Drugs that target specific gene alterations have proven beneficial in the treatment of cancer. Because cancer cells have multiple resistance mechanisms, it is important to understand the downstream pathways of the target genes and monitor the pharmacodynamic markers associated with therapeutic efficacy. We performed a transcriptome analysis to characterize the response of various cancer cell lines to a selective fibroblast growth factor receptor (FGFR) inhibitor (CH5183284/Debio 1347), a mitogen-activated protein kinase kinase (MEK) inhibitor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFR and MEK inhibition produced similar expression patterns, and the extracellular signal–regulated kinase (ERK) gene signature was altered in several FGFR inhibitor–sensitive cell lines. Consistent with these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR inhibitor–sensitive cell line. Because the mitogen-activated protein kinase (MAPK) pathway functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR inhibitor efficacy in a collection of cell lines with the ERK signature and identified dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Although a MEK inhibitor suppressed the MAPK pathway, most FGFR inhibitor–sensitive cell lines are insensitive to MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of FGFR inhibitor efficacy in FGFR-addicted cancers. Mol Cancer Ther; 14(12); 2831–9. ©2015 AACR.
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ERK Signal Suppression and Sensitivity to FGFR Inhibitor
FGFR Aberrations in Cancer
Purpose: Molecular profiling may have prognostic and predictive value, and is increasingly used in the clinical setting. There are more than a dozen fibroblast growth factor receptor (FGFR) inhibitors in development. Optimal therapeutic application of FGFR inhibitors requires knowledge of the rates and types of FGFR aberrations in a variety of cancer types.
Experimental Design: We analyzed frequencies of FGFR aberrations in 4,853 solid tumors that were, on physician request, tested in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Foundation Medicine) using next-generation sequencing (182 or 236 genes), and analyzed by N-of-One.
Results: FGFR aberrations were found in 7.1% of cancers, with the majority being gene amplification (66% of the aberrations), followed by mutations (26%) and rearrangements (8%). FGFR1 (mostly amplification) was affected in 3.5% of 4,853 patients; FGFR2 in 1.5%; FGFR3 in 2.0%; and FGFR4 in 0.5%. Almost every type of malignancy examined showed some patients with FGFR aberrations, but the cancers most commonly affected were urothelial (32% FGFR-aberrant); breast (18%); endometrial (~13%), squamous lung cancers (~13%), and ovarian cancer (~9%). Among 35 unique FGFR mutations seen in this dataset, all but two are found in COSMIC. Seventeen of the 35 are known to be activating, and 11 are transforming.
Conclusions: FGFR aberrations are common in a wide variety of cancers, with the majority being gene amplifications or activating mutations. These data suggest that FGFR inhibition could be an important therapeutic option across multiple tumor types. Clin Cancer Res; 22(1); 259–67. ©2015 AACR.
Afatinib + Cetuximab First-line in EGFR-Mutant Lung Cancer
Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFR-mutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFRT790M). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFRT790M-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy.
Experimental Design: Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-naïve EGFRL858R-induced lung adenocarcinomas delayed tumor relapse and drug-resistance compared with single-agent TKIs.
Results: Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFRT790M mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFRT790M.
Conclusions: These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-naïve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted. Clin Cancer Res; 22(2); 426–35. ©2015 AACR.
Down-regulation of PAR1 activity with a pHLIP-based allosteric antagonist induces cancer cell death
Even though abnormal expression of G protein-coupled receptors (GPCRs) and of their ligands is observed in many cancer cells of various origins, only a few anti-cancer compounds directly act on their signalling. One promising approach to modulate their activity consists of targeting the receptor cytoplasmic surfaces interacting with the associated G-proteins using peptides mimicking the intracellular loops of the receptor. Thus, to be fully effective, the peptide mimics must be selectively targeted to the tumour while sparing healthy tissues, translocated across the cell membrane and stay anchored to the cytoplasmic leaflet of the plasma membrane. In the present study, we introduce a novel way to selectively target and inhibit the activity of a GPCR in cancer cells under acidic conditions, such as those found in solid tumours. We find that the conjugation of a peptide fragment derived from the third intracellular loop (i3) of the protease-activated receptor 1 (PAR1) to a peptide that can selectively target tumours solely based on their acidity [pH(Low) Insertion Peptide (pHLIP)], produces a construct capable of effectively down-regulating PAR1 activity in a concentration- and pH-dependent manner and of inducing a potent cytotoxic effect in a panel of cancer cells that is proportional to the relative level of receptor expression at the cell surface. This strategy not only allows for a more selective targeting and specific intracellular delivery than current approaches, but also offers new possibilities for developing novel anti-cancer drugs targeting GPCRs.
Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells
Boronic Acid for the Traceless Delivery of Proteins into Cells
Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity
Octreotide-Mediated Tumor-Targeted Drug Delivery via a Cleavable Doxorubicin–Peptide Conjugate
Glycocalyx Engineering with a Recycling Glycopolymer that Increases Cell Survival In Vivo
Abstract
Synthetic glycopolymers that emulate cell-surface mucins have been used to elucidate the role of mucin overexpression in cancer. However, because they are internalized within hours, these glycopolymers could not be employed to probe processes that occur on longer time scales. In this work, we tested a panel of glycopolymers bearing a variety of lipids to identify those that persist on cell membranes. Strikingly, we found that cholesterylamine (CholA) anchored glycopolymers are internalized into vesicles that serve as depots for delivery back to the cell surface, allowing for the display of cell-surface glycopolymers for at least ten days, even while the cells are dividing. As with native mucins, the cell-surface display of CholA-anchored glycopolymers influenced the focal adhesion distribution. Furthermore, we show that these mimetics enhance the survival of nonmalignant cells in a zebrafish model of metastasis. CholA-anchored glycopolymers therefore expand the application of glycocalyx engineering in glycobiology.
Keepin’ it sweet! A panel of glycopolymers bearing a variety of lipids were used to identify constructs that persist on cell membranes. Cholesterylamine-anchored glycopolymers are internalized but recycle to allow for the continuous display of cell-surface glycopolymers for ten days. Furthermore, these mimetics enhance the survival of nonmalignant cells in a zebrafish model of metastasis.
Nanotechnology Approaches for the Delivery of Exogenous siRNA for HIV Therapy
In-Membrane Chemical Modification (IMCM) for Site-Specific Chromophore Labeling of GPCRs
Abstract
We present in-membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G-protein-coupled receptors (GPCRs) with minimal mutagenesis. This method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site-specific introduction of chromophores for NMR and fluorescence spectroscopy in the human κ-opioid receptor (KOR) and the human A2A adenosine receptor (A2AAR). IMCM is applicable to a wide range of in vitro studies of GPCRs, including single-molecule spectroscopy, and is a promising platform for in-cell spectroscopy experiments.
Dyeing of exposure: In-membrane chemical modification (IMCM) enables site-specific labeling of natural cysteine residues in G-protein-coupled receptors with minimal or no mutagenesis by taking advantage of the natural protection of most cysteines by the membrane environment. IMCM is applicable to a wide range of in vitro studies of GPCRs and is a promising platform for in-cell spectroscopy experiments.
A Type of Auxiliary for Native Chemical Peptide Ligation beyond Cysteine and Glycine Junctions
Abstract
Native chemical ligation enables the chemical synthesis of proteins. Previously, thiol-containing auxiliary groups have been used to extend the reaction scope beyond N-terminal cysteine residues. However, the N-benzyl-type auxiliaries used so far result in rather low reaction rates. Herein, a new Nα-auxiliary is presented. Consideration of a radical fragmentation for cleavage led to the design of a new auxiliary group which is selectively removed under mildly basic conditions (pH 8.5) in the presence of TCEP and morpholine. Most importantly and in contrast to previously described auxiliaries, the 2-mercapto-2-phenethyl auxiliary is not limited to Gly-containing sites and ligations succeed at sterically demanding junctions. The auxiliary is introduced in high yield by on-resin reductive amination with commercially available amino acid building blocks. The synthetic utility of the method is demonstrated by the synthesis of two antimicrobial proteins, DCD-1L and opistoporin-2.
A radical change of the auxiliary design enables extended native chemical peptide ligation at sterically demanding ligation junctions. The 2-mercapto-2-phenethyl group is small and flexible and avoids α-branching. The auxiliary is introduced by reductive amination on a solid support and removed under mildly basic conditions in a radical desulfurization triggered fragmentation reaction.
Chemical Tools for the Study of Intramembrane Proteases
Rapid Additive-Free Selenocystine–Selenoester Peptide Ligation
Self-Assembly of a Functional Oligo(Aniline)-Based Amphiphile into Helical Conductive Nanowires
Double Strain-Promoted Macrocyclization for the Rapid Selection of Cell-Active Stapled Peptides
Abstract
Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein–protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide–alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.
More strain, more gain: A strained cyclodialkyne was used to staple diazidopeptides directly in the medium of a cell culture assay. This in situ approach is simple to conduct and enables combined stapling and screening for cell-active stapled peptides in a parallel, high-throughput format. The method was applied to the p53/MDM2 interaction as proof of principle, and a new inhibitor was identified and its crystal structure with MDM2 obtained.
A multiphase transitioning peptide hydrogel for suturing ultrasmall vessels.
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A multiphase transitioning peptide hydrogel for suturing ultrasmall vessels.
Nat Nanotechnol. 2016 Jan;11(1):95-102
Authors: Smith DJ, Brat GA, Medina SH, Tong D, Huang Y, Grahammer J, Furtmüller GJ, Oh BC, Nagy-Smith KJ, Walczak P, Brandacher G, Schneider JP
Abstract
Many surgeries are complicated by the need to anastomose, or reconnect, micrometre-scale vessels. Although suturing remains the gold standard for anastomosing vessels, it is difficult to place sutures correctly through collapsed lumen, making the procedure prone to failure. Here, we report a multiphase transitioning peptide hydrogel that can be injected into the lumen of vessels to facilitate suturing. The peptide, which contains a photocaged glutamic acid, forms a solid-like gel in a syringe and can be shear-thin delivered to the lumen of collapsed vessels (where it distends the vessel) and the space between two vessels (where it is used to approximate the vessel ends). Suturing is performed directly through the gel. Light is used to initiate the final gel-sol phase transition that disrupts the hydrogel network, allowing the gel to be removed and blood flow to resume. This gel adds a new tool to the armamentarium for micro- and supermicrosurgical procedures.
PMID: 26524396 [PubMed - indexed for MEDLINE]
Opportunity Knocks: Organic Chemistry for Fragment-Based Drug Discovery (FBDD)
What’s a good fragment? Fragment-based drug discovery is well-established within many pharmaceutical, biotech, and academic institutions for generating new drugs. In this Essay, the opportunities and challenges for organic chemists to design and synthesize new fragments are described.
The Supersized Class III Lanthipeptide Stackepeptin Displays Motif Multiplication in the Core Peptide
Construction of Lasso Peptide Fusion Proteins
Triple-Negative Breast Cancer in 2015
The research article by Dent and colleagues, which was published in the August 1, 2007, issue of Clinical Cancer Research, provided a clinical description of metastatic progression of triple-negative breast cancers. Finding successful treatment strategies for women with triple-negative breast cancer remains a challenge. Clin Cancer Res; 21(17); 3813–4. ©2015 AACR.
See related article by Dent et al., Clin Cancer Res 2007;13(15) August 1, 2007;4429–34
Synthesis of Spiroligomer-Containing Macrocycles.
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Related Articles |
Synthesis of Spiroligomer-Containing Macrocycles.
J Org Chem. 2015 Sep 18;80(18):8968-78
Authors: Zhao Q, Schafmeister CE
Abstract
We demonstrate the synthesis and characterization of the solution conformations of a collection of functionalized spiroligomer-based macrocycles. These macrocycles contain 14 independently controllable stereocenters and four independently controllable functional groups on a highly preorganized scaffold. These molecules are being developed to display complex, preorganized surfaces for binding proteins and to create enzyme-like active sites. In this work, we demonstrate the convergent synthetic approach to this new class of macrocycles and demonstrate that the conformational properties of these molecules can be changed by altering the configuration stereocenters within the backbone.
PMID: 26296148 [PubMed - indexed for MEDLINE]
Cancer cell surface induced peptide folding allows intracellular translocation of drug.
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Related Articles |
Cancer cell surface induced peptide folding allows intracellular translocation of drug.
J Control Release. 2015 Jul 10;209:317-26
Authors: Medina SH, Schneider JP
Abstract
Many lead molecules identified in drug discovery campaigns are eliminated from consideration due to poor solubility and low cell permeability. These orphaned molecules could have clinical value if solubilized and delivered properly. SVS-1 is a de novo designed peptide that preferentially folds at the surface of tumor cells, adopting a β-hairpin conformation that rapidly translocates into the cytoplasm, and ultimately nucleus, of cells. SVS-1 is stable in serum and small molecules attached to the peptide are effectively delivered to cancer cells via mechanisms involving physical translocation and, to a lesser extent, clathrin-dependent endocytosis. For example, ligating the model hydrophobic drug Paclitaxel (PTX) to SVS-1 improved its aqueous solubility by ~1000-fold and successfully delivered and released PTX to cancer cells in vitro and tumors in vivo without toxic adjuvants. These results suggest that SVS-1 can serve as a simple, effective delivery platform for molecules with poor solubility and permeability.
PMID: 25979324 [PubMed - indexed for MEDLINE]