Development of a conditional in vivo model to evaluate the efficacy of small molecule inhibitors for the treatment of Raf-transformed hematopoietic cells.

Conditionally active forms of the Raf proteins (Raf-1, B-Raf, and A-Raf) were created by ligating NH2-terminal truncated activated forms (Delta) to the estrogen receptor (ER) hormone-binding domain resulting in estradiol-regulated constructs (DeltaRaf: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 DeltaRaf:ER oncoproteins were isolated. The ability of FDC-P1, DeltaRaf-1:ER, DeltaA-Raf:ER, and DeltaB-Raf:ER cells to form tumors in severe combined immunodeficient mice was compared. Mice injected with DeltaRaf:ER cells were implanted with beta-estradiol pellets to induce the DeltaRaf:ER oncoprotein. Cytokine-dependent parental cell lines did not form tumors. Implantation of beta-estradiol pellets into mice injected with DeltaRaf:ER cells significantly accelerated tumor onset and tumor size. The recovered DeltaRaf:ER cells displayed induction of extracellular signal-regulated kinase (ERK) in response to beta-estradiol stimulation, indicating that they had retained conditional activation of ERK even when passed through a severe combined immunodeficient mouse. The DeltaRaf: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 DeltaRaf:ER system can serve as a preclinical model to evaluate the effects of signal transduction inhibitors which target the Raf and MEK proteins.