Control of JAK2 Ubiquitination and Stability By CBL Family E3 Ubiquitin Ligases in Hematopoietic Stem/Progenitor Cells and Myeloid Malignancies

Janus Kinase 2 (JAK2) is the central tyrosine kinase in the signaling pathway of an array of hematopoietic receptors including thrombopoietin receptor (TPOR or MPL) in hematopoietic stem cells (HSCs) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in myeloid progenitors. While JAK2 plays an essential role in normal hematopoietic development, uncontrolled JAK2 signaling results in hematopoietic malignancies, including myeloproliferative neoplasms (MPNs). JAK2 protein level and activity are tightly regulated to ensure its normal function in normal hematopoiesis and prevent malignant transformation. JAK2 levels have been proposed to be regulated by a number of E3 ubiquitin ligases but none of them, when depleted in vivo, increases JAK2 protein level in HSC and progenitors (HSPCs), expands HSC pool, or enhances multiple lineage hematopoiesis.

Here we report that JAK2 stability is regulated by the CBL family E3 ubiquitin ligases, c-CBL and CBL-b, via the adaptor protein LNK (also called SH2B3). We previously showed that LNK directly inhibits MPL/JAK2 signaling in HSPCs (J Clin Invest. 2008;118(8):2832-2844). c-Cbl^-/- mice phenocopy Lnk^-/- mice, exhibiting an augmented HSPC pool with superior transplantability and hypersensitivity to cytokines (Genes Dev. 2008;22(8):992-997). Importantly, CBLloss-of-function mutations are found in a wide range of myeloid malignancies with the most frequent occurrence in chronic and juvenile myelomonocytic leukemia (CMML and JMML), both of which bear poor prognosis with propensity for AML (acute myeloid leukemia) progression.

Depletion of c-CBL and CBL-b in human hematopoietic progenitor TF-1 cells increases JAK2 level, extends JAK2 half-life and enhances cytokine-dependent JAK2 signaling and cell growth. Overexpression of an E3 ligase-inactive CBLmutant shows similar results, indicating that the E3 ubiquitin ligase activity of CBL is crucial. Ubiquitination and subsequent degradation of JAK2 is triggered by phosphorylation upon TPO stimulation. CBL/CBL-b dual depletion, overexpression of E3-dead CBL, or LNK depletion abrogated JAK2 ubiquitination, indicating CBL family E3 ligases are critical for cytokine-induced JAK2 ubiquitination and degradation, via LNK.

Using a novel Tamoxifen-inducible double KO mouse model Cbl^f/f;Cbl-b^f/f;Cre^ERT2, we investigated the role of CBL proteins in JAK2 regulation in HSCs. We showed that in vitro excision of c-Cbl and Cbl-b in primary HSPCs increased JAK2 levels and signaling. Cbl/Cbl-b-null HSPCs phenocopy Lnk^-/- HSCs, exhibiting superior transplantability. In vivo excision of c-Cbl and Cbl-b in mice transplanted with total BM cells led to an aggressive CMML-like MPN, similarly to our previous publication (Proc Natl Acad Sci U S A. 2010;107(37):16274-16279). Importantly, treatment with JAK inhibitor Ruxolitinib, significantly reduced HSPCs and mitigated CMML development.

Finally, our mechanistic work was translated into human hematopoietic malignancies. We demonstrate that primary human CBL^mutAMLs exhibit elevated JAK2 protein levels and enhanced GM-CSF-induced STAT5 signaling, in comparison to CBL^WTAMLs. In addition, CBL^mutAMLs are more sensitive to JAKi than CBL^WTAMLs.

Taken together, our studies unravel a novel signaling axis, CBL-LNK-JAK2, in regulating JAK2 protein turnover and signaling in HSPCs and MPNs. Our results will likely reveal new therapeutic strategies in treating CBL^mut myeloid malignancies.