Friday, April 6, 2012



The activation of MAPK pathway is largely associated with multiple cancer cases. Amongst the various components of the MAPK pathway, the oncogenic mutations within RAS and BRAF affect the downstream kinases, during the course of clinical and preclinical development. The second generation BRAFV600E selective inhibitor – PLX-4032 was initially found to be highly efficient. This later led to the discovery of RAF265 on the same grounds.


Point mutations (somatic in nature) within the BRAF gene, are seen mostly in the cases of thyroid and colorectal cancers. These mutations are seen within the exon 11 and exon 15, of the kinase domain. A mutation which replaces the Val residue with Glu is most commonly seen in at least 90% cases. As a result the P-loop fails to interact with the activation section. This makes the kinase enzyme inactive. Sorafenib was the first FDA approved inhibitor of RAF kinase. Further search for the discovery of RAF selective inhibitors led to the discovery of PLX -4032, XL281 and finally RAF265. All the three different splice variants of RAF are inhibited by RAF265. Due to its inhibitory effects on VEGFR-2, it also acted as an angiogenesis inhibitor [1].


Two important pathways (PI3K and MAPK pathways) are stimulated by growth factors and these pathways play a vital role in intracellular functions like proliferation and survival. The transcription factors involved in the vital cellular functions are regulated by these pathways. Within the MAPK pathway, Ras molecule is very important as it regulates the expression of several nuclear proteins. Alterations in the PI3K pathway and KRAS mutations are associated with immortalization and proliferation of cells. They also offer resistance to various chemotherapeutic agents. Hence an effective inhibition of these two pathways proved to be an efficient mechanism to check the growth of cancer. RAF265 is an efficient inhibitor of RAF/VEGFR2 and Everolimus is a rapamycin inhibitor within mammals. Different cell lines showing KRAS, BRAF and PIK3CA mutations were taken and tested with these inhibitors individually. Everolimus inhibited the down-stream targets of mTOR in all the cell lines and proved to be anti-tumorous under both in vitro and in vivo conditions. RAF265 showed its action in only those cell lines which showed a mutation within the BRAF. When these inhibitors were applied in combination, the phosphorylation levels of S6, AKT and 4EBP1 were found to be decreased under in vitro conditions within HCT116 cells. The action of RAF265 was further enhanced by Everolimus within few cell lines like HCT116 and H460. However this combination did not work well in the following cell lines MDAMB231 and A549. These results suggest that a combination of RAF265 and Everolimus deregulates RAS activated MAPK and PI3K pathway. The cross inhibition of S6 and 4EBP1 may be the underlying mechanism behind this [2].

The tumour-selective death receptor ligand tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for the treatment of human cancer. However, many tumours have evolved mechanisms to resist TRAIL-induced apoptosis. A number of studies have demonstrated that aberrant PI(3)K-Akt-mTOR survival signalling may confer TRAIL resistance by altering the balance between pro- and anti-apoptotic proteins. Here, we show that neuroendocrine tumour (NET) cell lines of heterogeneous origin exhibit a range of TRAIL sensitivities and that TRAIL sensitivity correlates with the expression of FLIP(S), caspase-8, and Bcl-2. Neither single mTOR inhibition by everolimus nor dual mTOR/PI(3)K inhibition by NVP-BEZ235 was able to enhance TRAIL susceptibility in any of the tested cell lines. In contrast, dual PI(3)K-Akt-mTOR and Raf-MEK-Erk pathway inhibition by the IGF-1R inhibitor NVP-AEW541 effectively restored TRAIL sensitivity in NCI-H727 bronchus carcinoid cells. Furthermore, blocking Raf-MEK-Erk signalling by the novel Raf inhibitor Raf265 significantly enhanced TRAIL sensitivity in NCI-H727 and CM insulinoma cells. While having no effect on FLIP(S) or caspase-8 expression, Raf265 strongly decreased Bcl-2 levels in those cell lines susceptible to its TRAIL-sensitizing action. Taken together, our findings suggest that combinations of Raf-MEK-Erk pathway inhibitors and TRAIL might offer a novel therapeutic strategy in NET disease.

When PRKD3 was blocked the activity of RAF265 was enhanced. This prevents the activation of the MAPK pathway again. As a result apoptosis is stimulated and cell cycle gets halted [3]. When the MAPK pathway is blocked by Raf265, the sensitivity towards TRAIL was enhanced within insulinoma cells. Within the TRAIL sensitive cells the levels of Bcl2 were found to be reduced [4].


In summary RAF265 shows a specific action against mutation within BRAF. It stimulates the process of apoptosis by sensitizing the cells to TRAIL.
1. Pratilas CA and Solit DB. Targeting the Mitogen-Activated Protein Kinase Pathway: Physiological Feedback and Drug Response. Clin Cancer Res 2010 May 14; 16: 3329-3334.
2. Mordant P, Loriot Y, et al. Dependence on Phosphoinositide 3-Kinase and RAS-RAF Pathways Drive the Activity of RAF265, a Novel RAF/VEGFR2 Inhibitor, and RAD001 (Everolimus) in Combination. Mol Cancer Ther 2010 Feb; 9(2):358-68.
3. Chen J, Shen Q, et al. Protein kinase D3 sensitizes RAF inhibitor RAF265 in melanoma cells by preventing reactivation of MAPK signaling. Cancer Res 2011 Apr 28.
4. Zitzmann K, de Toni E, et al. The novel Raf inhibitor Raf265 decreases Bcl-2 levels and confers TRAIL-sensitivity to neuroendocrine tumour cells. Endocr Relat Cancer 2011 Mar 21; 18(2):277-85.

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