New Insight Puts Craf in Sight As a Therapeutic Target

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IN THE SPOTLIGHT new Insight Puts CRAf in sight as a Therapeutic Target

Ana Paula Rebocho and Richard Marais summary: By selectively depleting components of the RAF-MEK-ERK pathway in transgenic mice, it is now shown in 2 studies that CRAF is critical for signaling to MEK downstream of oncogenic Kras and that BRAF is not required. Cancer Discovery; 1(2); 98–9. ©2011 AACR.

Commentary on Karreth et al., p. 128(4).

The RAS proteins are small GTP-binding proteins that both studies, any tumors that did arise in the Craf, Mek1/ integrate extracellular signals and activate several downstream Mek2, or Erk1/Erk2 deleted mice were “escapers” in which pathways to control cell proliferation, differentiation, and the target gene(s) had not been deleted. survival. There are three RAS genes in humans (HRAS , KRAS , These data demonstrate that whereas Mek1 and Mek2, NRAS ) and gain-of-function mutations occur in these genes and Erk1 and Erk2, can compensate for each other’s loss in 15% to 30% of human cancers. Despite this prevalence, (in this model at least), Braf is unable to compensate for oncogenic RAS has so far proven to be an intractable the loss of Craf. This shows that Craf is responsible for therapeutic target, and thus drug discovery efforts have transmitting signals from oncogenic Ras to Mek and that largely focused on inhibiting the downstream pathways ( 1 ). Braf is not required ( Fig 1B ). In agreement with this, Blasco The RAF–MEK–ERK pathway in particular has attracted et al. ( 5 ) demonstrate that cells expressing oncogenic Kras a great deal of attention because RAF, MEK, and ERK are but lacking Craf do not display increased levels of apoptosis protein kinases that form a 3-tiered cascade that drives cell or senescence, suggesting that when Craf is lost, cells simply proliferation downstream of RAS ( Fig. 1A ), and furthermore, do not register the presence of the oncogenic Kras. protein kinases are known to be tractable therapeutic tar- Taken together, these data show that in this lung model, gets. Both RAF and MEK inhibitors are currently in late-stage signaling through Craf, Mek, and Erk is essential for tumor clinical development ( 2 , 3 ). However, there are three RAFs initiation by oncogenic Kras. It should be noted that these (ARAF, BRAF, and CRAF), two MEKs (MEK1, MEK2), and data fall a little short of validating Craf and the Mek/Erk two ERKs (ERK1, ERK2), and to date the role of these in- pathway as a therapeutic target because oncogenic Kras was dividual proteins in cancer cell signaling has not been fully expressed at the same time as Craf, Mek1/2, or Erk1/2 were explored. deleted. Thus, it is not possible to determine if the deleted To address the role of these individual proteins, Karreth proteins merely play a role in tumor initiation (in which et al. (ref. 4; in this issue of Cancer Discovery ) and Blasco case they are unlikely to be effective therapeutic targets) or et al. ( 5 ) used Cre-recombinase/loxP technology to selectively delete the individual genes for components of this pathway in the lungs of mice bearing cancer driven by on- Figure 1. Model of RAF-MEK-ERK signaling driven by RAS. A , in cogenic Kras. Karreth et al. ( 4 ) show that Braf depletion did normal cells, RAS signals through BRAF and CRAF to activate MEK1/2 not affect tumor burden, whereas Craf depletion signifi- and ERK1/2, which drive cell proliferation, differentiation, and cantly reduced tumor formation. Blasco et al. ( 5 ) made simi- survival. B , in transformed cells, oncogenic RAS signals through CRAF exclusively and BRAF is not engaged. However, the complexity to lar observations: Braf depletion had little effect, whereas signaling downstream of CRAF allows MEK1 and MEK2 to compensate Craf depletion caused a significant reduction in tumor bur- for each other and ERK1 and ERK2 to compensate for each other. den. Blasco et al. ( 5 ) went on to demonstrate that deletion of Mek1, Mek2, Erk1, or Erk2 alone did not affect tumor development, but when Mek1 and Mek2, or Erk1 and Erk2, A RAS B *RAS were concomitantly deleted, as with Craf, there was again a significant reduction in tumor formation. Importantly, in BRAF CRAF BRAF CRAF

Authors’ Affiliation: Signal Transduction Team, Division of Tumour Biology, The Institute of Cancer Research, London, United Kingdom MEK1 MEK2 MEK1 MEK2 Corresponding Author: Richard Marais, Signal Transduction Team, Division of Tumour Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom. Phone: 207-153-5171; ERK1 ERK2 ERK1 ERK2 Fax: 207-153-5078; E-mail: [email protected] doi: 10.1158/2159-8290.CD-11-0118 Proliferation, Tumor progression ©2011 American Association for Cancer Research. differentiation, etc.

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if they are also required for tumor maintenance (necessary both as therapeutic agents and as research tools. They also for therapeutic effect). However, we have previously estab- raise some interesting questions. For example, why does on- lished that oncogenic NRAS and KRAS signal exclusively cogenic Ras not signal through Braf? Is this because Braf is through CRAF to MEK in human melanoma cell lines (6), not available, or because signaling by oncogenic Ras through and it has been shown that Craf is required for tumor main- Braf is incompatible with tumor progression? What is the role tenance in a mouse skin carcinogenesis model (7). Thus, of Araf? And finally, can targeting Craf inhibit tumor growth although the new data do not fully validate Craf, overall in humans? These studies also serve as a timely reminder that the case for targeting this pathway in general and CRAF in even after all the years of study, we still appear to have much particular—either alone or in combination with targeting to learn about the subtleties of signaling through this cascade. other proteins/pathways—in RAS-mutant tumors is becom- ing increasingly compelling. Of course, the effectiveness of Disclosure of Potential Conflicts of Interest CRAF-selective drugs will need to be carefully judged. It No potential conflicts of interest were disclosed. has recently been shown that in the presence of oncogenic RAS, BRAF-selective and pan-RAF inhibitors drive paradox- Published online July 18, 2011. ical activation of the MEK/ERK pathway through induction of BRAF-CRAF and CRAF-CRAF dimers that then lead to CRAF hyperactivation (8–10). Clearly, it will be important References to determine if CRAF-selective inhibitors also drive this . 1 Downward J. Targeting RAS signalling pathways in cancer therapy. response. Nat Rev Cancer 2003;3:11–22. A neat twist in the Blasco et al. (5) study was the 2. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman demonstration that global deletion of Mek1 and Mek2 or JA, et al. Inhibition of mutated, activated BRAF in metastatic mela- Erk1 and Erk2 in the animals led to death within a few weeks noma. N Engl J Med 2010;363:809–19. due to multiple organ failure. These data show that this 3. Fremin C, Meloche S. From basic research to clinical development of MEK1/2 inhibitors for cancer therapy. J Hematol Oncol 2010;3:8. pathway is essential for the general fitness of the animals. 4. Karreth FA, Frese KK, DeNicola GM, Baccarini M, Tuveson DA. Of course, it is unlikely that ATP-competitive drugs will be C-Raf is required for the initiation of lung cancer by K-RasG12D. as efficient as gene deletion at inhibiting individual kinases, Cancer Discovery 2011;1:128–36. and complete protein loss may have the same effects as its 5. Blasco RB, Francoz S, Santamaria D, Canamero M, Dubus P, inhibition, but this observation does suggest that complete Charron J, et al. c-Raf, but not B-Raf, is essential for development of blockade of this pathway cannot be tolerated, which will K-Ras oncogene-driven non-small cell lung carcinoma. Cancer Cell need to be borne in mind in the clinical setting. In contrast 2011;19:652–63. 6. Dumaz N, Hayward R, Martin J, Ogilvie L, Hedley D, Curtin JA, et al. to Mek1/2 and Erk1/2, concomitant deletion of Braf and In melanoma, RAS mutations are accompanied by switching signal- Craf did not lead to death of the animals, suggesting that ing from BRAF to CRAF and disrupted cyclic AMP signaling. Cancer loss of these proteins does not result in complete block- Res 2006;66:9483–91. ade of the pathway and that other kinases must sustain 7. Ehrenreiter K, Kern F, Velamoor V, Meissl K, Galabova-Kovacs G, Mek/Erk signaling sufficiently for survival. Araf is of course Sibilia M, et al. Raf-1 addiction in Ras-induced skin carcinogenesis. a clear candidate for this role, but other kinases that can Cancer Cell 2009;16:149–60. 8. Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, phosphorylate Mek, such as Cot/Tpl2, may also perform Dhomen N, et al. Kinase-dead BRAF and oncogenic RAS cooperate this function. It is also possible that KSR (a distant relative to drive tumor progression through CRAF. Cell 2010;140:209–21. of RAF that was originally thought to be a pseudokinase, 9. Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, but was recently shown to phosphorylate MEK) may also Alvarado R, et al. RAF inhibitors prime wild-type RAF to activate the play a role (11). MAPK pathway and enhance growth. Nature 2010;464:431–5. The results presented by Karreth et al. (4) in this issue of 10. P oulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild- Cancer Discovery and Blasco et al. (5) provide unequivocal type BRAF. Nature 2010;464:427–30. evidence that Craf is responsible for coupling oncogenic Ras 11. Brennan DF, Dar AC, Hertz NT, Chao WC, Burlingame AL, Shokat to Mek in vivo, at least in some settings (Fig. 1B). These data KM, et al. A Raf-induced allosteric transition of KSR stimulates support the need for the development of CRAF-selective drugs, phosphorylation of MEK. Nature 2011;472:366–9.

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Ana Paula Rebocho and Richard Marais

Cancer Discovery 2011;1:98-99.

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