The Tyrosine Kinase-Binding and Proline-Rich Domains of Mutant CBL Are Essential for Leukemogenesis

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL are associated with myeloid malignancies, yet the molecular mechanisms by which this tumor suppressor becomes a dominant oncogene to initiate the leukemogenic process are unclear. In this study, we used systematic mutagenesis to delineate the importance of the various protein-protein interaction domains/motifs of a CBL mutant that is most frequently found in human leukemias, Y371H, in inducing leukemogenesis. We tested the impact of these secondary mutations on the ability of CBL-Y371H to impart hypersensitivity to cytokines and to upregulate the associated signaling pathways in the TF1 leukemia cell line model, and in mouse hematopoietic stem/progenitor cells from Cbl-null mice to mimic the lack of wildtype CBL expression in most of the mutant CBL bearing leukemia patients. The secondary mutations included: Cbl G306E, to abrogate the ability of the TKB domain of mutant Cbl to bind to activated tyrosine kinases; internal deletion of the proline-rich domain (AA 477-688) to abrogate interactions with SH3 domain containing partners; Cbl-Y700/731/774F triple phosphorylation site mutant predicted not to interact with the SH2 domain-containing partners; and Cbl-1-436 deletion construct lacking all C-terminal motifs. Analyses of stably expressed mutants in TF-1 cells for hypersensitivity to SCF1 demonstrated an essential role of an intact TKB domain and a particularly important role of the proline-rich domain. Transient retroviral expression in Cbl-null primary murine hematopoietic stem/progenitor cells confirmed these results. Deletion of the proline-rich domain led to substantially less tyrosine phosphorylation of the oncogenic mutant Cbl as well as of the c-Kit receptor upon SCF stimulation, and cells expressing this mutant lacked the sustained activation of Erk1/2 and Akt typically seen after SCF stimulation of the Cbl-Y371H-expressing cells. Together, our data provide conclusive evidence that interaction of leukemogenic mutant Cbl proteins with the upstream tyrosine kinase (via the TKB domain) and with partner proteins (via the proline-rich domain) provides a basic mechanism for gain of function phenotype of mutant Cbl proteins. Our studies suggest that identification of the leukemogenesis-critical partners of the proline-rich domain and targeting of the TKB-tyrosine kinase interface provide new therapeutic approaches against mutant Cbl-driven leukemias.