Petranel Ferrao

Effects of Mutant c-Kit in Early Myeloid Cells

Activating mutations in c-Kit, the receptor for Stem Cell Factor (SCF), have been identified in dysplasias and leukaemias of the mast cell lineage and have been shown to contribute to transformation in model systems. Early myeloid cells also normally express c-Kit and their survival, proliferation and differentiation is promoted by SCE It might therefore be expected that c-Kit mutations could also be involved in some acute and/or chronic myeloid leukaemias. We have found that mutant c-Kit (and normal c-Kit in the presence of SCF) provides a strong differentiation stimulus in normal and immortalised murine early myeloid cells. Since maturation of haemopoietic cells, with the exception of mast cells, results in down-regulation of c-Kit expression, the transforming effects of mutant receptor may be self-limiting in most lineages. This is consistent with the observation that multipotential progenitor cells from some patients with systemic mastocytosis express mutant c-Kit. However, c-Kit mutations have been observed in a few cases of myelodysplastic syndromes or AML without mast cell features. Oncogenesis involves multiple genetic changes and the phenotype of malignant haemopoietic cells expressing mutant c-Kit may be influenced by co-oncogenic events. For example mutations blocking the differentiative effect of mutant c-Kit might result in AML rather than mastocytosis. Thus the extent to which c-Kit mutations contribute to malignancies of early myeloid phenotype remains unknown, and resolution of this issue is complicated by the heterogeneity of this family of diseases.

Intracellular P-gp contributes to functional drug efflux and resistance in acute myeloid leukaemia

Drug compartmentalization as well as drug efflux can contribute to drug resistance. We demonstrate the presence of P-gp in intracellular vesicles in certain AML cell lines and show localization of DNR to a similar subcellular compartment(s) that can be altered in the presence of P-gp inhibitors. Analysis of leukaemic cell lines and 50 AML patient samples showed that the level of P-gp mRNA or total P-gp protein correlated better with drug efflux than surface P-gp protein, suggesting that intracellular P-gp may contribute to MDR in AML. Therefore, the level of total P-gp protein or mRNA may be a better indicator of MDR than surface P-gp protein. In addition, we provide evidence for a novel mechanism of drug sequestration in K562 myeloid leukaemic cells.

Association of paediatric mastocytosis with a polymorphism resulting in an amino acid substitution (M541L) in the transmembrane domain of c-KIT

Background  The receptor tyrosine kinase c‐KIT plays a key role in normal mast cell development. Point mutations in c‐KIT have been associated with sporadic or familial mastocytosis.

Constitutively active mutant D816VKit induces megakayocyte and mast cell differentiation of early haemopoietic cells from murine foetal liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF.

PI3 Kinase mediates transformation of hematopoietic cells by the V816 c-kit mutant

Abstract C-Kit is a receptor tyrosine kinase that binds stem cell factor. Substitution of valine for aspartic acid 816 (V816) constitutively activates human c-Kit and this mutation is found in patients with mastocytosis, leukemia and germ cells tumors. Transduction of immortalized murine progenitor cells with wild-type c-Kit (MIHC-Kit) results in stem cell factor-induced growth (SCF), while cells expressing V816 c-Kit (MIHC-V816) are factor-independent and tumorigenic. The mechanisms mediating transformation by V816 c-Kit are unknown. SCF activates Erk 1, Erk2 and P13 kinase in MIHC-Kit cells, and P13 kinase contributes to activation of Akt and Jnks. In MIHC-V816 cells, P13 kinase, Jnk 1 and Jnk 2 were activated, but Akt, Erk 1 and Erk 2 were not. Thus, V816 c-Kit constitutively activates P13 kinase, but not all signaling pathways activated by wild-type c-Kit. Further, pathways downstream of P13 kinase are altered in MIHC-V816 cells. Studies with a P13 kinase inhibitor and V816/F721 c-Kit, a mutant incapable of recruiting P13 kinase, indicate that constitutive activation of P13 kinase plays a role in factor-independent growth mediated by V816 c-Kit and is critical for tumorigenicity of MIHC. These data are the first to demonstrate the role of P13 kinase in transformation of hematopoietic cells by this oncogenic c-Kit mutant.