Development of a Conditional In vivo Model to Evaluate the Efficacy of Small Molecule Inhibitors for the Treatment of Raf-Transformed Hematopoietic Cells

Abstract

Conditionally active forms of the Raf proteins (Raf-1, B-Raf, and A-Raf) were created by ligating NH₂-terminal truncated activated forms (Δ) to the estrogen receptor (ER) hormone-binding domain resulting in estradiol-regulated constructs (ΔRaf:ER). These different Raf:ER oncoproteins were introduced into the murine FDC-P1 hematopoietic cell line, and cells that grew in response to the three ΔRaf:ER oncoproteins were isolated. The ability of FDC-P1, ΔRaf-1:ER, ΔA-Raf:ER, and ΔB-Raf:ER cells to form tumors in severe combined immunodeficient mice was compared. Mice injected with ΔRaf:ER cells were implanted with β-estradiol pellets to induce the ΔRaf:ER oncoprotein. Cytokine-dependent parental cell lines did not form tumors. Implantation of β-estradiol pellets into mice injected with ΔRaf:ER cells significantly accelerated tumor onset and tumor size. The recovered ΔRaf:ER cells displayed induction of extracellular signal-regulated kinase (ERK) in response to β-estradiol stimulation, indicating that they had retained conditional activation of ERK even when passed through a severe combined immunodeficient mouse. The ΔRaf:ER cells were very sensitive to induction of apoptosis by the mitogen-activated protein/ERK kinase (MEK) 1 inhibitor CI1040 whereas parental cells were much less affected, demonstrating that the MEK1 may be useful in eliminating Ras/Raf/MEK–transformed cells. Furthermore, the effects of in vivo administration of the MEK1 inhibitor were evaluated and this inhibitor was observed to suppress the tumorigenicity of the injected cells. This ΔRaf:ER system can serve as a preclinical model to evaluate the effects of signal transduction inhibitors which target the Raf and MEK proteins.