Porcupine (PORCN) is a membrane-bound protein of the endoplasmic reticulum, which modifies Wnt proteins by adding palmitoleic acid. This modification is essential for Wnt ligand secretion. Patients with mutated PORCN display various skeletal abnormalities likely stemming from disrupted Wnt signaling pathways during the chondrocyte differentiation. To uncover the mechanism of PORCN action during chondrogenesis, we used 2 different PORCN inhibitors, C59 and LGK974, in several model systems, including micromasses, 3D cell cultures, long bone tissue cultures, and zebrafish animal model. PORCN inhibitors enhanced cartilaginous extracellular matrix (ECM) production and accelerated chondrocyte differentiation, which resulted in the earlier induction of cellular hypertrophy as well as cartilaginous mass expansion in micromass cultures and cartilaginous organoids. In addition, both PORCN inhibitors expanded the hypertrophic zone and reduced the proliferative zone in the growth plate. This led to a significant increase in cartilaginous tissue and ultimately resulted in the elongation of tibias in the mouse organ cultures. Also, LGK974 treatment of Danio rerio embryos induced expansion of craniofacial cartilage width together with the shortening of the body axis, which was consistent with a phenomenon occurring upon inhibition of non-canonical Wnt signaling. By combining PORCN inhibition with exogenous Wnt proteins activating either canonical/β-catenin (WNT3a) or non-canonical (WNT5a) signaling, we propose that the key mechanism mediating pro-chondrogenic effects of PORCN inhibition is the removal of canonical ligands that prevent chondrocyte differentiation. In summary, our results provide evidence of the distinct role of PORCN in both the early and late stages of cartilage development. Further, our data demonstrate that PORCN inhibitors can be used in the experimental and clinical strategies that need to trigger chondrocyte differentiation and/or cartilage outgrowth.

• Keywords
• Wnt, cartilage, chondrogenesis, hypertrophy, jaw hypoplasia, micromass cultures, orofacial anomalies, porcupine, tibia,
• Publication type
• Journal Article MeSH

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.

• Keywords
• Axin, NanoBRET, Wnt pathway, alternative splicing, casein kinase 1 alpha (CK1α), casein kinase 1 alpha-like (CK1α-like), gene knockout, inhibitor, phosphorylation, β-catenin,
• MeSH
• Alternative Splicing MeSH
• beta Catenin * metabolism   genetics   MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Casein Kinase Ialpha * metabolism   genetics   MeSH
• Glycogen Synthase Kinase 3 metabolism   genetics   MeSH
• Glycogen Synthase Kinase 3 beta metabolism   genetics   MeSH
• Humans MeSH
• Wnt3A Protein metabolism   genetics   MeSH
• Wnt Signaling Pathway * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• beta Catenin * MeSH
• Casein Kinase Ialpha * MeSH
• Glycogen Synthase Kinase 3 MeSH
• Glycogen Synthase Kinase 3 beta MeSH
• Wnt3A Protein MeSH
• WNT3A protein, human MeSH Browser

The migratory properties of leukemic cells are commonly associated with their pathological potential and can significantly affect the disease progression. While the research in immunopathology mostly employed powerful indirect methods such as flow cytometry, these cells were rarely observed directly using live imaging microscopy. This is especially true for the malignant cells of the B-cell lineage, such as those originating from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In this study, we employed open-source image analysis tools to automatically and quantitatively describe the amoeboid migration of four B-cell leukemic and lymphoma cell lines and primary CLL cells. To avoid the effect of the shear stress of the medium on these usually non-adherent cells, we have confined the cells using a modified under-agarose assay. Surprisingly, the behavior of tested cell lines differed substantially in terms of basal motility or response to chemokines and VCAM1 stimulation. Since casein kinase 1 (CK1) was reported as a regulator of B-cell migration and a promoter of CLL, we looked at the effects of CK1 inhibition in more detail. Migration analysis revealed that CK1 inhibition induced rapid negative effects on the migratory polarity of these cells, which was quantitatively and morphologically distinct from the effect of ROCK inhibition. We have set up an assay that visualizes endocytic vesicles in the uropod and facilitates morphological analysis. This assay hints that the effect of CK1 inhibition might be connected to defects in polarized intracellular transport. In summary, 1) we introduce and validate a pipeline for the imaging and quantitative assessment of the amoeboid migration of CLL/MCL cells, 2) we provide evidence that the assay is sensitive enough to mechanistically study migration defects identified by the transwell assay, and 3) we describe the polarity defects induced by inhibition or deletion of CK1ε.

• Keywords
• B cells, amoeboid cell migration, casein kinase 1, chronic lymphocytic leukemia, live imaging, mantle cell lymphoma, uropod,
• Publication type
• Journal Article MeSH

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies characterized by the dependence on B-cell receptor (BCR) signaling and by the high expression of ROR1, the cell surface receptor for Wnt-5a. Both, BCR and ROR1 are therapeutic targets in these diseases and the understanding of their mutual cross talk is thus of direct therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src family participating in BCR signaling, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 in its kinase domain and aids the recruitment of the E3 ligase c-CBL. We show that ROR1 surface dynamics in migrating primary CLL cells as well as chemotactic properties of CLL cells were inhibited by Lyn inhibitor dasatinib. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of crosstalk between BCR and ROR1 signaling pathways.

• Keywords
• BCR, CLL, Lyn, Ror1, crosstalk, phosphorylation, signaling pathway,
• Publication type
• Journal Article MeSH

Dishevelled (DVL) is the central signal transducer in both Wnt/β-catenin-dependent and independent signalling pathways. DVL is required to connect receptor complexes and downstream effectors. Since proximal Wnt pathway components and DVL itself are upregulated in many types of cancer, DVL represents an attractive therapeutic target in the Wnt-addicted cancers and other disorders caused by aberrant Wnt signalling. Here, we discuss progress in several approaches for the modulation of DVL function and hence inhibition of the Wnt signalling. Namely, we sum up the potential of modulation of enzymes that control post-translational modification of DVL - such as inhibition of DVL kinases or promotion of DVL ubiquitination and degradation. In addition, we discuss research directions that can take advantage of direct interaction with the protein domains essential for DVL function: the inhibition of DIX- and DEP-domain mediated polymerization and interaction of DVL PDZ domain with its ligands.

• Keywords
• Casein kinase 1, DIX oligomerization, Dishevelled, PDZ inhibitors, Wnt signalling-related diseases,
• MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Phosphoproteins * metabolism   MeSH
• Humans MeSH
• Dishevelled Proteins * metabolism   MeSH
• Wnt Signaling Pathway * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Phosphoproteins * MeSH
• Dishevelled Proteins * MeSH

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

• Keywords
• HEK293, NIH3T3, RPE-1, Wnt/β-catenin, Wnt3a, cell signaling, ciliogenesis, primary cilia,
• Publication type
• Journal Article MeSH

The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials.

• Keywords
• AML, CK1α, CK1ε, CLL, MM, WNT pathway, casein kinase 1, inhibitors, leukemia, umbralisib,
• MeSH
• Molecular Targeted Therapy * MeSH
• Hematologic Neoplasms drug therapy   enzymology   pathology   MeSH
• Casein Kinase I antagonists & inhibitors   chemistry   metabolism   MeSH
• Humans MeSH
• Neoplastic Stem Cells drug effects   metabolism   pathology   MeSH
• Antineoplastic Agents pharmacology   therapeutic use   MeSH
• Wnt Signaling Pathway MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Casein Kinase I MeSH
• Antineoplastic Agents MeSH

High grade serous carcinoma of the ovary, fallopian tube, and peritoneum (HGSC) is the deadliest gynecological disease which results in a five-year survival rate of 30% or less. HGSC is characterized by the early and rapid development of metastases accompanied by a high frequency of ascites i.e. the pathological accumulation of fluid in peritoneum. Ascites constitute a complex tumor microenvironment and contribute to disease progression by largely unknown mechanisms. Methods: Malignant ascites obtained from HGSC patients who had undergone cytoreductive surgery were tested for their ability to induce WNT signaling in the Kuramochi cell line, a novel and clinically relevant in vitro model of HGSC. Next, cancer spheroids (the main form of metastatic cancer cells in ascites) were evaluated with respect to WNT signaling. Kuramochi cells were used to determine the role of individual WNT signaling branches in the adoption of metastatic stem cell-like behavior by HGSC cells. Furthermore, we analyzed genomic and transcriptomic data on WNT/Planar Cell Polarity (PCP) components retrieved from public cancer databases and corroborated with primary patient samples and validated antibodies on the protein level. Results: We have shown that ascites are capable of inducing WNT signaling in primary HGSC cells and HGSC cell line, Kuramochi. Importantly, patients whose ascites cannot activate WNT pathway present with less aggressive disease and a considerably better outcome including overall survival (OS). Functionally, the activation of non-canonical WNT/PCP signaling by WNT5A (and not canonical WNT/β-catenin signaling by WNT3A) promoted the metastatic stem-cell (metSC) like behavior (i.e. self-renewal, migration, and invasion) of HGSC cells. The pharmacological inhibition of casein kinase 1 (CK1) as well as genetic ablation (dishevelled 3 knock out) of the pathway blocked the WNT5A-induced effect. Additionally, WNT/PCP pathway components were differentially expressed between healthy and tumor tissue as well as between the primary tumor and metastases. Additionally, ascites which activated WNT/PCP signaling contained the typical WNT/PCP ligand WNT5A and interestingly, patients with high levels of WNT5A protein in their ascites exhibited poor progression-free survival (PFS) and OS in comparison to patients with low or undetectable ascitic WNT5A. Together, our results suggest the existence of a positive feedback loop between tumor cells producing WNT ligands and ascites that distribute WNT activity to cancer cells in the peritoneum, in order to promote their pro-metastatic features and drive HGSC progression. Conclusions: Our results highlight the role of WNT/PCP signaling in ovarian cancerogenesis, indicate a possible therapeutic potential of CK1 inhibitors for HGSC, and strongly suggest that the detection of WNT pathway inducing activity ascites (or WNT5A levels in ascites as a surrogate marker) could be a novel prognostic tool for HGSC patients.

• Keywords
• WNT signaling, ascites, casein kinase 1, high grade serous carcinoma of the ovary, fallopian tube and peritoneum, planar cell polarity pathway,
• MeSH
• Ascites metabolism   pathology   MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Survival Rate MeSH
• Biomarkers, Tumor metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Microenvironment physiology   MeSH
• Ovarian Neoplasms metabolism   mortality   pathology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Wnt Signaling Pathway * MeSH
• Neoplasm Grading MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers, Tumor MeSH