Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.

Discovery of Potent and Selective WDR5 Proteolysis Targeting Chimeras as Potential Therapeutics for Pancreatic Cancer.

As a core chromatin-regulatory scaffolding protein, WDR5 mediates numerous protein-protein interactions (PPIs) with other partner oncoproteins. However, small-molecule inhibitors that block these PPIs exert limited cell-killing effects. Here, we report structure-activity relationship studies in pancreatic ductal adenocarcinoma (PDAC) cells that led to the discovery of several WDR5 proteolysis-targeting chimer (PROTAC) degraders, including 11 (MS132), a highly potent and selective von Hippel-Lindau (VHL)-recruiting WDR5 degrader, which displayed positive binding cooperativity between WDR5 and VHL, effectively inhibited proliferation in PDAC cells, and was bioavailable in mice and 25, a cereblon (CRBN)-recruiting WDR5 degrader, which selectively degraded WDR5 over the CRBN neo-substrate IKZF1. Furthermore, by conducting site-directed mutagenesis studies, we determined that WDR5 K296, but not K32, was involved in the PROTAC-induced WDR5 degradation. Collectively, these studies resulted in a highly effective WDR5 degrader, which could be a potential therapeutic for pancreatic cancer and several potentially useful tool compounds.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

Structures of SARS-CoV-2 N7-methyltransferase with DOT1L and PRMT7 inhibitors provide a platform for new antivirals.

The RNA N7-methyltransferase (MTase) activity of SARS-CoV-2's nsp14 protein is essential for viral replication and is a target for the development of new antivirals. Nsp14 uses S-adenosyl methionine (SAM) as the methyl donor to cap the 5' end of the SARS-CoV-2 mRNA and generates S-adenosyl homocysteine (SAH) as the reaction byproduct. Due to the central role of histone MTases in cancer, many SAM/SAH analogs with properties of cell permeability have recently been developed for the inhibition of these MTases. We have succeeded in identifying two such compounds (SGC0946 and SGC8158) that display significant antiviral activity and bind to the SARS-CoV-2 nsp14 N7-MTase core. Unexpectedly, crystal structures of SGC0946 and SGC8158 with the SARS-CoV-2 nsp14 N7-MTase core identify them as bi-substrate inhibitors of the viral MTase, co-occupying both the SAM and RNA binding sites; positing novel features that can be derivatized for increased potency and selectivity for SARS-CoV-2 nsp14. Taken together, the high-resolution structures and the accompanying biophysical and viral replication data provide a new avenue for developing analogs of SGC0946 and SGC8158 as antivirals.

Signal-induced enhancer activation requires Ku70 to read topoisomerase1-DNA covalent complexes.

Enhancer activation serves as the main mechanism regulating signal-dependent transcriptional programs, ensuring cellular plasticity, yet central questions persist regarding their mechanism of activation. Here, by successfully mapping topoisomerase I-DNA covalent complexes genome-wide, we find that most, if not all, acutely activated enhancers, including those induced by 17ß-estradiol, dihydrotestosterone, tumor necrosis factor alpha and neuronal depolarization, are hotspots for topoisomerase I-DNA covalent complexes, functioning as epigenomic signatures read by the classic DNA damage sensor protein, Ku70. Ku70 in turn nucleates a heterochromatin protein 1 gamma (HP1γ)-mediator subunit Med26 complex to facilitate acute, but not chronic, transcriptional activation programs. Together, our data uncover a broad, unappreciated transcriptional code, required for most, if not all, acute signal-dependent enhancer activation events in both mitotic and postmitotic cells.

High-resolution structures of the SARS-CoV-2 N7-methyltransferase inform therapeutic development.

Emergence of SARS-CoV-2 coronavirus has led to millions of deaths globally. We present three high-resolution crystal structures of the SARS-CoV-2 nsp14 N7-methyltransferase core bound to S-adenosylmethionine (1.62 Å), S-adenosylhomocysteine (1.55 Å) and sinefungin (1.41 Å). We identify features of the methyltransferase core that are crucial for the development of antivirals and show SAH as the best scaffold for the design of antivirals against SARS-CoV-2 and other pathogenic coronaviruses.

Discovery of a dual WDR5 and Ikaros PROTAC degrader as an anti-cancer therapeutic.

WD repeat domain 5 (WDR5), an integral component of the MLL/KMT2A lysine methyltransferase complex, is critically involved in oncogenesis and represents an attractive onco-target. Inhibitors targeting protein-protein interactions (PPIs) between WDR5 and its binding partners, however, do not inhibit all of WDR5-mediated oncogenic functions and exert rather limited antitumor effects. Here, we report a cereblon (CRBN)-recruiting proteolysis targeting chimera (PROTAC) of WDR5, MS40, which selectively degrades WDR5 and the well-established neo-substrates of immunomodulatory drugs (IMiDs):CRBN, the Ikaros zinc finger (IKZF) transcription factors IKZF1 and IKZF3. MS40-induced WDR5 degradation caused disassociation of the MLL/KMT2A complex off chromatin, resulting in decreased H3K4me2. Transcriptomic profiling revealed that targets of both WDR5 and IMiDs:CRBN were significantly repressed by treatment of MS40. In MLL-rearranged leukemias, which exhibit IKZF1 high expression and dependency, co-suppression of WDR5 and Ikaros by MS40 is superior in suppressing oncogenesis to the WDR5 PPI inhibitor, to MS40's non-PROTAC analog controls (MS40N1 and MS40N2, which do not bind CRBN and WDR5, respectively), and to a matched VHL-based WDR5 PROTAC (MS169, which degrades WDR5 but not Ikaros). MS40 suppressed the growth of primary leukemia patient cells in vitro and patient-derived xenografts in vivo. Thus, dual degradation of WDR5 and Ikaros is a promising anti-cancer strategy.

A selective WDR5 degrader inhibits acute myeloid leukemia in patient-derived mouse models.

Interactions between WD40 repeat domain protein 5 (WDR5) and its various partners such as mixed lineage leukemia (MLL) and c-MYC are essential for sustaining oncogenesis in human cancers. However, inhibitors that block protein-protein interactions (PPIs) between WDR5 and its binding partners exhibit modest cancer cell killing effects and lack in vivo efficacy. Here, we present pharmacological degradation of WDR5 as a promising therapeutic strategy for treating WDR5-dependent tumors and report two high-resolution crystal structures of WDR5-degrader-E3 ligase ternary complexes. We identified an effective WDR5 degrader via structure-based design and demonstrated its in vitro and in vivo antitumor activities. On the basis of the crystal structure of an initial WDR5 degrader in complex with WDR5 and the E3 ligase von Hippel­Lindau (VHL), we designed a WDR5 degrader, MS67, and demonstrated the high cooperativity of MS67 binding to WDR5 and VHL by another ternary complex structure and biophysical characterization. MS67 potently and selectively depleted WDR5 and was more effective than WDR5 PPI inhibitors in suppressing transcription of WDR5-regulated genes, decreasing the chromatin-bound fraction of MLL complex components and c-MYC, and inhibiting the proliferation of cancer cells. In addition, MS67 suppressed malignant growth of MLL-rearranged acute myeloid leukemia patient cells in vitro and in vivo and was well tolerated in vivo. Collectively, our results demonstrate that structure-based design can be an effective strategy to identify highly active degraders and suggest that pharmacological degradation of WDR5 might be a promising treatment for WDR5-dependent cancers.

Simultaneous CK2/TNIK/DYRK1 inhibition by 108600 suppresses triple negative breast cancer stem cells and chemotherapy-resistant disease.

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Shape of promoter antisense RNAs regulates ligand-induced transcription activation.

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17ß-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.

SOX11 Inhibitors Are Cytotoxic in Mantle Cell Lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) is a fatal subtype of non-Hodgkin lymphoma. SOX11 transcription factor is overexpressed in the majority of nodal MCL. We have previously reported that B cell-specific overexpression of SOX11 promotes MCL pathogenesis via critically increasing BCR signaling in vivo. SOX11 is an attractive target for MCL therapy; however, no small-molecule inhibitor of SOX11 has been identified to date. Although transcription factors are generally considered undruggable, the ability of SOX11 to bind to the minor groove of DNA led us to hypothesize that there may exist cavities at the protein-DNA interface that are amenable to targeting by small molecules. EXPERIMENTAL DESIGN: Using a combination of in silico predictions and experimental validations, we report here the discovery of three structurally related compounds (SOX11i) that bind SOX11, perturb its interaction with DNA, and effect SOX11-specific anti-MCL cytotoxicity. RESULTS: We find mechanistic validation of on-target activity of these SOX11i in the inhibition of BCR signaling and the transcriptional modulation of SOX11 target genes, specifically, in SOX11-expressing MCL cells. One of the three SOX11i exhibits relatively superior in vitro activity and displays cytotoxic synergy with ibrutinib in SOX11-expressing MCL cells. Importantly, this SOX11i induces cytotoxicity specifically in SOX11-positive ibrutinib-resistant MCL patient samples and inhibits Bruton tyrosine kinase phosphorylation in a xenograft mouse model derived from one of these subjects. CONCLUSIONS: Taken together, our results provide a foundation for therapeutically targeting SOX11 in MCL by a novel class of small molecules.

Structure and mechanism of B-family DNA polymerase ζ specialized for translesion DNA synthesis.

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31' Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein-protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ's role in TLS and provide a framework for new cancer therapeutics.

A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent.

Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical-grade rigosertib, which is free of this impurity, does not exhibit tubulin-binding activity. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.

Structural insights into mutagenicity of anticancer nucleoside analog cytarabine during replication by DNA polymerase η.

Cytarabine (AraC) is the mainstay chemotherapy for acute myeloid leukemia (AML). Whereas initial treatment with AraC is usually successful, most AML patients tend to relapse, and AraC treatment-induced mutagenesis may contribute to the development of chemo-resistant leukemic clones. We show here that whereas the high-fidelity replicative polymerase Polδ is blocked in the replication of AraC, the lower-fidelity translesion DNA synthesis (TLS) polymerase Polη is proficient, inserting both correct and incorrect nucleotides opposite a template AraC base. Furthermore, we present high-resolution crystal structures of human Polη with a template AraC residue positioned opposite correct (G) and incorrect (A) incoming deoxynucleotides. We show that Polη can accommodate local perturbation caused by the AraC via specific hydrogen bonding and maintain a reaction-ready active site alignment for insertion of both correct and incorrect incoming nucleotides. Taken together, the structures provide a novel basis for the ability of Polη to promote AraC induced mutagenesis in relapsed AML patients.

Cryo-EM structure and dynamics of eukaryotic DNA polymerase δ holoenzyme.

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe-4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.

Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17ß-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

Dismissal of RNA Polymerase II Underlies a Large Ligand-Induced Enhancer Decommissioning Program.

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.

Structural basis for polymerase η-promoted resistance to the anticancer nucleoside analog cytarabine.

Cytarabine (AraC) is an essential chemotherapeutic for acute myeloid leukemia (AML) and resistance to this drug is a major cause of treatment failure. AraC is a nucleoside analog that differs from 2'-deoxycytidine only by the presence of an additional hydroxyl group at the C2' position of the 2'-deoxyribose. The active form of the drug AraC 5'-triphosphate (AraCTP) is utilized by human replicative DNA polymerases to insert AraC at the 3' terminus of a growing DNA chain. This impedes further primer extension and is a primary basis for the drug action. The Y-family translesion synthesis (TLS) DNA polymerase η (Polη) counteracts this barrier to DNA replication by efficient extension from AraC-terminated primers. Here, we provide high-resolution structures of human Polη with AraC incorporated at the 3'-primer terminus. We show that Polη can accommodate AraC at different stages of the catalytic cycle, and that it can manipulate the conformation of the AraC sugar via specific hydrogen bonding and stacking interactions. Taken together, the structures provide a basis for the ability of Polη to extend DNA synthesis from AraC terminated primers.

Eukaryotic DNA polymerases.

The eukaryotic DNA replication machinery is conserved from yeast to humans and requires the actions of multiple DNA polymerases. In addition to replicative DNA polymerases for duplication of the leading and lagging DNA strands, another group of specialized polymerases is required for DNA repair and/or translesion DNA synthesis (TLS). We emphasize here recent findings that accelerate our understanding of the structure and mechanisms of these remarkable enzymes. We also highlight growing evidence on the role of DNA polymerases in the origin of certain cancers, and paradoxically as emerging targets for cancer therapy.