Cell Biology
Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene*

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Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr → Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr → Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.

E3 ubiquitin ligase
leukemia
mutagenesis
oncogene
receptor tyrosine kinase
CBL
proline-Rich region
tyrosine kinase binding domain

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*

This work was supported in part by National Institutes of Health Grants CA87986, CA105489, CA99163, and CA116552 (to H. B.) and CA96844 and CA144027 (to V. B.), Department of Defense Grants W81XWH-07-1-0351 and W81XWH-11-1-0171 (to V. B.), Nebraska Department of Health and Human Services LB-506 Grant 2014-01 and LB606 18123-Y3 (to H. B.), National Institutes of Health Institutional Development Award P30 GM106397 (IDeA) from the NIGMS. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1

These authors contributed equally to this work.

2

Trainee under the NCI Cancer Biology Training Grant from the National Institutes of Health.

3

Supported in part by the “Dr. Raphael Bonita Fund.”

4

Recipients of University of Nebraska Medical Center graduate assistantships.