Tyrosine Kinase-Directed Ubiquitin Ligases Cbl and Cbl-b Enforce Hematopoietic Stem Cell Quiescence By Negatively Regulating c-Kit and FLT3

Maintenance of quiescent hematopoietic stem cells (HSCs) is essential for life-long hematopoiesis. A prominent category of HSC quiescence regulators that determine HSC fate by directly interacting with niche-derived growth factors are receptor tyrosine kinases (RTKs). Cbl and Cbl-b are E3 ubiquitin ligases that are directed to activated tyrosine kinases and negatively regulate a number of cellular activation pathways. Previously, we established a conditional Cbl and Cbl-b double knockout (DKO) mouse strain using MMTV-Cre to delete floxed Cbl on a Cbl-b-null background, and identified that Cbl and Cbl-b function redundantly in controlling the growth factor-induced proliferation of HSCs. These DKO mice developed a rapidly fatal myeloproliferative disorder (MPD) accompanied by expansion of the HSC compartment. However, how the negative regulatory functions of Cbl-family proteins are integrated into HSC homeostatic program and the mechanistic basis for their role remain unknown.

Here, we utilize mouse models to examine the functional role of Cbl and Cbl-b in regulating HSCs and its potential mechanistic basis. Transplant analyses revealed that DKO HSCs are the disease-initiating cells. However, in vitro serial colony-forming assays showed that DKO HSCs possess a reduced colony-forming ability despite their hyper-proliferative status. Cell cycle analyses demonstrated a smaller pool of quiescent long-term HSCs in DKO mice. Non-responder assays in vivo showed a reduced frequency of functional HSCs in the DKO LSK compartment and serial HSC transplantation demonstrated compromised reconstitution ability of DKO HSCs, especially at the 2^nd round of transplantation. Mechanistically, DKO HSCs exhibit sustained signaling in response to c-Kit and FLT3 ligands, especially via p-Akt but also p-Erk and p-S6. The ligand-induced cell surface c-Kit and FLT3 receptor downregulation was slower in DKO HSCs. Furthermore, while c-Kit and FLT3 ligands promote higher proliferation of DKO bone marrow cells, this hyper-proliferation leads to loss of colony-forming potential that is significantly reversed by respective kinase inhibitors.

Together, our data reveal a novel and physiologically essential role of Cbl and Cbl-b in the enforcement of HSC quiescence and protection against exhaustion by fine-tuning the signaling pathways downstream of tyrosine kinase-coupled receptors such as c-Kit and FLT3. These findings could have significant implications for hematological neoplasms associated with mutations of Cbl.