions becomes stabilized, where it can provide the transcriptional activating domain for Lef/Tcf DNA binding proteins. In skin epithelium, forced stabilization of β-catenin in epidermis promotes hair follicle morphogenesis, whereas conditional removal of β-catenin in hair progenitor cells specifies an epidermal fate. We now report that a single protein, a stabilized version of β-catenin lacking the COOH-terminal transactivation domain, acts in epidermis to promote hair fates and in hair cells to promote epidermal fate. This reveals fundamental differences in ways that epidermal and hair cells naturally respond to β-catenin signaling. In exploring the phenotype, we uncovered mechanistic insights into the complexities of Lef1/Tcf/β-catenin signaling. Importantly, how a cell will respond to the transgene product, where it will be localized, and whether it can lead to activation of endogenous β-catenin/Tcf/Lef complexes is specifically tailored to skin stem cells, their particular lineage and their relative stage of differentiation. Finally, by varying the level of β-catenin signaling during a cell fate program, the skin cell appears to be pliable, switching fates multiple times.
PMCID: PMC2173126  PMID: 12135986
Wnt signaling; epidermis; hair follicle; β-catenin; morphogenesis

Results 1-12 (12)