Scientific Papers

For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.

Activating mTOR mutations in a patient with an extraordinary response on a phase I trial of everolimus and pazopanib.

Cancer Discov. 2014 Mar 27.

Wagle, N., Grabiner, B.C.*, Van Allen, E.M., Hodis, E., Jacobus, S., Supko, J.G., Stewart, M., Choueiri, T.K., Gandhi, L., Cleary, J.M., Elfiky, A.A., Taplin, M.E., Stack, E.C., Signoretti, S., Loda, M., Shapiro, G.I., Sabatini, D.M.*, Lander, E.S., Gabriel, S.B., Kantoff, P.W., Garraway, L.A., and Rosenberg, J.E.

Understanding the genetic mechanisms of sensitivity to targeted anticancer therapies may improve patient selection, response to therapy, and rational treatment designs. One approach to increase this understanding involves detailed studies of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder in a phase I study of pazopanib and everolimus in advanced solid tumors. Whole-exome sequencing of a patient with a 14-month complete response on this trial revealed two concurrent mutations in mTOR, the target of everolimus. In vitro experiments demonstrate that both mutations are activating, suggesting a biologic mechanism for exquisite sensitivity to everolimus in this patient. The use of precision (or "personalized") medicine approaches to screen patients with cancer for alterations in the mTOR pathway may help to identify subsets of patients who may benefit from targeted therapies directed against mTOR.

 

Structure-activity relationships for withanolides as inducers of the cellular heat-shock response.

J Med Chem. 2014 Mar 25.

Wijeratne, E.M., Xu, Y.M., Scherz-Shouval, R.*, Marron, M.T., Rocha, D.D., Liu, M.X., Costa-Lotufo, L.V., Santagata, S., Lindquist, S.*, Whitesell, L.*, and Gunatilaka, A.A.

To understand the relationship of structure to the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; 1) and 36 analogues (2-37), and compared their cytotoxicity to cytoprotective heat shock-inducing activity (HSA). Analyzing structure-activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-epi-WA (28) to 33 enhanced, while introduction of beta-OH to WA at C-12 (29) and C-15 (30) decreased both activities. Introduction of beta-OAc to 4,27-diacetyl-WA (16) at C-15 (37) decreased HSA without affecting cytotoxicity, but at C-12 (36) it had minimal effect. Importantly, acetylation of 27-OH yielding 15 from 1, 16 from 14, and 35 from 34 enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to selectively enhance HSA, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms.

 

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells.

Nat Methods. 2014 Mar 23.

Petschnigg, J., Groisman, B., Kotlyar, M., Taipale, M.*, Zheng, Y., Kurat, C.F., Sayad, A., Sierra, J.R., Usaj, M.M., Snider, J., Nachman, A., Krykbaeva, I.*, Tsao, M.S., Moffat, J., Pawson, T., Lindquist, S.*, Jurisica, I., and Stagljar, I.

Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.

 

Global analyses of the effect of different cellular contexts on microRNA targeting.

Mol Cell. 2014 Mar 20;53(6):1031-43.

Nam, J.W.*, Rissland, O.S.*, Koppstein, D.*, Abreu-Goodger, C., Jan, C.H.*, Agarwal, V.*, Yildirim, M.A.*, Rodriguez, A., and Bartel, D.P.*

MicroRNA (miRNA) regulation clearly impacts animal development, but the extent to which development-with its resulting diversity of cellular contexts-impacts miRNA regulation is unclear. Here, we compared cohorts of genes repressed by the same miRNAs in different cell lines and tissues and found that target repertoires were largely unaffected, with secondary effects explaining most of the differential responses detected. Outliers resulting from differential direct targeting were often attributable to alternative 3' UTR isoform usage that modulated the presence of miRNA sites. More inclusive examination of alternative 3' UTR isoforms revealed that they influence approximately 10% of predicted targets when comparing any two cell types. Indeed, considering alternative 3' UTR isoform usage improved prediction of targeting efficacy significantly beyond the improvements observed when considering constitutive isoform usage. Thus, although miRNA targeting is remarkably consistent in different cell types, considering the 3' UTR landscape helps predict targeting efficacy and explain differential regulation that is observed.

 

ILCs in the zone.

Nat Immunol. 2014 Mar 19;15(4):313-4.

Victora, G.D.*

 

A diverse array of cancer-associated mTOR mutations are hyperactivating and can predict rapamycin sensitivity.

Cancer Discov. 2014 Mar 14.

Grabiner, B.C.*, Nardi, V., Birsoy, K.*, Possemato, R.*, Shen, K.*, Sinha, S.*, Jordan, A.*, Beck, A.H., and Sabatini, D.M. *

Genes encoding components of the PI3K-Akt-mTOR signaling axis are frequently mutated in cancer, but few mutations have been characterized in MTOR, the gene for the mTOR kinase. Using publicly available tumor genome sequencing data, we generated a comprehensive catalog of mTOR pathway mutations in cancer, identifying 33 MTOR mutations that confer pathway hyperactivation. The mutations cluster in six distinct regions in the C-terminal half of mTOR and occur in multiple cancer types, with one cluster particularly prominent in kidney cancer. The activating mutations do not affect mTOR complex assembly, but a subset reduces binding to the mTOR inhibitor Deptor. mTORC1 signaling in cells expressing various activating mutations remains sensitive to pharmacological mTOR inhibition, but is partially resistant to nutrient deprivation. Lastly, cancer cell lines with hyperactivating MTOR mutations display heightened sensitivity to rapamycin both in culture and as in vivo xenografts, suggesting that such mutations confer mTOR pathway dependency.

 

Early BCR events and antigen capture, processing, and loading on MHC Class II on B cells.
Front Immunol. 2014 Mar 10;5:92.

Avalos, A.M.*, and Ploegh, H.L.*

B cells are efficient antigen-presenting cells (APCs), relying on antigen uptake through the B cell receptor (BCR). The mechanism of antigen recognition remains a topic of debate; while the prevalent view holds that antigens need to be multivalent for BCR activation, monovalent antigens can also initiate B cell responses. In this review, we describe the steps required for antigen uptake, processing, and loading of peptides onto MHC Class II compartments in B cells for efficient presentation to CD4 T cells, with a special focus in the initial steps of BCR recognition of antigen.

 

The adaptor protein p66Shc inhibits mTOR-dependent anabolic metabolism.

Sci Signal. 2014 Feb 18;7(313):ra17.

Soliman, M.A., Rahman, A.M.A., Lamming, D.A.*, Birsoy, K.*, Pawling, J., Frigolet, M.E., Lu, H.G., Fantus, I.G., Pasculescu, A., Zheng, Y., Sabatini, D.M.*, Dennis, J.W., and Pawson, T.

Adaptor proteins link surface receptors to intracellular signaling pathways and potentially control the way cells respond to nutrient availability. Mice deficient in p66Shc, the most recently evolved isoform of the Shc1 adaptor proteins and a mediator of receptor tyrosine kinase signaling, display resistance to diabetes and obesity. Using quantitative mass spectrometry, we found that p66Shc inhibited glucose metabolism. Depletion of p66Shc enhanced glycolysis and increased the allocation of glucose-derived carbon into anabolic metabolism, characteristics of a metabolic shift called the Warburg effect. This change in metabolism was mediated by the mammalian target of rapamycin (mTOR) because inhibition of mTOR with rapamycin reversed the glycolytic phenotype caused by p66Shc deficiency. Thus, unlike the other isoforms of Shc1, p66Shc appears to antagonize insulin and mTOR signaling, which limits glucose uptake and metabolism. Our results identify a critical inhibitory role for p66Shc in anabolic metabolism.

 

Critical role for IL-1 beta in DNA damage-induced mucositis.

Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):E702-11.

Kanarek, N., Grivennikov, S.I., Leshets, M., Lasry, A., Alkalay, I., Horwitz, E., Shaul, Y.D.*, Stachler, M., Voronov, E., Apte, R.N., Pagano, M., Pikarsky, E., Karin, M., Ghosh, S., and Ben-Neriah, Y.

beta-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-kappa B inhibitor I kappa B. To appreciate tissue-specific roles of beta-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1 beta as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1 beta is induced by DNA damage via an NF-kappa B-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-kappa B, with failure to express the endogenous IL-1 beta receptor antagonist IL-1Ra upon fourallele loss. Antibody neutralization of IL-1 beta prevents epithelial tight junction dysfunction and alleviates mucositis in beta-TrCP-deficient mice. IL-1 beta antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

 

LoQAtE-Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast.

Nucleic Acids Res. 2014 Jan;42(Database issue):D726-30.

Breker, M., Gymrek, M.*, Moldavski, O., and Schuldiner, M.

Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating environments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called 'LoQAtE' (Localizaiton and Quantitation Atlas of the yeast proteomE), available online at http://www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localization shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H2O2 and nitrogen starvation) and two genetic mutations [in the chaperonin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of localization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspectives on the significant role of proteins while combating external and internal fluctuations.

 
*Author affiliated with Whitehead Institute for Biomedical Research

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