Publications by authors named "Jarrod A Marto"

156 Publications

Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase.

Mol Cell 2021 Dec 15. Epub 2021 Dec 15.

Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. Electronic address:

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.
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http://dx.doi.org/10.1016/j.molcel.2021.11.033DOI Listing
December 2021

PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors.

Anal Chem 2021 10 4;93(41):13791-13799. Epub 2021 Oct 4.

Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, United States.

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.
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http://dx.doi.org/10.1021/acs.analchem.1c02349DOI Listing
October 2021

Exploring Ligand-Directed -Acyl--alkylsulfonamide-Based Acylation Chemistry for Potential Targeted Degrader Development.

ACS Med Chem Lett 2021 Aug 21;12(8):1302-1307. Epub 2021 Jul 21.

Department of Chemical and Systems Biology, ChEM-H, Stanford Cancer Institute, School of Medicine, Stanford University, Stanford, California 94305, United States.

Ligand-directed bioconjugation strategies have been used for selective protein labeling in live cells or tissue samples in applications such as live-cell imaging. Here we hypothesized that a similar strategy could be used for targeted protein degradation. To test this possibility, we developed a series of CDK2-targeting -acyl--alkylsulfonamide (NASA)-containing acylation probes. The probes featured three components: a CDK2 homing ligand, a CRL4 E3 ligase recruiting ligand, and a NASA functionality. We determined that upon target binding, NASA-mediated reaction resulted in selective functionalization of Lys89 on purified or native CDK2. However, we were unable to observe CDK2 degradation, which is in contrast to the efficient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable efficient protein degradation remains an open question.
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http://dx.doi.org/10.1021/acsmedchemlett.1c00285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8366001PMC
August 2021

Small molecule inhibition of deubiquitinating enzyme JOSD1 as a novel targeted therapy for leukemias with mutant JAK2.

Leukemia 2022 Jan 29;36(1):210-220. Epub 2021 Jul 29.

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

Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell's intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN.
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http://dx.doi.org/10.1038/s41375-021-01336-9DOI Listing
January 2022

Chemoproteomic methods for covalent drug discovery.

Chem Soc Rev 2021 Aug;50(15):8361-8381

Department of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA. and Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.

Covalent drugs constitute cornerstones of modern medicine. The past decade has witnessed growing enthusiasm for development of covalent inhibitors, fueled by clinical successes as well as advances in analytical techniques associated with the drug discovery pipeline. Among these, mass spectrometry-based chemoproteomic methods stand out due to their broad applicability from focused analysis of electrophile-containing compounds to surveying proteome-wide inhibitor targets. Here, we review applications of both foundational and cutting-edge chemoproteomic techniques across target identification, hit discovery, and lead characterization/optimization in covalent drug discovery. We focus on the practical aspects necessary for the general drug discovery scientist to design, interpret, and evaluate chemoproteomic experiments. We also present three case studies on clinical stage molecules to further showcase the real world significance and future opportunities of these methodologies.
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http://dx.doi.org/10.1039/d1cs00231gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8328943PMC
August 2021

Identification and validation of selective deubiquitinase inhibitors.

Cell Chem Biol 2021 12 14;28(12):1758-1771.e13. Epub 2021 Jun 14.

Department of Cancer Biology and the Linde Program in Cancer Chemical 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) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes.
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http://dx.doi.org/10.1016/j.chembiol.2021.05.012DOI Listing
December 2021

BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage.

Nat Commun 2021 06 10;12(1):3542. Epub 2021 Jun 10.

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

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.
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http://dx.doi.org/10.1038/s41467-021-23716-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192922PMC
June 2021

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

Nat Chem Biol 2021 09 10;17(9):954-963. Epub 2021 May 10.

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

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
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http://dx.doi.org/10.1038/s41589-021-00786-7DOI Listing
September 2021

Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma.

Eur J Med Chem 2021 Oct 20;221:113481. Epub 2021 Apr 20.

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

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.
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http://dx.doi.org/10.1016/j.ejmech.2021.113481DOI Listing
October 2021

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

J Cell Biol 2021 01;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

A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus.

Nat Commun 2020 12 1;11(1):6135. Epub 2020 Dec 1.

Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis. Using RNA-seq profiling of the intima of lesions, here we identify a macrophage-specific lncRNA MAARS (Macrophage-Associated Atherosclerosis lncRNA Sequence). Aortic intima expression of MAARS increases by 270-fold with atherosclerotic progression and decreases with regression by 60%. MAARS knockdown reduces atherosclerotic lesion formation by 52% in LDLR mice, largely independent of effects on lipid profile and inflammation, but rather by decreasing macrophage apoptosis and increasing efferocytosis in the vessel wall. MAARS interacts with HuR/ELAVL1, an RNA-binding protein and important regulator of apoptosis. Overexpression and knockdown studies verified MAARS as a critical regulator of macrophage apoptosis and efferocytosis in vitro, in an HuR-dependent manner. Mechanistically, MAARS knockdown alters HuR cytosolic shuttling, regulating HuR targets such as p53, p27, Caspase-9, and BCL2. These findings establish a mechanism by which a macrophage-specific lncRNA interacting with HuR regulates apoptosis, with implications for a broad range of vascular disease states.
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http://dx.doi.org/10.1038/s41467-020-19664-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708640PMC
December 2020

Discovery of a Selective, Covalent IRAK1 Inhibitor with Antiproliferative Activity in MYD88 Mutated B-Cell Lymphoma.

ACS Med Chem Lett 2020 Nov 9;11(11):2238-2243. Epub 2020 Oct 9.

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

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas, including Waldenström's macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01 with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a highly selective probe of IRAK1 for further development.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667833PMC
November 2020

, a DNA damage response gene, is required for Notch-mediated induction of squamous cell differentiation.

Elife 2020 09 16;9. Epub 2020 Sep 16.

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

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including , which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. is epistatic to , a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation.
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http://dx.doi.org/10.7554/eLife.58081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529455PMC
September 2020

Discovery of Covalent MKK4/7 Dual Inhibitor.

Cell Chem Biol 2020 12 10;27(12):1553-1560.e8. Epub 2020 Sep 10.

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

MKK4/7 are kinases that phosphorylate JNKs and regulate the MAPK signaling pathway. Their overexpression has been associated with tumorigenesis and aggressiveness in cancers such as breast, prostate, non-small cell lung, and pediatric leukemia, making them a potential target for inhibitor development. Here, we report the discovery, development, and validation of a dual MKK4/7 inhibitor, BSJ-04-122, that covalently targets a conserved cysteine located before the DFG motif and displays excellent kinome selectivity. BSJ-04-122 exhibits potent cellular target engagement and induces robust target-specific downstream effects. The combination of the dual MKK4/7 inhibitor with a selective, covalent JNK inhibitor demonstrated an enhanced antiproliferative activity against triple-negative breast cancer cells. Taken together, the results show that BSJ-04-122 represents a pharmacological probe for MKK4/7 and credential covalent targeting as a way to explore the therapeutic potential of these kinases.
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http://dx.doi.org/10.1016/j.chembiol.2020.08.014DOI Listing
December 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

Rationally Designed Covalent BCL6 Inhibitor That Targets a Tyrosine Residue in the Homodimer Interface.

ACS Med Chem Lett 2020 Jun 3;11(6):1269-1273. Epub 2020 Apr 3.

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

B-cell lymphoma 6 (BCL6) is a transcriptional repressor frequently deregulated in lymphoid malignancies. BCL6 engages with number of corepressors, and these protein-protein interactions are being explored as a strategy for drug development. Here, we report the development of an irreversible BCL6 inhibitor that uses a sulfonyl fluoride to covalently react with the hydroxyl group of Tyrosine 58 located in the lateral groove. exhibits significantly improved inhibitory activity compared to that of its reversible parental compound and displays sustained target engagement and antiproliferative activity in cells. therefore represents an example of a tyrosine-directed covalent inhibitor of BCL6 which demonstrates advantages relative to reversible targeting.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294706PMC
June 2020

Discovery of MFH290: A Potent and Highly Selective Covalent Inhibitor for Cyclin-Dependent Kinase 12/13.

J Med Chem 2020 07 25;63(13):6708-6726. Epub 2020 Jun 25.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Genetic depletion of cyclin-dependent kinase 12 (CDK12) or selective inhibition of an analog-sensitive CDK12 reduces DNA damage repair gene expression, but selective inhibition of endogenous CDK12 is difficult. Here, we report the development of MFH290, a novel cysteine (Cys)-directed covalent inhibitor of CDK12/13. MFH290 forms a covalent bond with Cys-1039 of CDK12, exhibits excellent kinome selectivity, inhibits the phosphorylation of serine-2 in the C-terminal domain (CTD) of RNA-polymerase II (Pol II), and reduces the expression of key DNA damage repair genes. Importantly, these effects were demonstrated to be CDK12-dependent as mutation of Cys-1039 rendered the kinase refractory to MFH290 and restored Pol II CTD phosphorylation and DNA damage repair gene expression. Consistent with its effect on DNA damage repair gene expression, MFH290 augments the antiproliferative effect of the PARP inhibitor olaparib.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01929DOI Listing
July 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

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

Long noncoding RNA integrates a DNA-PK-mediated DNA damage response and vascular senescence.

Sci Transl Med 2020 02;12(531)

Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 () is highly expressed in the vascular endothelium and decreases during lesion progression. knockdown accelerated atherosclerotic lesion formation by 2.4-fold in mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by knockdown. expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.
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http://dx.doi.org/10.1126/scitranslmed.aaw1868DOI Listing
February 2020

Development of a covalent inhibitor of gut bacterial bile salt hydrolases.

Nat Chem Biol 2020 03 10;16(3):318-326. Epub 2020 Feb 10.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.
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http://dx.doi.org/10.1038/s41589-020-0467-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036035PMC
March 2020

Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway.

Cancer Cell 2020 01;37(1):104-122.e12

Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.
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http://dx.doi.org/10.1016/j.ccell.2019.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146079PMC
January 2020

Structure-Based Design of a Potent and Selective Covalent Inhibitor for SRC Kinase That Targets a P-Loop Cysteine.

J Med Chem 2020 02 30;63(4):1624-1641. Epub 2020 Jan 30.

Department of Biological Chemistry and Molecular Pharmacology , Harvard Medical School , Boston , Massachusetts 02115 , United States.

SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P-loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) as a starting point, we developed covalent inhibitor , which discriminates SRC from other covalent targets of including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound exhibited sustained inhibition of SRC signaling both and . Moreover, exhibited potent antiproliferative effects in nonsmall cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493195PMC
February 2020

TRIP13 regulates DNA repair pathway choice through REV7 conformational change.

Nat Cell Biol 2020 01 8;22(1):87-96. Epub 2020 Jan 8.

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

DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
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http://dx.doi.org/10.1038/s41556-019-0442-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336368PMC
January 2020

STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.

Elife 2020 Jan 8;9. Epub 2020 Jan 8.

Broad Institute of Harvard and MIT, Cambridge, United States.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.
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http://dx.doi.org/10.7554/eLife.53003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6984821PMC
January 2020

Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling.

Sci Signal 2019 11 5;12(606). Epub 2019 Nov 5.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.
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http://dx.doi.org/10.1126/scisignal.aay2369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212804PMC
November 2019

Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.

Nature 2019 11 3;575(7783):545-550. Epub 2019 Oct 3.

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

RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
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http://dx.doi.org/10.1038/s41586-019-1660-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014971PMC
November 2019
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