Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1–c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.

Direct inhibition of transcription factor complexes remains a central challenge in the discipline of ligand discovery. In general, these proteins lack surface involutions suitable for high-affinity binding by small molecules. Here we report the design of synthetic, cell-permeable, stabilized α-helical peptides that target a critical protein-protein interface in the NOTCH transactivation complex. We demonstrate that direct, high-affinity binding of the hydrocarbon-stapled peptide SAHM1 prevents assembly of the active transcriptional complex. Inappropriate NOTCH activation is directly implicated in the pathogenesis of several disease states, including T-cell acute lymphoblastic leukaemia (T-ALL). The treatment of leukaemic cells with SAHM1 results in genome-wide suppression of NOTCH-activated genes. Direct antagonism of the NOTCH transcriptional program ©2009 Macmillan Publishers Limited. All rights reservedCorrespondence and requests for materials should be addressed to J.E.B. (james_bradner@dfci.harvard.edu) or G.L.V. (gregory_verdine@harvard.edu). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions R.E.M., G.L.V. and J.E.B. conceptualized the study, designed the experiments, interpreted data, and wrote the manuscript. Design, synthesis and biological characterization of SAHM peptides was performed by R.E.M. C.D.B., J.C.A. and S.C.B. contributed key reagents and analysed data. R.E.M., M.C., T.N.D., J.C.A., A.L.K., D.G.G. and J.E.B. established the bioluminescent T-ALL model, designed and performed in vivo experiments and analysed data.Author Information All microarray data has been deposited to the Gene Expression Omnibus at the National Center for Biotechnology Information under accession numbers GSE18198 and GSE18351. Reprints and permissions information is available at www.nature.com/reprints. The authors declare competing financial interests: details accompany the full-text HTML version of the paper at www.nature.com/nature. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 October 7. Ligand binding to the extracellular domain of NOTCH1 initiates sequential proteolytic processing events catalysed respectively by an ADAM family metalloprotease and a γ-secretase complex, resulting in cytoplasmic release of the intracellular domain of NOTCH1 (ICN1)4 -6. ICN1 then translocates to the nucleus and loads onto the DNA-bound transcription factor CSL7. The engagement of ICN1 with CSL creates a long, shallow groove along the interface of the two proteins that serves as a binding surface for coactivator proteins of the mastermind-like (MAML) family8 , 9. The resulting ICN-CSL-MAML ternary complex then recruits the core transcription machinery, effecting activation of NOTCH-dependent target genes.The duration and strength of NOTCH signalling is normally tightly controlled. Whereas lossof-function mut...

Notch signaling plays multiple roles to direct diverse decisions regarding cell fate during T cell development. During helper T (Th) cell differentiation, Notch is involved in generating optimal Th2 cell responses. Here, we present data investigating how Notch mediates Th2 cell differentiation. Notch showed a CD4(+) T cell intrinsic role in promoting IL-4 expression that required GATA-3. In the absence of Notch signals, Gata3 expression was markedly diminished. Introduction of an activated allele of Notch1 into CD4(+) T cells led to the specific and direct upregulation of a developmentally regulated Gata3 transcript that included the exon 1a sequences. Furthermore, Notch acted in parallel with GATA-3 to synergistically activate IL-4 expression. Together, these data implicate Gata3 as a direct transcriptional Notch target that acts in concert with Notch signaling to generate optimal Th2 cell responses.

The conformational space sampled by the two-domain protein calmodulin has been explored by an approach based on four sets of NMR observables obtained on Tb 3؉ -and Tm 3؉ -substituted proteins. The observables are the pseudocontact shifts and residual dipolar couplings of the C-terminal domain when lanthanide substitution is at the N-terminal domain. Each set of observables provides independent information on the conformations experienced by the molecule. It is found that not all sterically allowed conformations are equally populated. Taking the N-terminal domain as the reference, the C-terminal domain preferentially resides in a region of space inscribed in a wide elliptical cone. The axis of the cone is tilted by Ϸ30°with respect to the direction of the N-terminal part of the interdomain helix, which is known to have a flexible central part in solution. The C-terminal domain also undergoes rotation about the axis defined by the C-terminal part of the interdomain helix. Neither the extended helix conformation initially observed in the solid state for free calcium calmodulin nor the closed conformation(s) adopted by calcium calmodulin either alone or in its adduct(s) with target peptide(s) is among the most preferred ones. These findings are unique, both in terms of structural information obtained on a biomolecule that samples multiple conformations and in terms of the approach developed to achieve the results. The same approach is in principle applicable to other multidomain proteins, as well as to multiple interaction modes between two macromolecular partners. C almodulin (CaM) is a paradigm case in structural biology. The following brief survey of the history of the structural and dynamic studies on this protein serves the double purpose of putting the present findings in proper perspective and of acknowledging those pieces of previous information that were essential to allow the present approach to be developed and to yield novel structural information.CaMs are two-domain proteins belonging to the large family of EF-hand proteins (1-3). They contain Ϸ150 amino acid residues, organized into two domains of Ϸ70 aa each and connected by a short linker. Each domain is made up of two special helix-loop-helix motifs (EF-hand motifs) that can bind a calcium ion in the loop. The two loops are held close to one another by two short antiparallel ␤-strands forming a threehydrogen bond stretch of ␤-sheet. The function of CaM in cell cytoplasm is that of responding to sudden rises of calcium concentration by binding up to four calcium ions in the four EF-hand loops, by changing conformation because of metal binding, and by thus becoming able to recognize, bind to, and activate, a number of proteins and enzymes (1,(4)(5)(6)(7)(8). Early x-ray data (9) showed the four-calcium (Ca 2 ) N (Ca 2 ) C CaM form (subscripts N and C refer to the calcium atoms bound by the N-and C-terminal domains, respectively) to have a dumbbell shape, with helix 4, the last helix of the N-terminal domain, and helix 5, the first helix of the C-termi...