Abstract
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.
Highlights
CBL and CBL-B, but not CBL-C, contain an extensive C-terminal region that includes a proline-rich region (PRR) for interactions with SH3 domain-containing proteins such as Grb2, Nck, CIN85, and Src family protein-tyrosine kinases (PTKs) [2,3,4,5,6] and several tyrosine residues that undergo phosphorylation by associated PTKs to form binding sites for interactions with SH2 domain-containing signaling intermediates: Tyr(P)-700 mediates binding to Vav family of Rho/Rac/Cdc42 guanine nucleotide exchange factors; Tyr(P)-731 mediates binding to PI3-kinase via its p85 subunit, and Tyr(P)-774 mediates binding to Crk family adaptors, which in turn promote the interaction with C3G, a guanine nucleotide exchange factor for Ras-related small GTPases Rap1 (6 –9)
Whether the gain-of-function of mutant CBL reflects the relative increase in the dosage of mutant CBL protein relative to that of CBL-B, the remaining wild type CBL family member expressed in hematopoietic stem cells or a true gain-of-function that can be observed in the absence of any wild type CBL and CBL-B expression is unknown
We introduced retroviruses that code for wild type (WT) CBL or the leukemia-associated CBL-Y371H mutant (Fig. 1A) as well as GFP into primary mouse CBL-null or CBL/CBL-B-null hematopoietic stem/progenitor cells (HSPCs), selected the infected cells by FACS based on GFP expression (Fig. 1B), and assessed the extent of cytokine hyper-responsiveness imparted by mutant CBL expression
Summary
Investigations in a variety of cell biological models have elucidated the basic biochemical mechanisms by which CBL proteins function as negative regulators of PTK signaling [1, 12]. A key additional feature of CBL mutations found in myeloid leukemias is that the remaining wild type (WT) CBL allele is replaced by the mutant allele, resulting in the frequently observed acquired uniparental disomy at the 11q-23 locus where the CBL gene is located [15, 27, 30] This has led to suggestions that mutant CBL proteins may function through a gain-of-function mechanism and/or by competing with the remaining wild type CBL family member CBL-B [34], because CBL-C is not expressed in hematopoietic lineages [35]. By engineering second-site mutations or deletions in key structural motifs/domains of the CBL-Y371H mutant to disable its protein-protein interactions, we identify a critical role of the proline-rich region in its ability to promote cytokine hypersensitivity in murine primary hematopoietic stem/progenitor cells (HSPCs), which we confirm and extend by expressing these engineered mutants in the TF-1 human leukemia cell line. Coupled with an essential requirement of an intact TKB domain of mutant CBL, our results provide a model for how mutant CBL functions as a dominant oncogene
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