ABSTRACT
Casein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1-induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition.
Subject(s)
Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase Idelta/antagonists & inhibitors , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Animals , Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/metabolism , Casein Kinase Idelta/genetics , Casein Kinase Idelta/metabolism , Cell Line, Tumor , Drug Delivery Systems , HEK293 Cells , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mice , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , PiperidinesABSTRACT
Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1(-/-) mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1(-/-);Wnt5a(-/-) mice revealed a greater loss of Nurr1(+) cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson's disease.
Subject(s)
Dopaminergic Neurons/physiology , Mesencephalon/growth & development , Neurogenesis/physiology , Stem Cells/cytology , Wnt Proteins/metabolism , Wnt Signaling Pathway/physiology , Wnt1 Protein/metabolism , Analysis of Variance , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Count , Cell Differentiation/physiology , Dopaminergic Neurons/metabolism , Immunohistochemistry , LIM-Homeodomain Proteins/metabolism , Mice , Mice, Knockout , Nerve Tissue Proteins/metabolism , Neurogenesis/genetics , Parkinson Disease/metabolism , Parkinson Disease/therapy , Stem Cells/metabolism , Transcription Factors/metabolism , Wnt-5a Protein , Wnt1 Protein/deficiencyABSTRACT
Near-haploid acute lymphoblastic leukemia is rare subgroup of the disease, which is very important due to very poor prognosis and resistance to treatment including novel monoclonal antibodies and CAR-T therapy.
ABSTRACT
Lrp6 is generally described as a receptor required for signal transduction in the Wnt/beta-catenin pathway. Wnt5a, however, is a Wnt ligand that usually does not activate Wnt/beta-catenin but rather activates noncanonical Wnt signaling. We have previously shown that Lrp6 can inhibit noncanonical Wnt5a/Wnt11 signaling and that Lrp5/6 loss-of-function produces noncanonical gain-of function defects, which can be rescued by loss of Wnt5a. Here, we describe other phenotypes found in Wnt5a/Lrp6 compound mutant mice, including a worsening of individual Wnt5a or Lrp6 loss of function phenotypes. Lrp6 haploinsufficiency in a Wnt5a-/- background caused spina bifida and exacerbated posterior truncation. Wnt5a-/-Lrp6-/- embryos displayed presomitic mesoderm morphogenesis, somitogenesis, and neurogenesis defects, which are much more severe than in either of the single mutants. Interestingly these results reveal a further level of complexity in processes in which Wnt5a and LRP6 cooperate, or oppose each other, during mouse development.
Subject(s)
Embryonic Development/genetics , LDL-Receptor Related Proteins/metabolism , Mesoderm/embryology , Neural Tube/embryology , Phenotype , Signal Transduction/physiology , Wnt Proteins/metabolism , Animals , DNA Primers/genetics , Embryonic Development/physiology , Genotype , Immunohistochemistry , In Situ Hybridization , LDL-Receptor Related Proteins/genetics , Low Density Lipoprotein Receptor-Related Protein-6 , Mice , Mice, Knockout , Wnt Proteins/genetics , Wnt-5a ProteinABSTRACT
Lrp5/6 are crucial coreceptors for Wnt/beta-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/beta-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6-/- mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.
Subject(s)
LDL-Receptor Related Proteins/chemistry , LDL-Receptor Related Proteins/metabolism , Receptors, LDL/chemistry , Receptors, LDL/metabolism , Signal Transduction , Wnt Proteins/metabolism , Xenopus Proteins/metabolism , Animals , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Embryonic Development/drug effects , Enzyme Activation/drug effects , Gene Deletion , Heart/embryology , Heart Defects, Congenital/embryology , Heart Defects, Congenital/metabolism , Heterozygote , Low Density Lipoprotein Receptor-Related Protein-5 , Low Density Lipoprotein Receptor-Related Protein-6 , Mice , Mice, Mutant Strains , Neural Tube Defects/metabolism , Oligonucleotides, Antisense/pharmacology , Phenotype , Protein Binding/drug effects , Protein Structure, Tertiary , Receptors, LDL/deficiency , Signal Transduction/drug effects , Wnt-5a Protein , Xenopus/embryology , Xenopus/metabolism , beta Catenin/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9-10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5-E13.5. Analysis of Wnt5a-/- mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a-/- mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.