Abstract
Stem cell leukemia/lymphoma syndrome (SCLL) develops as a result of the ligand-independent, constitutive activation of FGFR1 kinase as a result of chromosome translocations that lead to chimeric FGFR1 kinases. This disorder typically presents as a chronic myeloproliferative disease accompanied by B-cell and T-cell lymphomas with rapid progression to AML. The BCR-FGFR1 variant of SCLL is relatively rare, with only ~20 cases reported to date but is perhaps one of the more aggressive forms of the disease. While the BCR-FGFR1 rearrangement clearly activates the FGFR1 kinase domain through oligomerization, which is also the case for the other FGFR1 chimeric kinases. The more aggressive progression of BCR-FGFR1-driven disease, however, may be a result of the effects of unique domains within the BCR component of the chimeric protein.
To investigate the relative involvement of the BCR GEF subdomain for BCR-FGFR1 driven SCLL, we created deletion mutant isoforms of the chimeric protein and evaluated their relative contribution to primary bone marrow cells proliferation and colony-formation in vitro, and SCLL development in a syngeneic, in vivo transduction and transplantation model. Our data demonstrates that, in fact, the GEF domain acts to suppress the potency of the BCR-FGFR1 kinase, since deletion constructs lead to an enhanced bone marrow cell proliferation and granulocyte/macrophage or pre-B-lymphocytes colony formation in in vitro assay, and much more aggressive progression of SCLL in vivo with a shorter latency period and severe lymph node infiltration. Histological analysis of the GEF deletion tumors compared with BCR-FGFR1 shows increased lymphocytes in the peripheral blood, increased hypercellularity in the bone marrow, greater disorganization in the spleen and increased infiltration in the liver. The immunofluorescence staining results shows besides the common B220+IgM- immunophenotype in both the full length and deletion construct group mice, there is a significantly higher levels of Sca1+Kit+ cells, indicating a more stem cell-like phenotype in the latter group.
To investigate differences in molecular signaling pathways in the cells expressing the different BCR-FGFR1 derivatives, we performed reverse phase protein array (RPPA) analysis comparing relative protein levels of well-characterized signal transduction proteins between BaF3 cells expressing either BCR-FGFR1 or the GEF deletion. In this analysis we observed decreases in Rock1 and Pten levels and an increase in activated Akt in the cells with the GEF deletion, which was validated by western blot in both the SCLL cell lines and primary tumors from mice transplanted with bone marrow cells expressing either the full length BCR1-FGFR1 or the GEF deletion construct. To further investigate the function of RhoA in leukemogenesis, we downregulated RhoA in BaF3 cells expressing BCR1-FGFR1 using shRNAs. In these cells, suppression of RhoA expression led to reduced levels of the GTP-active form, with proportional increases in pAkt and increased cell proliferation, comparable with cells transduced with the GEF deletion construct. The functional role of RhoA was further validated by a rescue experiment with constitutively active or inactive RhoA mutant constructs.
To relate these observations to SCLL cells, we created BBC2 cells that either overexpressed RhoA or, through expression of shRNA, showed reduced levels of RhoA. In these cells the inverse functional relationship between RhoA expression and pAKT and p21 levels was confirmed. Analysis of cell growth rates shows that knockdown of RhoA leads to increased cell viability compared with empty vector transduced cells and reduced cell viability when RhoA is overexpressed. In vivo engraftment data showed that the disease in these mice with RhoA knock down developed more rapidly than for the parental BBC2 cells expressing the BCR-FGFR1 kinase. This data further confirmed the direct involvement of RhoA signaling in the BCR-FGFR1 driven SCLL.
Taken together, our studies demonstrated that, the GEF domain acts to suppress the potency of the BCR-FGFR1 kinase, and this attenuation of leukemogenesis is related to enhanced RhoA activation, PTEN activation and reduced pAKT activation as a consequence.
Disclosures
No relevant conflicts of interest to declare.
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