21 research outputs found

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

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    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

    Multiple cancer pathways regulate telomere protection

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    Telomeres are considered as universal anti-cancer targets, as telomere maintenance is essential to sustain indefinite cancer growth. Mutations in telomerase, the enzyme that maintains telomeres, are among the most frequently found in cancer. In addition, mutations in components of the telomere protective complex, or shelterin, are also found in familial and sporadic cancers. Most efforts to target telomeres have focused in telomerase inhibition; however, recent studies suggest that direct targeting of the shelterin complex could represent a more effective strategy. In particular, we recently showed that genetic deletion of the TRF1 essential shelterin protein impairs tumor growth in aggressive lung cancer and glioblastoma (GBM) mouse models by direct induction of telomere damage independently of telomere length. Here, we screen for TRF1 inhibitory drugs using a collection of FDA-approved drugs and drugs in clinical trials, which cover the majority of pathways included in the Reactome database. Among other targets, we find that inhibition of several kinases of the Ras pathway, including ERK and MEK, recapitulates the effects of Trf1 genetic deletion, including induction of telomeric DNA damage, telomere fragility, and inhibition of cancer stemness. We further show that both bRAF and ERK2 kinases phosphorylate TRF1 in vitro and that these modifications are essential for TRF1 location to telomeres in vivo Finally, we use these new TRF1 regulatory pathways as the basis to discover novel drug combinations based on TRF1 inhibition, with the goal of effectively blocking potential resistance to individual drugs in patient-derived glioblastoma xenograft models.We thank the Confocal Microscopy, Protein Engineering, Mass Spectrometry,Comparative Pathology, and Mouse Facility Units at CNIO. MAB laboratory is funded by SAF 2013-45111-R from MINECO,Fundación Botín and Banco Santander, Worldwide Cancer Research 16-1177. LB is a fellow of the La Caixa-Severo Ochoa International PhD Programme.S

    An allosteric cross-talk between the activation loop and the ATP binding site regulates the activation of Src kinase

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    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe

    Modulation of telomere protection by the PI3K/AKT pathway

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    Telomeres and the insulin/PI3K pathway are considered hallmarks of aging and cancer. Here, we describe a role for PI3K/AKT in the regulation of TRF1, an essential component of the shelterin complex. PI3K and AKT chemical inhibitors reduce TRF1 telomeric foci and lead to increased telomeric DNA damage and fragility. We identify the PI3Kα isoform as responsible for this TRF1 inhibition. TRF1 is phosphorylated at different residues by AKT and these modifications regulate TRF1 protein stability and TRF1 binding to telomeric DNA in vitro and are important for in vivo TRF1 telomere location and cell viability. Patient-derived breast cancer PDX mouse models that effectively respond to a PI3Kα specific inhibitor, BYL719, show decreased TRF1 levels and increased DNA damage. These findings functionally connect two of the major pathways for cancer and aging, telomeres and the PI3K pathway, and pinpoint PI3K and AKT as novel targets for chemical modulation of telomere protection.We are indebted to D. Megias for microscopy analysis, to D. Calvo for protein purification as well as to J. Muñoz and F. García for LC/MS/MS analysis. The research was funded by project SAF2013-45111-R of Societal Changes Program of the Spanish Ministry of Economics and Competitiveness (MINECO) co-financed through the European Fund of Regional Development (FEDER), Fundación Botín, Banco Santander (Santander Universities Global Division) and Worldwide Cancer Research (WCR 16-1177).S

    Tumor regression and resistance mechanisms upon CDK4 and RAF1 inactivation in KRAS/P53 mutant lung adenocarcinomas.

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    KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors.We thank S. Ortega for the generation of the Cdk4FxKD mouse model; and M. San Roman, R. Villar, M. C. Gonzalez, A. Lopez, N. Cabrera, P. Villanueva, J. Condo, J. Klett, A. Cebria, A. Otero, O. Dominguez, G. Luengo, G. Garaulet, F. Mulero, and D. Megias for excellent technical support. This work was supported by European Research Council Grant ERC-2015-AdG/695566, THERACAN, Spanish Ministry of Science, Innovation, and Universities Grant RTC-2017-6576-1, and the Autonomous Community of Madrid Grant B2017/BMD-3884 iLUNG-CM (to M.B.); Spanish Ministry of Science, Innovation, and Universities Grant RTI2018-094664B-I00 (to M.B. and M.M.); and National Natural Science Foundation of China Grant 31771469 (to H.W.). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. L.E.-B. is the recipient of an FPI fellowship from the Spanish Ministry of Economy and Competitiveness. F.F.-G., M.S., and P.N. were supported by an FPU fellowships from the Spanish Ministry of Education.S

    Towards a Molecular Understanding of the Link between Imatinib Resistance and Kinase Conformational Dynamics.

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    Due to its inhibition of the Abl kinase domain in the BCR-ABL fusion protein, imatinib is strikingly effective in the initial stage of chronic myeloid leukemia with more than 90% of the patients showing complete remission. However, as in the case of most targeted anti-cancer therapies, the emergence of drug resistance is a serious concern. Several drug-resistant mutations affecting the catalytic domain of Abl and other tyrosine kinases are now known. But, despite their importance and the adverse effect that they have on the prognosis of the cancer patients harboring them, the molecular mechanism of these mutations is still debated. Here by using long molecular dynamics simulations and large-scale free energy calculations complemented by in vitro mutagenesis and microcalorimetry experiments, we model the effect of several widespread drug-resistant mutations of Abl. By comparing the conformational free energy landscape of the mutants with those of the wild-type tyrosine kinases we clarify their mode of action. It involves significant and complex changes in the inactive-to-active dynamics and entropy/enthalpy balance of two functional elements: the activation-loop and the conserved DFG motif. What is more the T315I gatekeeper mutant has a significant impact on the binding mechanism itself and on the binding kinetics

    Free energy of the DFG flip transition.

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    <p>Free energy surfaces of Abl, Src (adapted from Ref. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004578#pcbi.1004578.ref029" target="_blank">29</a>]), and Abl drug-resistant mutants projected on the distances between DFG Asp<sub>404</sub> and Lys<sub>295</sub> (CV1) and DFG Phe<sub>405</sub> and Ile<sub>293</sub> (Leu<sub>137</sub> in Src) (CV2). The free energy minima corresponding to DFG-in conformations are labeled “IN”, while “OUT” correspond to DFG-out conformations. The contour lines are drawn every 1 kcal/mol.</p

    Free energy of imatinib (un-)binding to Abl and to the T315I ‘gatekeeper’ mutant.

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    <p>Free energy surfaces associated to the binding of imatinib to WT Abl (top panel) and the T315I Abl “gatekeeper” mutant (bottom panel). The deepest energy minima correspond to the crystallographic binding pose and are labeled A. On the way out, B and B’ correspond to an intermediate state (metastable in WT Abl) where imatinib is in between the DFG and the <i>α</i>C helix. States C and C’ correspond to the “external binding pose”. Interestingly in Abl T315I there are two exit channels and both have an higher barrier than in the WT. The contour lines are drawn every 2 kcal/mol.</p

    Free energy of the A-loop opening.

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    <p>Free energy surfaces of Abl, Src, and drug-resistant mutants projected on the optimal path describing the conformational change of the A-loop from open to closed in Src (CV1) and Abl (CV2). The free energy minima corresponding to an extended A-loop active-like conformation are labeled “A”, “B” is used for A-loop semi-closed (inactive) conformations and “C” for fully closed A-loop conformations. The contour lines are drawn every 1 kcal/mol.</p
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