Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy

Abstract

Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling.

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

“…Next, we expressed the WT CBL or CBL-Y371H mutant in CBL/CBL-B DKO HSPCs and assessed their responsiveness to SCF or thrombopoietin, as above. As we have shown previously (36,37), CBL/CBL-B DKO HSPCs by themselves show a dramatically heightened response to cytokines (Fig. 1, F-H).…”

“…5A). As expected (16,37), SCF stimulation induced a higher and more sustained AKT and ERK1/2 phosphorylation in CBL-Y371H-expressing TF-1 cells compared with the empty vector or WT CBL-expressing control cells. Importantly, the TF-1 cells expressing the PRRdeleted CBL-Y371H mutant failed to exhibit the enhanced and sustained AKT and ERK1/2 phosphorylation (Fig.…”

“…Our recent studies in murine hematopoietic stem cells have highlighted the importance of stem cell factor (SCF)/c-KIT receptor signaling as an important target of CBL proteins (37). Furthermore, aberrations of c-KIT signaling are well established in myeloid leukemia (41).…”

“…derived from mouse models of mutant CBL leukemogenesis and leukemic cell lines have demonstrated that cytokine hypersensitivity promoted by mutant CBL proteins or by loss of CBL/ CBL-B expression is associated with increased downstream signaling (16,32,37,39,40,(42)(43)(44). Given our findings that the PRR of mutant CBL is essential for its ability to promote cytokine hyper-responsiveness, we assessed the impact of PRR deletion on the ability of the CBL-Y371H mutant to promote the hyper-activation of signaling in response to SCF stimulation.…”

“…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). Consistent with this idea, mouse hematopoietic stem cell knock-out of CBL and CBL-B together (36,37) or a CBL RF domain mutant knock-in on a CBL-null background (16) leads to a myeloproliferative disease similar to human leukemic disease caused by CBL/ CBL-B mutations. How the leukemia-associated CBL mutants function as dominant oncogenes, however, remains unclear.…”