High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3 ؉ multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand-associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand. (Blood. 2012;120(19): 4049-4057) IntroductionThe Flt3 receptor tyrosine kinase is expressed at high levels on most myeloid and lymphoblastic leukemias, 1 and for many years it has been considered a potential target for compounds that inhibit its kinase activity. 2,3 However, it has been difficult to test this possibility because of a lack of both suitable animal models and Flt3 inhibitors with the desired potency, specificity, and pharmacokinetic properties. Recently, we characterized a mouse with an inactivating knock-in mutation in the RING finger domain of the c-Cbl E3 ubiquitin ligase that has provided a model for studying myeloid malignancies driven by enhanced Flt3 signaling. This mouse develops a myeloproliferative disease (MPD) progressing to lethal leukemia that is characterized by markedly elevated WBC counts, splenomegaly, and extensive myeloid cell invasion into peripheral organs. 4 Analysis of Lin Ϫ Sca-1 ϩ c-Kit ϩ (LSK) cells in the BM revealed a marked expansion of cells expressing high levels of Flt3 compared with LSK cells from wild-type and c-Cbl-deficient mice. 4 Furthermore, the Flt3 ϩ LSK cells (defined as multipotent progenitors, MPPs) 5 showed enhanced Flt3 signaling to the Erk and PI3K pathways. When these mice were mated to Flt3 ligand-deficient mice, the doubly mutant mice did not develop leukemia, 4 indicating that Flt3 signaling is an essential component for driving disease development.Mutations in c-Cbl have been identified in 5%-20% of patients within the World Health Organization (WHO) groupings of myelodysplastic syndrome (MDS) and myelodysplastic/myeloproliferative neoplasm (MDS/MPN). 6-14 MDS/MPN patients are further classified into chronic myelomonocytic leukemia, juvenile ...

The RING finger type E3 ubiquitin ligase, Cbl-b, is abundantly expressed in bone marrow-derived mast cells (BMMCs) and functions as a potent negative regulator of signalling responses from the high-affinity IgE receptor (FcvarepsilonRI). To determine the contribution of Cbl-b E3 ligase activity we generated knockin mice with a loss-of-function mutation in the RING finger domain. We find the mice to be healthy and, unlike equivalent c-Cbl RING finger mutant mice, produce homozygous offspring at the expected frequency. Comparative analyses of BMMCs from Cbl-b knockout and Cbl-b RING finger mutant mice revealed that both showed similarly enhanced FcvarepsilonRI signalling compared to wild-type cells for most parameters examined. A notable exception was a markedly higher level of activation of IkappaB kinase (IKK) in Cbl-b knockout BMMC compared to RING finger mutant-derived cells. In addition BMMCs from the Cbl-b RING finger mutant did not retard FcvarepsilonRI internalization to the extent observed for knockout cells. Most striking however was the finding that RING finger mutant mast cells do not produce the very high levels of TNF-alpha, IL-6, and MCP-1 evident in Cbl-b knockout cultures following FcvarepsilonRI activation. Thus the ability of Cbl-b to function as a negative regulator of FcvarepsilonRI signalling that promotes inflammatory cytokine production is largely independent of the RING finger domain.

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCRdirected thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

The Ets transcription factor Fli-1 is preferentially expressed in hematopoietic tissues and cells, including immature T cells, but the role of Fli-1 in T cell development has not been closely examined. To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development. We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo. Surprisingly, Fli-1 overexpression in vivo eventuated in development of pre-T cell lymphoblastic leukaemia/lymphoma (pre-T LBL). Known Fli-1 target genes such as the pro-survival Bcl-2 family members were not found to be upregulated. In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours. These data show a novel function for Fli-1 in T cell development and leukaemogenesis and provide a new mouse model of pre-T LBL to identify treatment options that target the Fli-1 and Notch1 signalling pathways.