Publications by authors named "Loren D Walensky"

81 Publications

Characterizing Native and Hydrocarbon-Stapled Enfuvirtide Conformations with Ion Mobility Mass Spectrometry and Hydrogen-Deuterium Exchange.

J Am Soc Mass Spectrom 2021 Mar 3;32(3):753-761. Epub 2021 Feb 3.

Metrology, National Research Council Canada, 1200 Montreal Road, Ottawa, Ontario K1A 0R6, Canada.

The number of approved peptide therapeutics, as well as those in development, has been increasing in recent years. Frequently, the biological activity of such peptides is elicited through the adoption of secondary structural elements upon interaction with their cellular target. However, many therapeutic peptides are unstructured in solution and accordingly exhibit a poor bioavailability due to rapid proteolysis . To combat this degradation, numerous naturally occurring peptides with therapeutic properties contain stabilizing features, such as N-to-C cyclization or disulfide bonds. Recently, hydrocarbon stapling via non-native amino acid substitution followed by ring-closing metathesis has been shown to induce a dramatic stabilization of α-helical peptides. Identifying the ideal staple location along the peptide backbone is a critical developmental step, and methods to streamline this optimization are needed. Mass spectrometry-based methods such as ion mobility (IM) and hydrogen-deuterium exchange (HDX) can detect multiple discrete peptide conformations, a significant advantage over bulk spectroscopic techniques. In this study we use IM-MS and HDX-MS to demonstrate that the native 36-residue enfuvirtide peptide is highly dynamic in solution and the conformational ensemble populated by stabilized constructs depends heavily on the staple location. Further, our measurements yielded results that correlate well with the average α-helical content measured by circular dichroism. The MS-based approaches described herein represent sensitive and potentially high-throughput methods for characterizing and identifying optimally stapled peptides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jasms.0c00453DOI Listing
March 2021

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival.

J Cell Biol 2021 Jan;220(1)

Department of Neurobiology, Harvard Medical School, Boston, MA.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.202005051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721913PMC
January 2021

Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.

Nat Chem Biol 2020 11 17;16(11):1218-1226. Epub 2020 Aug 17.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-020-0625-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904387PMC
November 2020

Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation.

Nat Metab 2020 05 11;2(5):432-446. Epub 2020 May 11.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet β-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42255-020-0199-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568475PMC
May 2020

A redox switch regulates the structure and function of anti-apoptotic BFL-1.

Nat Struct Mol Biol 2020 09 13;27(9):781-789. Epub 2020 Jul 13.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-020-0458-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544158PMC
September 2020

Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization.

Mol Cell 2020 07 12;79(1):68-83.e7. Epub 2020 Jun 12.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAX) for analysis. Here, we report the production and characterization of a full-length BAX that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAX enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAX, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472837PMC
July 2020

Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering.

Cell Chem Biol 2020 06 14;27(6):647-656.e6. Epub 2020 May 14.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405809PMC
June 2020

Identification of a Structural Determinant for Selective Targeting of HDMX.

Structure 2020 07 30;28(7):847-857.e5. Epub 2020 Apr 30.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2020.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427690PMC
July 2020

Hydrocarbon-Stitched Peptide Agonists of Glucagon-Like Peptide-1 Receptor.

ACS Chem Biol 2020 06 27;15(6):1340-1348. Epub 2020 May 27.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, United States.

Glucagon-like peptide 1 (GLP-1) is a natural peptide agonist of the GLP-1 receptor (GLP-1R) found on pancreatic β-cells. Engagement of the receptor stimulates insulin release in a glucose-dependent fashion and increases β-cell mass, two ideal features for pharmacologic management of type 2 diabetes. Thus, intensive efforts have focused on developing GLP-1-based peptide agonists of GLP-1R for therapeutic application. A primary challenge has been the naturally short half-life of GLP-1 due to its rapid proteolytic degradation . Whereas mutagenesis and lipidation strategies have yielded clinical agents, we developed an alternative approach to preserving the structure and function of GLP-1 by all-hydrocarbon , + 7 stitching. This particular "stitch" is especially well-suited for reinforcing and protecting the structural fidelity of GLP-1. Lead constructs demonstrate striking proteolytic stability and potent biological activity . Thus, we report a facile approach to generating alternative GLP-1R agonists for glycemic control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.0c00308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366314PMC
June 2020

Site-Dependent Cysteine Lipidation Potentiates the Activation of Proapoptotic BAX.

Cell Rep 2020 03;30(10):3229-3239.e6

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

BCL-2 family proteins converge at the mitochondrial outer membrane to regulate apoptosis and maintain the critical balance between cellular life and death. This physiologic process is essential to organism homeostasis and relies on protein-protein and protein-lipid interactions among BCL-2 family proteins in the mitochondrial lipid environment. Here, we find that trans-2-hexadecenal (t-2-hex), previously implicated in regulating BAX-mediated apoptosis, does so by direct covalent reaction with C126, which is located on the surface of BAX at the junction of its α5/α6 core hydrophobic hairpin. The application of nuclear magnetic resonance spectroscopy, hydrogen-deuterium exchange mass spectrometry, specialized t-2-hex-containing liposomes, and BAX mutational studies in mitochondria and cells reveals structure-function insights into the mechanism by which covalent lipidation at the mitochondria sensitizes direct BAX activation. The functional role of BAX lipidation as a control point of mitochondrial apoptosis could provide a therapeutic strategy for BAX modulation by chemical modification of C126.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.02.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343539PMC
March 2020

Mind the gap: Expediting gender parity in MD-PhD admissions.

JCI Insight 2020 02 27;5(4). Epub 2020 Feb 27.

Harvard/MIT MD-PhD Program and.

The 2018 National MD-PhD Program Outcomes Study highlighted the critical need to increase MD-PhD trainee diversity and close the gender gap in MD-PhD enrollment. This Association of American Medical Colleges imperative prompted us to evaluate trends in female matriculation from our institutional MD-PhD program compared with national data. Based on a 10-year review of Harvard/MIT Medical Scientist Training Program admissions, we observed a sharp and sustained increase in female matriculants for the past 5 years that is well above the national average. We report our experience with achieving gender parity among matriculants of our MD-PhD program, identify the specific stage of the admissions process where the gender balance acutely shifted, and attribute the increase in female matriculation to concrete administrative changes that were put into place just prior to the observed gender balance shift. These changes included increasing the number of faculty participants in application screening and awardee selection and establishing gender balance among faculty decision makers. We believe that adopting basic administrative practices geared toward increasing the diversity of perspectives among admissions faculty has the potential to expedite gender parity of MD-PhD matriculants nationwide and could eventually help achieve gender balance in the national physician-scientist workforce.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.136037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101133PMC
February 2020

Design of stapled antimicrobial peptides that are stable, nontoxic and kill antibiotic-resistant bacteria in mice.

Nat Biotechnol 2019 10 19;37(10):1186-1197. Epub 2019 Aug 19.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

The clinical translation of cationic α-helical antimicrobial peptides (AMPs) has been hindered by structural instability, proteolytic degradation and in vivo toxicity from nonspecific membrane lysis. Although analyses of hydrophobic content and charge distribution have informed the design of synthetic AMPs with increased potency and reduced in vitro hemolysis, nonspecific membrane toxicity in vivo continues to impede AMP drug development. Here, we analyzed a 58-member library of stapled AMPs (StAMPs) based on magainin II and applied the insights from structure-function-toxicity measurements to devise an algorithm for the design of stable, protease-resistant, potent and nontoxic StAMP prototypes. We show that a lead double-stapled StAMP named Mag(i+4)1,15(A9K,B21A,N22K,S23K) can kill multidrug-resistant Gram-negative pathogens, such as colistin-resistant Acinetobacter baumannii in a mouse peritonitis-sepsis model, without observed hemolysis or renal injury in murine toxicity studies. Inputting the amino acid sequences alone, we further generated membrane-selective StAMPs of pleurocidin, CAP18 and esculentin, highlighting the generalizability of our design platform.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-019-0222-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437984PMC
October 2019

Targeting BAX to drug death directly.

Authors:
Loren D Walensky

Nat Chem Biol 2019 07 17;15(7):657-665. Epub 2019 Jun 17.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

BCL-2 family protein interactions regulate apoptosis, a critical process that maintains tissue homeostasis but can cause a host of human diseases when deregulated. Venetoclax is the first FDA-approved drug to reactivate apoptosis in cancer by selectively targeting an anti-apoptotic BCL-2 family member. The drug's activity relies on an 'inhibit the inhibitor' mechanism, whereby blockade of a key surface groove on BCL-2 disables its capacity to neutralize pro-apoptotic effectors, such as BAX, a chief executioner protein of the apoptotic pathway. A series of physiologic and pharmacologic regulatory sites that mediate the activation or inhibition of BAX have recently been identified, providing blueprints for the development of alternative apoptosis modulators to block pathologic cell survival or avert unwanted cell death by drugging BAX directly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-019-0306-6DOI Listing
July 2019

Targeting BAX to drug death directly.

Authors:
Loren D Walensky

Nat Chem Biol 2019 07 17;15(7):657-665. Epub 2019 Jun 17.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

BCL-2 family protein interactions regulate apoptosis, a critical process that maintains tissue homeostasis but can cause a host of human diseases when deregulated. Venetoclax is the first FDA-approved drug to reactivate apoptosis in cancer by selectively targeting an anti-apoptotic BCL-2 family member. The drug's activity relies on an 'inhibit the inhibitor' mechanism, whereby blockade of a key surface groove on BCL-2 disables its capacity to neutralize pro-apoptotic effectors, such as BAX, a chief executioner protein of the apoptotic pathway. A series of physiologic and pharmacologic regulatory sites that mediate the activation or inhibition of BAX have recently been identified, providing blueprints for the development of alternative apoptosis modulators to block pathologic cell survival or avert unwanted cell death by drugging BAX directly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-019-0306-6DOI Listing
July 2019

MDM2 and MDM4 Are Therapeutic Vulnerabilities in Malignant Rhabdoid Tumors.

Cancer Res 2019 05 12;79(9):2404-2414. Epub 2019 Feb 12.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.

Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered and , the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of . We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth , with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-18-3066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497578PMC
May 2019

Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer.

Cell Rep 2018 09;24(13):3393-3403.e5

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells. Because cancers harbor a diversity of resistance mechanisms and typically require multi-agent treatment, we further investigated BFL-1/A1 co-dependencies by mining a genome-scale CRISPR-Cas9 screen. We identified ataxia-telangiectasia-mutated (ATM) kinase as a BFL-1/A1 co-dependency in acute myeloid leukemia (AML), which informed the validation of BFL-1/A1 and ATM inhibitor co-treatment as a synergistic approach to subverting apoptotic resistance in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.08.089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365304PMC
September 2018

Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in wild-type Ewing sarcoma.

J Exp Med 2018 08 25;215(8):2137-2155. Epub 2018 Jul 25.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA

Ewing sarcoma is a pediatric cancer driven by EWS-ETS transcription factor fusion oncoproteins in an otherwise stable genomic background. The majority of tumors express wild-type , and thus, therapies targeting the p53 pathway would benefit most patients. To discover targets specific for wild-type Ewing sarcoma, we used a genome-scale CRISPR-Cas9 screening approach and identified and validated , , and as druggable dependencies. The stapled peptide inhibitor of MDM2 and MDM4, ATSP-7041, showed anti-tumor efficacy in vitro and in multiple mouse models. The USP7 inhibitor, P5091, and the Wip1/ inhibitor, GSK2830371, decreased the viability of Ewing sarcoma cells. The combination of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic action on the p53 pathway. The effects of the inhibitors, including the specific USP7 inhibitor XL-188, were rescued by concurrent knockout, highlighting the essentiality of intact p53 for the observed cytotoxic activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20171066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080915PMC
August 2018

Dynamic Regulation of Long-Chain Fatty Acid Oxidation by a Noncanonical Interaction between the MCL-1 BH3 Helix and VLCAD.

Mol Cell 2018 03;69(5):729-743.e7

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid β-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1 alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2018.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916823PMC
March 2018

Iterative optimization yields Mcl-1-targeting stapled peptides with selective cytotoxicity to Mcl-1-dependent cancer cells.

Proc Natl Acad Sci U S A 2018 01 16;115(5):E886-E895. Epub 2018 Jan 16.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;

Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1-binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1-dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or "staple" that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation workflow applicable to a host of challenging biomedical targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1712952115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798337PMC
January 2018

Crystal Structures of Anti-apoptotic BFL-1 and Its Complex with a Covalent Stapled Peptide Inhibitor.

Structure 2018 01 21;26(1):153-160.e4. Epub 2017 Dec 21.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2017.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947960PMC
January 2018

Paclitaxel Reduces Axonal Bclw to Initiate IPR1-Dependent Axon Degeneration.

Neuron 2017 Oct;96(2):373-386.e6

Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA; Departments of Cancer Biology and Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of many cancer treatments. The hallmark of CIPN is degeneration of long axons required for transmission of sensory information; axonal degeneration causes impaired tactile sensation and persistent pain. Currently the molecular mechanisms of CIPN are not understood, and there are no available treatments. Here we show that the chemotherapeutic agent paclitaxel triggers CIPN by altering IP receptor phosphorylation and intracellular calcium flux, and activating calcium-dependent calpain proteases. Concomitantly paclitaxel impairs axonal trafficking of RNA-granules and reduces synthesis of Bclw (bcl2l2), a Bcl2 family member that binds IPR1 and restrains axon degeneration. Surprisingly, Bclw or a stapled peptide corresponding to the Bclw BH4 domain interact with axonal IPR1 and prevent paclitaxel-induced degeneration, while Bcl2 and Bclx cannot do so. Together these data identify a Bclw-IPR1-dependent cascade that causes axon degeneration and suggest that Bclw-mimetics could provide effective therapy to prevent CIPN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2017.09.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680044PMC
October 2017

Direct Activation of BAX by BTSA1 Overcomes Apoptosis Resistance in Acute Myeloid Leukemia.

Cancer Cell 2017 10;32(4):490-505.e10

Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address:

The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2017.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793879PMC
October 2017

Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor.

Nat Cell Biol 2017 Oct 25;19(10):1226-1236. Epub 2017 Sep 25.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Direct interactions between pro- and anti-apoptotic BCL-2 family members form the basis of cell death decision-making at the outer mitochondrial membrane (OMM). Here we report that three anti-apoptotic BCL-2 proteins (MCL-1, BCL-2 and BCL-XL) found untethered from the OMM function as transcriptional regulators of a prosurvival and growth program. Anti-apoptotic BCL-2 proteins engage a BCL-2 homology (BH) domain sequence found in SUFU (suppressor of fused), a tumour suppressor and antagonist of the GLI DNA-binding proteins. BCL-2 proteins directly promote SUFU turnover, inhibit SUFU-GLI interaction, and induce the expression of the GLI target genes BCL-2, MCL-1 and BCL-XL. Anti-apoptotic BCL-2 protein/SUFU feedforward signalling promotes cancer cell survival and growth, and can be disabled with BH3 mimetics-small molecules that target anti-apoptotic BCL-2 proteins. Our findings delineate a chemical strategy for countering drug resistance in GLI-associated tumours and reveal unanticipated functions for BCL-2 proteins as transcriptional regulators.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb3616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657599PMC
October 2017

Allosteric sensitization of proapoptotic BAX.

Nat Chem Biol 2017 Sep 10;13(9):961-967. Epub 2017 Jul 10.

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675120PMC
September 2017

gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development.

FASEB J 2017 08 26;31(8):3540-3554. Epub 2017 Apr 26.

Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA;

Low nephron endowment at birth has been associated with an increased risk for developing hypertension and chronic kidney disease. We demonstrated in an earlier study that conditional deletion of the microRNA (miRNA)-processing enzyme Dicer from nephron progenitors results in premature depletion of the progenitors and increased expression of the proapoptotic protein Bim (also known as Bcl-2L11). In this study, we generated a compound mouse model with conditional deletion of both and , to determine the biologic significance of increased Bim expression in -deficient nephron progenitors. The loss of partially restored the number of nephron progenitors and improved nephron formation. The number of progenitors undergoing apoptosis was significantly reduced in kidneys with loss of a single allele, or both alleles, of compared to mutant kidneys. Furthermore, 2 miRNAs expressed in nephron progenitors ( and regulated Bim levels and Together, these data suggest that miRNA-mediated regulation of Bim controls nephron progenitor survival during nephrogenesis, as one potential means of regulating nephron endowment.-Cerqueira, D. M., Bodnar, A. J., Phua, Y. L., Freer, R., Hemker, S. L., Walensky, L. D., Hukriede, N. A., Ho, J. gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201700010RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503708PMC
August 2017

Challenges in Targeting a Basic Helix-Loop-Helix Transcription Factor with Hydrocarbon-Stapled Peptides.

ACS Chem Biol 2016 11 4;11(11):3146-3153. Epub 2016 Oct 4.

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute , Boston, Massachusetts 02215, United States.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein-protein and protein-DNA interactions mediated by α-helices. Thus, α-helical decoys have been proposed as potential targeted therapies for pathologic bHLH transcription. Here, we developed a library of stabilized α-helices of OLIG2 (SAH-OLIG2) to test the capacity of hydrocarbon-stapled peptides to disrupt OLIG2 homodimerization, which drives the development and chemoresistance of glioblastoma multiforme, one of the deadliest forms of human brain cancer. Although stapling successfully reinforced the α-helical structure of bHLH constructs of varying length, sequence-specific dissociation of OLIG2 dimers from DNA was not achieved. Re-evaluation of the binding determinants for OLIG2 self-association and stability revealed an unanticipated role of the C-terminal domain. These data highlight potential pitfalls in peptide-based targeting of bHLH transcription factors given the liabilities of their positively charged amino acid sequences and multifactorial binding determinants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.6b00465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5534327PMC
November 2016

Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors.

Cell Chem Biol 2016 Sep 8;23(9):1123-1134. Epub 2016 Sep 8.

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical α-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055752PMC
http://dx.doi.org/10.1016/j.chembiol.2016.07.022DOI Listing
September 2016

Biophysical determinants for cellular uptake of hydrocarbon-stapled peptide helices.

Nat Chem Biol 2016 10 22;12(10):845-52. Epub 2016 Aug 22.

Department of Pediatric Oncology, Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

Hydrocarbon-stapled peptides are a class of bioactive alpha-helical ligands developed to dissect and target protein interactions. While there is consensus that stapled peptides can be effective chemical tools for investigating protein regulation, their broader utility for therapeutic modulation of intracellular interactions remains an active area of study. In particular, the design principles for generating cell-permeable stapled peptides are empiric, yet consistent intracellular access is essential to in vivo application. Here, we used an unbiased statistical approach to determine which biophysical parameters dictate the uptake of stapled-peptide libraries. We found that staple placement at the amphipathic boundary combined with optimal hydrophobic and helical content are the key drivers of cellular uptake, whereas excess hydrophobicity and positive charge at isolated amino acid positions can trigger membrane lysis at elevated peptide dosing. Our results provide a design roadmap for maximizing the potential to generate cell-permeable stapled peptides with on-mechanism cellular activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055751PMC
http://dx.doi.org/10.1038/nchembio.2153DOI Listing
October 2016

Allosteric inhibition of antiapoptotic MCL-1.

Nat Struct Mol Biol 2016 06 9;23(6):600-7. Epub 2016 May 9.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

MCL-1 is an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Although drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small-molecule covalent modification of C286 at a new interaction site distant from the BH3-binding groove. Our structure-function analyses revealed that the BH3 binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in mouse cells. Thus, we characterize an allosteric mechanism for disrupting the antiapoptotic BH3 binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.3223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900187PMC
June 2016