Activity of pemetrexed in pre-clinical chordoma models and humans.

Chordomas are rare slow growing tumors, arising from embryonic remnants of notochord with a close predilection for the axial skeleton. Recurrence is common and no effective standard medical therapy exists. Thymidylate synthase (TS), an intracellular enzyme, is a key rate-limiting enzyme of DNA biosynthesis and repair which is primarily active in proliferating and metabolically active cells. Eighty-four percent of chordoma samples had loss of TS expression which may predict response to anti-folates. Pemetrexed suppresses tumor growth by inhibiting enzymes involved in folate metabolism, resulting in decreased availability of thymidine which is necessary for DNA synthesis. Pemetrexed inhibited growth in a preclinical mouse xenograft model of human chordoma. We report three cases of metastatic chordoma that had been heavily treated previously with a variety of standard therapies with poor response. In two cases, pemetrexed was added and objective responses were observed on imaging with one patient on continuous treatment for > 2 years with continued shrinkage. One case demonstrated tumor growth after treatment with pemetrexed. The two cases which had a favorable response had a loss of TS expression, whereas the one case with progressive disease had TS present. These results demonstrate the activity of pemetrexed in recurrent chordoma and warrant a prospective clinical trial which is ongoing (NCT03955042).

Mapping the landscape of genetic dependencies in chordoma.

Identifying the spectrum of genes required for cancer cell survival can reveal essential cancer circuitry and therapeutic targets, but such a map remains incomplete for many cancer types. We apply genome-scale CRISPR-Cas9 loss-of-function screens to map the landscape of selectively essential genes in chordoma, a bone cancer with few validated targets. This approach confirms a known chordoma dependency, TBXT (T; brachyury), and identifies a range of additional dependencies, including PTPN11, ADAR, PRKRA, LUC7L2, SRRM2, SLC2A1, SLC7A5, FANCM, and THAP1. CDK6, SOX9, and EGFR, genes previously implicated in chordoma biology, are also recovered. We find genomic and transcriptomic features that predict specific dependencies, including interferon-stimulated gene expression, which correlates with ADAR dependence and is elevated in chordoma. Validating the therapeutic relevance of dependencies, small-molecule inhibitors of SHP2, encoded by PTPN11, have potent preclinical efficacy against chordoma. Our results generate an emerging map of chordoma dependencies to enable biological and therapeutic hypotheses.

Emerging target discovery and drug repurposing opportunities in chordoma.

The development of effective and personalized treatment options for patients with rare cancers like chordoma is hampered by numerous challenges. Biomarker-guided repurposing of therapies approved in other indications remains the fastest path to redefining the treatment paradigm, but chordoma's low mutation burden limits the impact of genomics in target discovery and precision oncology efforts. As our knowledge of oncogenic mechanisms across various malignancies has matured, it's become increasingly clear that numerous properties of tumors transcend their genomes - leading to new and uncharted frontiers of therapeutic opportunity. In this review, we discuss how the implementation of cutting-edge tools and approaches is opening new windows into chordoma's vulnerabilities. We also note how a convergence of emerging observations in chordoma and other cancers is leading to the identification and evaluation of new therapeutic hypotheses for this rare cancer.

Regulation of Kinase Activity in the Caenorhabditis elegans EGF Receptor, LET-23.

In the active HER receptor dimers, kinases play distinct roles; one is the catalytically active kinase and the other is its allosteric activator. This specialization enables signaling by the catalytically inactive HER3, which functions exclusively as an allosteric activator upon heterodimerization with other HER receptors. It is unclear whether the allosteric activation mechanism evolved before HER receptors functionally specialized. We determined the crystal structure of the kinase domain of the only EGF receptor in Caenorhabditis elegans, LET-23. Our structure of a non-human EGFR kinase reveals autoinhibitory features conserved in the human counterpart. Strikingly, mutations within the putative allosteric dimer interface abrogate activity of the isolated LET-23 kinase and of the full-length receptor despite these regions being only partially conserved with human EGFR. Our results indicate that ancestral EGFRs have built-in features that poise them for allosteric activation that could facilitate emergence of the catalytically dead, yet functional, orthologs.

EGFR Ligands Differentially Stabilize Receptor Dimers to Specify Signaling Kinetics.

Epidermal growth factor receptor (EGFR) regulates many crucial cellular programs, with seven different activating ligands shaping cell signaling in distinct ways. Using crystallography and other approaches, we show how the EGFR ligands epiregulin (EREG) and epigen (EPGN) stabilize different dimeric conformations of the EGFR extracellular region. As a consequence, EREG or EPGN induce less stable EGFR dimers than EGF-making them partial agonists of EGFR dimerization. Unexpectedly, this weakened dimerization elicits more sustained EGFR signaling than seen with EGF, provoking responses in breast cancer cells associated with differentiation rather than proliferation. Our results reveal how responses to different EGFR ligands are defined by receptor dimerization strength and signaling dynamics. These findings have broad implications for understanding receptor tyrosine kinase (RTK) signaling specificity. Our results also suggest parallels between partial and/or biased agonism in RTKs and G-protein-coupled receptors, as well as new therapeutic opportunities for correcting RTK signaling output.

Deletion Mutations Keep Kinase Inhibitors in the Loop.

Effective clinical application of conformationally selective kinase inhibitors requires tailoring drug choice to the tumor's activating mutation(s). In this issue of Cancer Cell, Foster et al. (2016) describe how activating deletions in BRAF, EGFR, and HER2 cause primary resistance to common inhibitors, suggesting strategies for improved inhibitor selection.

The Dark Side of Cell Signaling: Positive Roles for Negative Regulators.

Cell signaling is dominated by analyzing positive responses to stimuli. Signal activation is balanced by negative regulators that are generally considered to terminate signaling. Rather than exerting only negative effects, however, many such regulators play important roles in enhancing cell-signaling control. Considering responses downstream of selected cell-surface receptors, we discuss how receptor internalization affects signaling specificity and how rapid kinase/phosphatase and GTP/GDP cycles increase responsiveness and allow kinetic proofreading in receptor signaling. We highlight the blurring of distinctions between positive and negative signals, recasting signal termination as the response to a switch-like transition into a new cellular state.

Ligand regulation of a constitutively dimeric EGF receptor.

Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

Putting together structures of epidermal growth factor receptors.

Numerous crystal structures have been reported for the isolated extracellular region and tyrosine kinase domain of the epidermal growth factor receptor (EGFR) and its relatives, in different states of activation and bound to a variety of inhibitors used in cancer therapy. The next challenge is to put these structures together accurately in functional models of the intact receptor in its membrane environment. The intact EGFR has been studied using electron microscopy, chemical biology methods, biochemically, and computationally. The distinct approaches yield different impressions about the structural modes of communication between extracellular and intracellular regions. They highlight possible differences between ligands, and also underline the need to understand how the receptor interacts with the membrane itself.

Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.

Lipid and membrane mimetic environments modulate spin label side chain configuration in the outer membrane protein A.

In the present work, the factors that determine EPR line shapes from spin labels at the protein-hydrocarbon interface of a ß-barrel membrane protein are examined. The EPR spectra from hydrocarbon facing sites in the outer membrane protein A (OmpA) are highly dependent upon the detergent or lipid into which OmpA is reconstituted. In general, line shapes at these sites are correlated with the solvent accessibility in the supporting amphiphile. A notable exception is CHAPS, which yields rigid limit EPR line shapes for labels at every position along a transmembrane ß-strand in OmpA. EPR line shapes from the surface of OmpA are not strongly influenced by steric interference with neighboring side chains, but are modulated by solutes that should interact with hydrophobic surfaces. These results suggest that differences in EPR spectra in different supporting environments are not the result of differences in protein dynamics but are a result of different configurations or rotameric states that are assumed by the label. This conclusion is supported by distance measurements across the OmpA ß-barrel, which indicate that labels yielding more motionally restricted line shapes interact more closely with the protein surface. These results have implications for the use of spin-label-derived distance constraints in protein structure determination and demonstrate that spin labels on membrane proteins provide a highly sensitive probe for the environment surrounding a membrane protein.

Molecular origin of electron paramagnetic resonance line shapes on ß-barrel membrane proteins: the local solvation environment modulates spin-label configuration.

In this work, electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography were used to examine the origins of EPR line shapes from spin-labels at the protein-lipid interface on the ß-barrel membrane protein BtuB. Two atomic-resolution structures were obtained for the methanethiosulfonate spin-label derivatized to cysteines on the membrane-facing surface of BtuB. At one of these sites, position 156, the label side chain resides in a pocket formed by neighboring residues; however, it extends from the protein surface and yields a single-component EPR spectrum in the crystal that results primarily from fast rotation about the fourth and fifth bonds linking the spin-label to the protein backbone. In lipid bilayers, site 156 yields a multicomponent spectrum resulting from different rotameric states of the labeled side chain. Moreover, changes in the lipid environment, such as variations in bilayer thickness, modulate the EPR spectrum by modulating label rotamer populations. At a second site, position 371, the labeled side chain interacts with a pocket on the protein surface, leading to a highly immobilized single-component EPR spectrum that is not sensitive to hydrocarbon thickness. This spectrum is similar to that seen at other sites that are deep in the hydrocarbon, such as position 170. This work indicates that the rotameric states of spin-labels on exposed hydrocarbon sites are sensitive to the environment at the protein-hydrocarbon interface, and that this environment may modulate weak interactions between the labeled side chain and the protein surface. In the case of BtuB, lipid acyl chain packing is not symmetric around the ß-barrel, and EPR spectra from labeled hydrocarbon-facing sites in BtuB may reflect this asymmetry. In addition to facilitating the interpretation of EPR spectra of membrane proteins, these results have important implications for the use of long-range distance restraints in protein structure refinement that are obtained from spin-labels.

Conformational exchange in a membrane transport protein is altered in protein crystals.

Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B(12). Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.