Publications by authors named "Sirano Dhe-Paganon"

96 Publications

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

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cell 2020 Nov;183(5):1219-1233.e18

Transplantation Research Center, Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.
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http://dx.doi.org/10.1016/j.cell.2020.10.045DOI Listing
November 2020

The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma.

Nat Struct Mol Biol 2020 09 3;27(9):836-845. Epub 2020 Aug 3.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA.

Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.
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http://dx.doi.org/10.1038/s41594-020-0466-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714695PMC
September 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.
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http://dx.doi.org/10.1038/s42255-020-0199-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568475PMC
May 2020

Identification of a potent and selective covalent Pin1 inhibitor.

Nat Chem Biol 2020 09 1;16(9):979-987. Epub 2020 Jun 1.

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

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.
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http://dx.doi.org/10.1038/s41589-020-0550-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442691PMC
September 2020

Loss of heterozygosity of essential genes represents a widespread class of potential cancer vulnerabilities.

Nat Commun 2020 05 20;11(1):2517. Epub 2020 May 20.

Departments of Cancer Biology, Boston, MA, USA.

Alterations in non-driver genes represent an emerging class of potential therapeutic targets in cancer. Hundreds to thousands of non-driver genes undergo loss of heterozygosity (LOH) events per tumor, generating discrete differences between tumor and normal cells. Here we interrogate LOH of polymorphisms in essential genes as a novel class of therapeutic targets. We hypothesized that monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles. We identified 5664 variants in 1278 essential genes that undergo LOH in cancer and evaluated the potential for each to be targeted using allele-specific gene-editing, RNAi, or small-molecule approaches. We further show that allele-specific inactivation of either of two essential genes (PRIM1 and EXOSC8) reduces growth of cells harboring that allele, while cells harboring the non-targeted allele remain intact. We conclude that LOH of essential genes represents a rich class of non-driver cancer vulnerabilities.
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http://dx.doi.org/10.1038/s41467-020-16399-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239950PMC
May 2020

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

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

Discovery and Structure-Activity Relationship Study of ()-5-Methylenethiazolidin-4-one Derivatives as Potent and Selective Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors.

J Med Chem 2020 05 27;63(9):4880-4895. Epub 2020 Apr 27.

Department of Cancer Biology, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, Massachusetts 02215, United States.

Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 Å X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure-activity relationship led to the development of compound , replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -β by the inhibitors in HEK 293T cells.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292230PMC
May 2020

Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism.

Sci Rep 2020 03 24;10(1):5324. Epub 2020 Mar 24.

Department of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.
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http://dx.doi.org/10.1038/s41598-020-62076-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093416PMC
March 2020

Structure-Activity Relationship Study of Covalent Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors.

ACS Med Chem Lett 2020 Mar 3;11(3):346-352. Epub 2019 Nov 3.

Department of Cancer Biology, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, Massachusetts 02215, United States.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure-activity relationship (SAR) study of this scaffold, resulting in compound , which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound represents a highly selective pan-PI5P4K covalent lead molecule.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074221PMC
March 2020

Targeting the PI5P4K Lipid Kinase Family in Cancer Using Covalent Inhibitors.

Cell Chem Biol 2020 05 3;27(5):525-537.e6. Epub 2020 Mar 3.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.
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http://dx.doi.org/10.1016/j.chembiol.2020.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286548PMC
May 2020

Recurrent SMARCB1 Mutations Reveal a Nucleosome Acidic Patch Interaction Site That Potentiates mSWI/SNF Complex Chromatin Remodeling.

Cell 2019 11 20;179(6):1342-1356.e23. Epub 2019 Nov 20.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.
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http://dx.doi.org/10.1016/j.cell.2019.10.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175411PMC
November 2019

Structural Implications of STAT3 and STAT5 SH2 Domain Mutations.

Cancers (Basel) 2019 Nov 8;11(11). Epub 2019 Nov 8.

Centre for Medicinal Chemistry, University of Toronto at Mississauga, Mississauga, ON L5L 1C6, Canada.

Src Homology 2 (SH2) domains arose within metazoan signaling pathways and are involved in protein regulation of multiple pleiotropic cascades. In signal transducer and activator of transcription (STAT) proteins, SH2 domain interactions are critical for molecular activation and nuclear accumulation of phosphorylated STAT dimers to drive transcription. Sequencing analysis of patient samples has revealed the SH2 domain as a hotspot in the mutational landscape of STAT proteins although the functional impact for the vast majority of these mutations remains poorly characterized. Despite several well resolved structures for SH2 domain-containing proteins, structural data regarding the distinctive STAT-type SH2 domain is limited. Here, we review the unique features of STAT-type SH2 domains in the context of all currently reported STAT3 and STAT5 SH2 domain clinical mutations. The genetic volatility of specific regions in the SH2 domain can result in either activating or deactivating mutations at the same site in the domain, underscoring the delicate evolutionary balance of wild type STAT structural motifs in maintaining precise levels of cellular activity. Understanding the molecular and biophysical impact of these disease-associated mutations can uncover convergent mechanisms of action for mutations localized within the STAT SH2 domain to facilitate the development of targeted therapeutic interventions.
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http://dx.doi.org/10.3390/cancers11111757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895964PMC
November 2019

Dual Inhibition of TAF1 and BET Bromodomains from the BI-2536 Kinase Inhibitor Scaffold.

ACS Med Chem Lett 2019 Oct 13;10(10):1443-1449. Epub 2019 Sep 13.

Department of Cancer Biology, Dana Farber Cancer Institute, Boston Massachusetts 02115, United States.

Recent reports have highlighted the dual bromodomains of TAF1 (TAF1(1,2)) as synergistic with BET inhibition in cellular cancer models, engendering interest in TAF/BET polypharmacology. Here, we examine structure activity relationships within the BI-2536 PLK1 kinase inhibitor scaffold, previously reported to bind BRD4. We examine binding by this ligand to TAF1(2) and apply structure guided design strategies to discriminate binding to both the PLK1 kinase and BRD4(1) bromodomain while retaining activity on TAF1(2). Through this effort we discover potent dual inhibitors of TAF1(2)/BRD4(1), as well as biased derivatives showing marked TAF1 selectivity. We resolve X-ray crystallographic data sets to examine the mechanisms of the observed TAF1 selectivity and to provide a resource for further development of this scaffold.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792166PMC
October 2019

Structural and functional consequences of the STAT5B driver mutation.

Nat Commun 2019 06 7;10(1):2517. Epub 2019 Jun 7.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, L5L 1C6, Canada.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5B, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5B in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5B-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5B patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5B crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5B can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5B, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5B activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.
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http://dx.doi.org/10.1038/s41467-019-10422-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555848PMC
June 2019

Arsenic targets Pin1 and cooperates with retinoic acid to inhibit cancer-driving pathways and tumor-initiating cells.

Nat Commun 2018 08 9;9(1):3069. Epub 2018 Aug 9.

Department of Medicine, Division of Translational Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA.

Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) combination safely cures fatal acute promyelocytic leukemia, but their mechanisms of action and efficacy are not fully understood. ATRA inhibits leukemia, breast, and liver cancer by targeting isomerase Pin1, a master regulator of oncogenic signaling networks. Here we show that ATO targets Pin1 and cooperates with ATRA to exert potent anticancer activity. ATO inhibits and degrades Pin1, and suppresses its oncogenic function by noncovalent binding to Pin1's active site. ATRA increases cellular ATO uptake through upregulating aquaporin-9. ATO and ATRA, at clinically safe doses, cooperatively ablate Pin1 to block numerous cancer-driving pathways and inhibit the growth of triple-negative breast cancer cells and tumor-initiating cells in cell and animal models including patient-derived orthotopic xenografts, like Pin1 knockout, which is substantiated by comprehensive protein and microRNA analyses. Thus, synergistic targeting of Pin1 by ATO and ATRA offers an attractive approach to combating breast and other cancers.
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http://dx.doi.org/10.1038/s41467-018-05402-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085299PMC
August 2018

Islet-Derived eATP Fuels Autoreactive CD8 T Cells and Facilitates the Onset of Type 1 Diabetes.

Diabetes 2018 10 31;67(10):2038-2053. Epub 2018 Jul 31.

Division of Nephrology, Boston Children's Hospital, Harvard Medical School, Boston, MA

Extracellular ATP (eATP) activates T cells by engaging the P2X7R receptor. We identified two loss-of-function P2X7R mutations that are protective against type 1 diabetes (T1D) and thus hypothesized that eATP/P2X7R signaling may represent an early step in T1D onset. Specifically, we observed that in patients with newly diagnosed T1D, P2X7R is upregulated on CD8 effector T cells in comparison with healthy control subjects. eATP is released at high levels by human/murine islets in vitro in high-glucose/inflammatory conditions, thus upregulating P2X7R on CD8 T cells in vitro. P2X7R blockade with oxidized ATP reduces the CD8 T cell-mediated autoimmune response in vitro and delays diabetes onset in NOD mice. Autoreactive CD8 T-cell activation is highly dependent upon eATP/P2X7R-mediated priming, while a novel sP2X7R recombinant protein abrogates changes in metabolism and the autoimmune response associated with CD8 T cells. eATP/P2X7R signaling facilitates the onset of autoimmune T1D by fueling autoreactive CD8 cells and therefore represents a novel targeted therapeutic for the disorder.
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http://dx.doi.org/10.2337/db17-1227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905486PMC
October 2018

P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes.

J Clin Invest 2018 08 16;128(8):3490-3503. Epub 2018 Jul 16.

International Center for Type 1 Diabetes, Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, "L. Sacco" Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy.

Purinergic receptor-7 (P2X7R) signaling controls Th17 and Th1 generation/differentiation, while NOD-like receptor P3 (NLRP3) acts as a Th2 transcriptional factor. Here, we demonstrated the existence of a P2X7R/NLRP3 pathway in T cells that is dysregulated by a P2X7R intracellular region loss-of-function mutation, leading to NLRP3 displacement and to excessive Th17 generation due to abrogation of the NLRP3-mediated Th2 program. This ultimately resulted in poor outcomes in cardiac-transplanted patients carrying the mutant allele, who showed abnormal Th17 generation. Transient NLRP3 silencing in nonmutant T cells or overexpression in mutant T cells normalized the Th profile. Interestingly, IL-17 blockade reduced Th17 skewing of human T cells in vitro and abrogated the severe allograft vasculopathy and abnormal Th17 generation observed in preclinical models in which P2X7R was genetically deleted. This P2X7R intracellular region mutation thus impaired the modulatory effects of P2X7R on NLRP3 expression and function in T cells and led to NLRP3 dysregulation and Th17 skewing, delineating a high-risk group of cardiac-transplanted patients who may benefit from personalized therapy.
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http://dx.doi.org/10.1172/JCI94524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063506PMC
August 2018

Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands.

Nat Chem Biol 2018 04 5;14(4):405-412. Epub 2018 Mar 5.

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

The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.
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http://dx.doi.org/10.1038/s41589-018-0010-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866761PMC
April 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.
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http://dx.doi.org/10.1016/j.str.2017.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947960PMC
January 2018

Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7.

Cell Chem Biol 2017 12 19;24(12):1490-1500.e11. Epub 2017 Oct 19.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5-10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors.
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http://dx.doi.org/10.1016/j.chembiol.2017.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749250PMC
December 2017

Inhibition of USP10 induces degradation of oncogenic FLT3.

Nat Chem Biol 2017 Dec 2;13(12):1207-1215. Epub 2017 Oct 2.

Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.
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http://dx.doi.org/10.1038/nchembio.2486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314479PMC
December 2017

MELK is not necessary for the proliferation of basal-like breast cancer cells.

Elife 2017 09 19;6. Epub 2017 Sep 19.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, United States.

Thorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth. This discrepancy to previous findings illuminated selectivity issues of the widely used MELK inhibitor OTSSP167, and potential off-target effects of MELK-targeting short hairpins. The different genetic and chemical tools developed here allow for the identification and validation of any causal roles MELK may play in cancer biology, which will be required to guide future MELK drug discovery efforts. Furthermore, our study provides a general framework for preclinical target validation.
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http://dx.doi.org/10.7554/eLife.26693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605198PMC
September 2017

Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands.

Angew Chem Int Ed Engl 2017 05 18;56(21):5738-5743. Epub 2017 Apr 18.

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

The bromodomain-containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl-lysine recognition. We have therefore created the first BRD9-directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10- to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non-BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.
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http://dx.doi.org/10.1002/anie.201611281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967236PMC
May 2017

Transcription control by the ENL YEATS domain in acute leukaemia.

Nature 2017 03 1;543(7644):270-274. Epub 2017 Mar 1.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.
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http://dx.doi.org/10.1038/nature21688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497220PMC
March 2017

Leveraging Gas-Phase Fragmentation Pathways for Improved Identification and Selective Detection of Targets Modified by Covalent Probes.

Anal Chem 2016 12 30;88(24):12248-12254. Epub 2016 Nov 30.

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School , Boston, Massachusetts 02115, United States.

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.
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http://dx.doi.org/10.1021/acs.analchem.6b03394DOI Listing
December 2016

p53-related protein kinase confers poor prognosis and represents a novel therapeutic target in multiple myeloma.

Blood 2017 03 12;129(10):1308-1319. Epub 2017 Jan 12.

Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA.

p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcriptase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-type-p53 MM.1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM.
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http://dx.doi.org/10.1182/blood-2016-09-738500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345734PMC
March 2017

Design and characterization of bivalent BET inhibitors.

Nat Chem Biol 2016 Dec 24;12(12):1089-1096. Epub 2016 Oct 24.

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

Cellular signaling is often propagated by multivalent interactions. Multivalency creates avidity, allowing stable biophysical recognition. Multivalency is an attractive strategy for achieving potent binding to protein targets, as the affinity of bivalent ligands is often greater than the sum of monovalent affinities. The bromodomain and extraterminal domain (BET) family of transcriptional coactivators features tandem bromodomains through which BET proteins bind acetylated histones and transcription factors. All reported antagonists of the BET protein BRD4 bind in a monovalent fashion. Here we describe, to our knowledge for the first time, a bivalent BET bromodomain inhibitor-MT1-which has unprecedented potency. Biophysical and biochemical studies suggest MT1 is an intramolecular bivalent BRD4 binder that is more than 100-fold more potent, in cellular assays, than the corresponding monovalent antagonist, JQ1. MT1 significantly (P < 0.05) delayed leukemia progression in mice, as compared to JQ1. These data qualify a powerful chemical probe for BET bromodomains and a rationale for further development of multidomain inhibitors of epigenetic reader proteins.
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http://dx.doi.org/10.1038/nchembio.2209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117811PMC
December 2016