Abstract 21: Selective and potent inhibitors of the mutant B-Raf pathway paradoxically stimulate the MAPK pathway in wild-type B-Raf cells
Authors: Carnahan Josette , Beltran Pedro , Babij Carol , Le Quynh , Rose Mark J. , Vonderfecht Steven , Kim Joseph L. , Smith Adrian L. , Broome Martin A. , Fernando Manory , Kha Hue , Belmontes Brian , Radinsky Robert , Kendall Richard , Burgess Teresa L. 
Published: February 2011

B-Raf is a member of the Raf family of serine/threonine kinases, which also include A-Raf and C-Raf. While all 3 Raf kinases stimulate the MAPK signaling cascade, B-Raf is the most catalytically active. B-Raf hyperactivity through mutation or over-expression is reported in many solid and hematologic malignancies. An especially high frequency (60%) of activating B-Raf mutations is found in melanoma, with the kinase domain mutant (V600E) accounting for greater than 95% of these. The identification and development of B-Raf inhibitors that selectively block signaling of the V600EB-Raf dependent MAPK pathway may have therapeutic value in these and other malignancies driven by activation of B-Raf. We report here on the characterization of novel, potent and selective small molecule Raf kinase inhibitors demonstrating potent cellular activity. Compound 1 (Smith, DeMorin et al. 2009) exhibits excellent potency in cells harboring V600EB-Raf (P-ERK IC50 ranging from 1-14nM) however, cells that contain wild type B-Raf are significantly less sensitive to inhibition of MAPK signaling by compound 1 (P-ERK IC50 ranging from 260nM to &gt; 1μM). This shift in potency between V600EB-Raf versus wtB-Raf cell lines is also observed with cellular viability. In contrast to cells harboring B-Raf mutation, exposure to selected Raf inhibitors resulted in a dose-dependent, and sustained activation of MAPK signaling, increased Raf basal kinase activity and heterodimer stabilization in all wild type B-Raf cell lines examined . In the mutant KRAS MIA PaCa-2 and A549, Raf inhibition by selected compounds led to entry into the cell cycle, 80% increased proliferation, 40% increased colony formation and significantly stimulated tumor growth in vivo (Beltran et al, AACR 2010). Inhibition with structurally distinct Raf inhibitors or isoform specific siRNA knock-down of Raf demonstrated that these effects were mediated directly through Raf. Either A-Raf or C-Raf, mediated the Raf-inhibitor-induced MAPK pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in both malignant and normal cells such as HUVEC cells. Indeed mouse treatment with any of the efficacious inhibitors resulted in hyperplasia observed in the epithelial layer of mouse esophagus and stomach. These data suggest that mutant versus wild type B-Raf MAPK pathways are distinctly regulated in cells. An implication of these results is that certain Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients.

Smith, A. L., F. F. DeMorin, et al. (2009). “Selective Inhibitors of the Mutant B-Raf Pathway: Discovery of a Potent and Orally Bioavailable Aminoisoquinoline.” Journal of Medicinal Chemistry 52(20): 6189-6192.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 21.