Abstract

Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, KRAS, and NRAS are mutated in JMML patients, leading to aberrant regulation of RAS signaling. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated the mechanism by which CBL-Y371H, a linker region mutant, and CBL-C384R, a RING finger mutant, lead to enhanced GM-CSF signaling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor βc subunit in response to stimulation, although expression of total GM-CSFR βc was lower. This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Incubation with the JAK2 inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR βc in cells expressing CBL mutants, whereas treatment with the SRC kinase inhibitor dasatinib resulted in equalization of GM-CSFR βc phosphorylation signal between wild type CBL and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

Highlights

  • A subset of juvenile myelomonocytic leukemia (JMML) patients harbor mutations in the E3 ubiquitin ligase CBL

  • Inhibition of proteasomal and lysosomal degradation stabilized JAK2 to comparable levels in all three cell lines (Fig. 3C, middle). These results indicate that the expression of CBL-Y371H and CBL-C384R mutants affects the degradation of JAK2, leading to elevated levels, which in turn contribute to increased JAK2 phosphorylation and potentially signaling in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation

  • The majority of CBL mutations identified clustered in the linker region and RING finger, both of which play an important role in the E3 ligase activity of CBL

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Summary

Introduction

A subset of juvenile myelomonocytic leukemia (JMML) patients harbor mutations in the E3 ubiquitin ligase CBL. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor ␤c subunit in response to stimulation, expression of total GM-CSFR ␤c was lower This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways

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