The receptor tyrosine kinases FLT3 and KIT are highly expressed on the surface of leukemic blasts in most patients with acute myeloid leukemia. Although about one third of patients display activating mutations in FLT3 (and more rarely in KIT), the majority of patients have no mutations in FLT3 or KIT. Previously, we demonstrated that Cbl functions as the E3 ligase for both FLT3 and KIT, and that ligase-inactivating mutations of Cbl stabilize FLT3 and KIT on the cell surface by preventing endocytosis and degradation (Sargin et al, Blood 2007). Furthermore, we demonstrated that expression of E3-ligase deficient Cbl mutants led to the development of a myeloproliferative disease in a murine bone marrow transplantation model (Bandi et al, Blood 2009). However, Cbl mutations are rarely found in AML.
Here, we investigated the role of the Cbl regulators suppressors of T-cell signaling 1 and 2(STS1 and STS2) in stabilizing wild-type FLT3 and KIT on the cell surface of hematopoietic stem and progenitor cells (HSPCs). STS1 is ubiquitously expressed, while STS2 expression is restricted to the hematopoietic tissue. STS1 and STS2 constitutively bind to Cbl, while their binding to FLT3 and KIT is dependent on ligand-activation by FL and SCF, respectively. Interestingly, STS1 (but not STS2) functions as a tyrosine phosphatase for both ligand-activated FLT3 and KIT. This required the PGM domain of STS1, as PGM point mutant of STS1 did not dephosphorylate FLT3 or KIT. In line with this, knockdown of STS1 using stably expressing shRNA constructs showed a significant hyperphosphorylation of FLT3 and KIT.
By using STS1/STS2 single and double knockout mice, we analyzed the effects of STS1 and STS2 on hematopoietic stem and progenitor cells in vivo. We found that deficiency of STS1 causes an increase of both absolute number and frequency of LSK (lineage marker-, KIT+, Sca1+) cells, which contain HSPCs. This phenotype was even more pronounced in STS1 and STS2 double knockout (dKO) mice, and is mainly attributable to the short term hematopoietic stem cell (ST-HSC) and multipotent progenitor (MPP) cell population, as defined by both standard and SLAM markers. Colony assays using primary bone marrow cells revealed a significantly higher colony forming ability in STS1-KO and dKO cells compared to wild type (wt) cells, particularly after serial replating. A careful analysis of the cells derived from methylcellulose culture revealed an increased proportion of immature (Mac1- CD48+ CD16/32-) cells in STS1-KO and dKO cells. Competitive repopulation assays showed an advantage for dKO cells when compared to wt, suggesting that the LT-HSC compartment is also affected. Even more pronounced were the differences in CFU-S assays (colony forming units spleen), displaying significantly more colonies of dKO compared to wt donor cells, functionally demonstrating a significantly increased ST-HSC / MPP population in dKO donors. A detailed analysis of the downstream signaling events demonstrated that loss of STS1 specifically causes an activated PI3-Kinase / AKT pathway.
In summary, our data demonstrates that STS1 functions as a phosphatase of FLT3 and KIT and, using genetic mouse models, indicates a critical role in the maintenance and proliferation of long-term and short-term hematopoietic stem cells. This may also affect sensitivity to kinase inhibitors.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.