We thank Dr. Shiva Malek and her colleagues (Genentech Inc.) for sharing their results with us before publication. We also thank M. San Roman, R. Villar, M.C. Gonzalez, A. Lopez, N. Cabrera, P. Villanueva, J. Condo, O. Dominguez, and S. Ortega for excellent technical support. This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN); the Spanish Ministry of Science, Innovation, and Universities (RTC-2017-6576-1 and RTI2018094664-B-I00) and the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM) to M.B., as well as by a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00) to M.B. and M.M. M.B. is a recipient of an Endowed Chair from the AXA Research Fund. M.S., P.N., and F.F.-G. were supported by FPU fellowships from the Spanish Ministry of Education. L.E.-B. was a recipient of an FPI fellowship from the Spanish Ministry of Economy and Competitiveness. S.G.-A. is a recipient of a postdoctoral fellowship from the Asociacion Espanola Contra el Cancer (AECC).S

Barbacid, Mariano

Caleiras, E

Caleiras, Eduardo

Drosten, Matthias

Esteban-Burgos, Esteban-Burgos L

Esteban-Burgos, Laura

Fernández-García, Fernando

Garcia-Alonso, Sara

Guerra, Carmen

Martinez Torrecuadrada, Jorge Luis

Martinez-Torrecuadrada, Jorge

Musteanu, Mónica

Nieto, Patricia

Sanclemente, Manuel

Santamaria, David

Cancer Cell

English

REPISALUD

llLetterRAF1 kinase activity is dispensablefor KRAS/p53 mutant lung tumor progressionManuel Sanclemente,1 Patricia Nieto,1,5 Sara Garcia-Alonso,1 Fernando Fernández-Garcı́a,1 Laura Esteban-Burgos,1Carmen Guerra,1 Matthias Drosten,1 Eduardo Caleiras,2 Jorge Martinez-Torrecuadrada,3 David Santamarı́a,1,6Monica Musteanu,1,7,4,* and Mariano Barbacid1,4,*1Molecular Oncology, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain2Biotechnology, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain3Structural Biology Programs, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain4Senior author5Present address: Asociación Española Contra el Cáncer (AECC), 28040 Madrid, Spain6Present address: University of Bordeaux, INSERM U1218, ACTION Laboratory, IECB, 33607 Pessac, France7Present address: Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid, 28040Madrid, Spain*Correspondence: mmusteanu@cnio.es (M.M.), mbarbacid@cnio.es (M.B.)https://doi.org/10.1016/j.ccell.2021.01.008KRAS mutant tumors account for atleast one-fourth of all human cancers.Yet, almost 40 years after their identifi-cation in human tumors, no effectivetherapies have been developed, withthe possible exception of those tumorscarrying G12C mutation (Ostrem et al.,2013). Ironically, KRAS oncogenes pri-marily signal via the MAPK pathwaymade up of druggable kinases. Unfortu-nately, none of the kinase inhibitorsdeveloped during the last decade haveshown anti-tumor efficacy (Drosten andBarbacid, 2020). Previous studies usinggenetically engineered mice have re-vealed that complete inactivation ofMAPK signaling is incompatible withadult life (Blasco et al., 2011). Moreover,genetic interrogation of the putativetherapeutic properties of each of the ki-nases has revealed that only ablation ofRAF1 results in significant levels of tu-mor regression of advanced lung ade-nocarcinomas induced by Kras andTrp53 mutations (Sanclemente et al.,2018). RAF1 ablation also resulted incomplete regression of a subset ofpancreatic ductal adenocarcinomas, incombination with the EGF receptor(Blasco et al., 2019). Although thesetherapeutic responses have beenachieved by ablating RAF1 expression,it appears that the most direct way totranslate these observations to a clinicalsetting would be to develop selective ki-nase inhibitors against RAF1.To interrogate the suitability of thistherapeutic strategy, we have generatedmouse strains that express conditionalknockin alleles that encode two indepen-294 Cancer Cell 39, March 8, 2021 ª 2021 Eldent RAF1 kinase dead isoforms,RAF1D468A and RAF1K375M (Figure S1A).Both of these mutations completelyabolish RAF1 kinase activity, asdetermined by the lack of phosphoryla-tion of their natural substrate MEK1(Figure S1B). Surprisingly, systemicexpression of these kinase isoforms un-der the control of the endogenous Raf1locus in mice bearing advanced Kras/Trp53 tumors (Sanclemente et al., 2018)failed to induce significant levels of tumorregression (Figure S1C). Indeed, tumor-bearing mice in which RAF1 expressionwas replaced by expression of theRAF1D468A isoform upon Cre-mediatedrecombination behaved exactly thesame as mice that retained expressionof the wild-type protein (Figure S1C).Cre-mediated recombination efficacy ofthis conditional Raf1 kinase-dead allelewas over 90% in all cases analyzed, indi-cating that most tumor cells expressedthe RAF1D468A isoform rather than thewild-type protein. In the case of theRAF1K375M isoform, tumor progressionwas more moderate than in control ani-mals, and we observed limited levels oftumor regression in a small percentage(15%) of animals (Figure S1C). Cre-medi-ated recombination efficacy of this con-ditional Raf1 kinase-dead allele wasover 80%, indicating that most tumorcells expressed the RAF1K375M isoform(Figure S1C). The slightly differential re-sults obtained with these two isoformsis due to the fact that RAF1K375M ispartially unstable, resulting in overallexpression levels significantly lowerthan those of either RAF1D468A or wild-sevier Inc.type RAF1 proteins (Figure S1C). Theseresults, taken together, conclusivelyshow that the contribution of RAF1 toKras/Trp53-driven tumors is not medi-ated by its kinase activity.Further analysis of these kinase-deadisoforms revealed that the partially unsta-ble RAF1K375M isoform is not phosphory-lated in S338 and S621, an event thoughtto be mediated in cis and required forproper protein stability (Figure S1D) (No-ble et al., 2008). However, both residuesare phosphorylated in RAF1D468A-ex-pressing cell lines, indicating that theirphosphorylation must be mediated byother kinases, since this isoform doesnot have kinase activity. Therefore, thelack of S338 and S621 phosphorylationin RAF1K375M might be a consequenceof conformational changes rather than ofthe absence of kinase activity. Finally, alltumors expressing RAF1D468A orRAF1K375M retained significant levels ofMEK1 phosphorylation regardless of thedifferential properties of these RAF1 ki-nase-dead isoforms (Figures S1C andS1D), thus indicating that elimination ofRAF1 kinase activity has little effect, ifany, on MAPK signaling, an activity likelyto be sustained by BRAF.Previous studies have indicated that,in addition to its role in MAPK signaling,RAF1 has anti-apoptotic activity. This ef-fect is mediated, at least in part, by itsability to inhibit the pro-apoptotic kinasesASK1 and MST2. Moreover, in vitrostudies suggested that this anti-apoptotic activity is likely to be kinase in-dependent (Chen et al., 2001; O’Neillet al., 2004). To interrogate whether theLetterllanti-proliferative effect of ablating RAF1is mediated by these kinases, weblocked proliferation of human A549lung adenocarcinoma cells as well as ofcells obtained from a patient-derivedxenograft (PDX) model, PDX-dc1, withlentiviral vectors expressing two inde-pendent shRNAs against RAF1 (Sancle-mente et al., 2018). Co-infection of theseRAF1 shRNAs with two independentshRNAs against ASK1 or MST2 that effi-ciently blocked expression of these pro-apoptotic kinases (Figure S1E) restoredthe proliferative properties of thesehuman lung tumor cells (Figure S1F).Re-expression of a cDNA encoding themurine RAF1 protein, whose sequencescould not be recognized by the humanRAF1 shRNAs, restored proliferation ofthe A549 and PDX-dc1 cells to levelssimilar to those observed upon co-infec-tion with ASK1 or MST2 shRNAs. Similarresults were obtained expressing themurine RAF1K375M and RAF1D468A ki-nase-dead isoforms in PDX-dc1 cells,thus adding additional evidence to sup-port the concept that the antitumor effectobserved upon RAF1 ablation is inde-pendent of its kinase activity. Theseresults demonstrate that the anti-prolifer-ative effect of silencing RAF1 expressionin human lung adenocarcinoma cells ismediated by the pro-apoptotic proper-ties of ASK1 and MST2.As illustrated here, RAF1K375M retainedanti-apoptotic activity similar to that of thewild-type RAF1 protein, as determined byits inability to induce cleaved caspase-3 inKras/Trp53 mouse tumor cells. Technicaldifficulties derived from the limitedrecombination efficiency exhibited bythe endogenous Cre recombinase pre-vented us from interrogating whether theRAF1D468A isoform also retained anti-apoptotic activity. More importantly, theanti-proliferative effect of ablating RAF1in two independent lung human tumorcell lines, A549 and PDX-dc1, could beefficiently reverted by downregulatingthe expression of the pro-apoptoticASK1 and MST2 kinases. These results,taken together, illustrate that the thera-peutic effect observed upon RAF1 abla-tion in Kras/Trp53 mutant lung tumors ismediated by loss of its anti-apoptoticproperties rather than of its kinase cata-lytic activity.These observations have importantimplications for the design of effectivetherapeutic strategies to block progres-sion of KRAS mutant human cancers.They also help to explain, at least inpart, the poor results obtained so far inthe clinic with RAF inhibitors that blockeither their kinase activity or other fea-tures that regulate their involvement inMAPK signaling. Indeed, recent studiesfrom our laboratory using some of theseRAF kinase inhibitors, including thepanRAF inhibitors MLN2480 andLSN3074753; a RAF1-selective inhibi-tor, GW5074; and the paradox breakerPLX8394, have also failed to show sig-nificant therapeutic activity on Kras/Trp53 mutant lung tumors (Esteban-Burgos et al., 2020).In summary, these studies stronglysuggest that current therapeutic strate-gies based on inhibition of RAF1 kinaseactivity are unlikely to produce anti-tumorresults in the clinic. Instead, pharmaco-logical targeting of RAF1 will require novelstrategies that prevent the anti-apoptoticactivity of RAF1, either by blocking itsinteraction with the ASK1 or MST2 ki-nases (Chen et al., 2001; O’Neill et al.,2004) or, more directly, by inhibitingRAF1 expression with selective RAF1 de-graders. The limited toxicities resultingfrom systemic ablation of RAF1 expres-sion in adult mice (Sanclemente et al.,2018) suggest that the increasedapoptotic activity that may result fromloss of RAF1 expression has limited effecton normal tissues. Yet, toxicities in miceand humans may not be the same.The last decade has witnessed atremendous surge in the use of degronchemistry to inactivate targets whosecontribution to tumor development isnot mediated by classical enzymatic ac-tivities (Lai and Crews, 2017). Indeed, asignificant number of cancer genesencode undruggable proteins such asKRAS, with the possible exception ofthose carrying G12C mutations (Ostremet al., 2013). The development of phar-macologically viable proteolysis-target-ing chimeras (PROTACs) might be a suit-able therapeutic option to block RAF1expression via proteasome-mediateddegradation. These bifunctional chemo-types do not require binding to function-ally relevant domains, thus offering wideroptions than catalytic inhibitors or inhibi-tors of protein-protein interactions. Un-veiling the complete structure of RAF1should provide critical information notonly to design PROTACs capable ofinducing its degradation but also to iden-tify potential vulnerabilities that mayresult in RAF1 degradation. Hopefully,pharmacological phenocopying of the re-sults obtained in genetically engineeredmouse tumor models will provide thera-peutic benefit to patients suffering fromKRAS mutant cancers in the not-too-distant future.SUPPLEMENTAL INFORMATIONSupplemental information can be found online athttps://doi.org/10.1016/j.ccell.2021.01.008.ACKNOWLEDGMENTSWe thank Dr. Shiva Malek and her colleagues(Genentech Inc.) for sharing their results with usbefore publication. We also thank M. San Roman,R. Villar, M.C. González, A. Lopez, N. Cabrera, P.Villanueva, J. Condo, O. Domı́nguez, and S. Ortegafor excellent technical support. This work was sup-ported by grants from the European ResearchCouncil (ERC-2015-AdG/695566, THERACAN);the Spanish Ministry of Science, Innovation, andUniversities (RTC-2017-6576-1 and RTI2018-094664-B-I00) and the Autonomous Communityof Madrid (B2017/BMD-3884 iLUNG-CM) toM.B., as well as by a grant from the Spanish Minis-try of Science, Innovation and Universities(RTI2018-094664-B-I00) to M.B. and M.M. M.B.is a recipient of an Endowed Chair from the AXAResearch Fund. M.S., P.N., and F.F.-G. were sup-ported by FPU fellowships from the Spanish Minis-try of Education. L.E.-B. was a recipient of an FPIfellowship from the Spanish Ministry of Economyand Competitiveness. S.G.-A. is a recipient of apostdoctoral fellowship from the Asociación Espa-ñola Contra el Cáncer (AECC).REFERENCESBlasco, R.B., Francoz, S., Santamarı́a, D.,Cañamero, M., Dubus, P., Charron, J., Baccarini,M., and Barbacid, M. (2011). c-Raf, but not B-Raf, is essential for development of K-Ras onco-gene-driven non-small cell lung carcinoma.Cancer Cell 19, 652–663.Blasco, M.T., Navas, C., Martin-Serrano, G.,Grana-Castro, O., Lechuga, C.G., Martin-Diaz, L.,Djurec, M., Li, J., Morales-Cacho, L., Esteban-Burgos, L., et al. (2019). Complete regression ofadvanced pancreatic ductal adenocarcinomasupon combined inhibition of EGFR and C-RAF.Cancer Cell 35, 573–587.Chen, J., Fujii, K., Zhang, L., Roberts, T., and Fu, H.(2001). Raf-1 promotes cell survival by antago-nizing apoptosis signal-regulating kinase 1 througha MEK-ERK independent mechanism. Proc. Natl.Acad. Sci. USA 98, 7783–7788.Drosten, M., and Barbacid, M. (2020). Targetingthe MAPK pathway in KRAS-driven tumors.Cancer Cell 37, 543–550.Esteban-Burgos, L., Wang, H., Nieto, P., Zheng, J.,Blanco-Aparicio, C., Varela, C., Gómez-López, G.,Fernández-Garcı́a, F., Sanclemente, M., Guerra,C., et al. (2020). Tumor regression and resistanceCancer Cell 39, March 8, 2021 295Letterllmechanisms upon CDK4 and RAF1 inactivation inKRAS/P53 mutant lung adenocarcinomas. Proc.Natl. Acad. Sci. USA 117, 24415–24426.Lai, A.C., and Crews, C.M. (2017). Induced proteindegradation: an emerging drug discovery para-digm. Nat. Rev. Drug Discov. 16, 101–114.Noble, C., Mercer, K., Hussain, J., Carragher, L.,Giblett, S., Hayward, R., Patterson, C., Marais,R., and Pritchard, C.A. (2008). CRAF autophos-296 Cancer Cell 39, March 8, 2021phorylation of serine 621 is required to prevent itsproteasome-mediated degradation. Mol. Cell 31,862–872.O’Neill, E., Rushworth, L., Baccarini, M., andKolch, W. (2004). Role of the kinase MST2 in sup-pression of apoptosis by the proto-oncogeneproduct Raf-1. Science 306, 2267–2270.Ostrem, J.M., Peters, U., Sos, M.L., Wells, J.A.,and Shokat, K.M. (2013). K-Ras(G12C) inhibitorsallosterically control GTP affinity and effector inter-actions. Nature 503, 548–551.Sanclemente, M., Francoz, S., Esteban-Burgos, L.,Bousquet-Mur, E., Djurec, M., Lopez-Casas, P.P.,Hidalgo, M., Guerra, C., Drosten, M., Musteanu,M., and Barbacid, M. (2018). c-RAF ablation in-duces regression of advanced Kras/Trp53 mutantlung adenocarcinomas by a mechanism indepen-dent of MAPK signaling. Cancer Cell 33, 217–228.

RAF1 kinase activity is dispensable for KRAS/p53 mutant lung tumor progression.

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RAF1 kinase activity is dispensable for KRAS/p53 mutant lung tumor progression.

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