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1.
Genes Dev ; 31(10): 1007-1023, 2017 05 15.
Article in English | MEDLINE | ID: mdl-28611190

ABSTRACT

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b Importantly, primary human CBL mutated (CBLmut ) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies.


Subject(s)
Janus Kinase 2/metabolism , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/physiopathology , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing , Animals , Cell Line, Tumor , Cell Proliferation/genetics , Cytokines/metabolism , Enzyme Stability , Hematopoietic Stem Cells/enzymology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Janus Kinase 2/genetics , Leukemia, Myeloid, Acute/genetics , Membrane Proteins , Mice , Mutation , Proteolysis , Proto-Oncogene Proteins c-cbl/genetics , Signal Transduction/genetics , Ubiquitination
2.
Nat Commun ; 12(1): 6393, 2021 11 04.
Article in English | MEDLINE | ID: mdl-34737297

ABSTRACT

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.


Subject(s)
Glycogen Storage Disease Type II/enzymology , alpha-Glucosidases/metabolism , Animals , Autophagy , Enzyme Replacement Therapy , Female , Glycogen Storage Disease Type II/therapy , Liver/enzymology , Male , Mice , alpha-Glucosidases/genetics
3.
Nat Commun ; 7: 11226, 2016 Apr 18.
Article in English | MEDLINE | ID: mdl-27088444

ABSTRACT

Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Carrier Proteins/metabolism , Cytokines/metabolism , Janus Kinase 1/metabolism , Membrane Proteins/metabolism , Signal Transduction , Animals , CD4-Positive T-Lymphocytes/cytology , Carrier Proteins/genetics , Cell Proliferation , Cells, Cultured , Colitis/genetics , Colitis/metabolism , Female , Immunoblotting , Intercellular Signaling Peptides and Proteins , Lymphocyte Count , Male , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Proteolysis , Proteomics/methods , Reverse Transcriptase Polymerase Chain Reaction
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