A Small Molecule Disruptor of Rb/Raf-1 Interaction Inhibits Cell Proliferation, Angiogenesis, and Growth of Human Tumor Xenografts in Nude Mice

Abstract

Although it is well established that cyclin-dependent kinases phosphorylate and inactivate Rb, the Raf-1 kinase physically interacts with Rb and initiates the phosphorylation cascade early in the cell cycle. We have identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251), that potently and selectively disrupts the Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E, and Rb/HDAC binding. The selective inhibition of Rb/Raf-1 binding suppressed the ability of Rb to recruit Raf-1 to proliferative promoters and inhibited E2F1-dependent transcriptional activity. RRD-251 inhibited anchorage-dependent and anchorage-independent growth of human cancer cells and knockdown of Rb with short hairpin RNA or forced expression of E2F1 rescued cells from RRD-251-mediated growth arrest. P.o. treatment of mice resulted in significant tumor growth suppression only in tumors with functional Rb, and this was accompanied by inhibition of angiogenesis, inhibition of proliferation, decreased phosphorylated Rb levels, and inhibition of Rb/Raf-1 but not Rb/E2F1 binding in vivo. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents. [Cancer Res 2008;68(10):3810-8]

Raf kinases in cancer–roles and therapeutic opportunities

“…This interaction results in the recruitment of the Rb/ C-Raf complex to proliferative promoters and increases E2F1-dependent transcriptional activity, counteracting the antiproliferative function of Rb (Wang et al, 1998;Dasgupta et al, 2006;Kinkade et al, 2008).…”

“…C-Raf can promote endothelial cell survival by either MEK-dependent or -independent pathways, including ASK-1 inhibition (Alavi et al, 2003(Alavi et al, , 2007. In addition, selective disruption of the interaction between C-Raf and Rb inhibits the development of tumor-associated microvessels and suppresses the growth of tumor xenografts (Dasgupta et al, 2004;Kinkade et al, 2008). Thus, several C-Raf-dependent pathways can contribute to angiogenesis.…”

“…Thus, MEK/ERK is undoubtedly a target of activated Raf in tumorigenesis. In the case of C-Raf, other targets potentially contributing to cell transformation have been proposed, such as the nuclear factor-kB pathway (Baumann et al, 2000), Rb (Kinkade et al, 2008) and BAD (Polzien et al, 2009), all reviewed by Niault and Baccarini (2010). In addition, C-Raf can inhibit apoptosis by binding to, and inhibiting, the stress-induced kinase ASK-1 (Chen et al, 2001) and the homolog of Drosophila's Hippo, the MST-2 kinase (O'Neill et al, 2004;Matallanas et al, 2007); and finally, C-Raf interferes, by direct binding, with the activity of the cytoskeleton-based Rho effector Rok-a (also known as ROCK2), resulting in defects in cell migration, apoptosis and differentiation (Ehrenreiter et al, 2005(Ehrenreiter et al, , 2009Piazzolla et al, 2005) (Figure 2).…”

Protein-protein Interactions: Network Analysis and Applications in Drug Discovery

“…An ELISA-based HTS identified RRD-251 ( Figure 4A) as a potent and selective small molecule disruptor of Rb-RAF1 interaction. Its potential as anti-cancer drug was underscored by the fact that peroral treatment of tumor-bearing xenografted mice significantly suppressed tumor growth by inhibiting angiogenesis and cell proliferation [28].…”

Lysine methylation regulates the pRb tumour suppressor protein

“…The primers used were as follows: DHFR forward: 5-GCCTAAGCT GCGCAA GTGGT-3; DHFR reverse: 5-GTCTCCGTTCTTGCCAAT CC-3; TS forward primer 5-CTG CCAGCTGTACCAGAC AT-3; TS reverse primer 5-ATGTGCATCTCCCAAAGTG T-3 as described (Kinkade et al, 2008;Vandromme et al, 2008).…”