Robust Repression Research Articles

ORF48 is required for optimal lytic replication of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes persistent infection in the host by encoding a vast network of proteins that aid immune evasion. One of these targeted innate immunity pathways is the cGAS-STING pathway, which inhibits the reactivation of KSHV from latency. Previously, we identified multiple cGAS/STING inhibitors encoded by KSHV, suggesting that the counteractions of this pathway by viral proteins are critical for maintaining a successful KSHV life cycle. However, the detailed mechanisms of how these viral proteins block innate immunity and facilitate KSHV lytic replication remain largely unknown. In this study, we report that ORF48, a previously identified negative regulator of the cGAS/STING pathway, is required for optimal KSHV lytic replication. We used both siRNA and deletion-based systems to evaluate the importance of intact ORF48 in the KSHV lytic cycle. In both systems, loss of ORF48 resulted in defects in lytic gene transcription, lytic protein expression, viral genome replication and infectious virion production. ORF48 genome deletion caused more robust and global repression of the KSHV transcriptome, possibly due to the disruption of RTA promoter activity. Mechanistically, overexpressed ORF48 was found to colocalize and interact with endogenous STING in HEK293 cells. Endogenous ORF48 and STING interactions were also detected in reactivated iSLK.219 cells. Compared with the control cell line, HUVEC cells stably expressing ORF48 exhibited repressed STING-dependent innate immune signaling upon ISD or diABZI treatment. However, the loss of ORF48 in our iSLK-based lytic system failed to induce IFNβ production, suggesting a redundant role of ORF48 on STING signaling during the KSHV lytic phase. Thus, ORF48 is required for optimal KSHV lytic replication through additional mechanisms that need to be further explored.

Engineered transcription activator-like effector dimer proteins confer DNA loop-dependent gene repression comparable to Lac repressor.

Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes.

Small-RNA-guided histone modifications and somatic genome elimination in ciliates.

Transposable elements and other repeats are repressed by small-RNA-guided histone modifications in fungi, plants and animals. The specificity of silencing is achieved through base-pairing of small RNAs corresponding to the these genomic loci to nascent noncoding RNAs, which allows the recruitment of histone methyltransferases that methylate histone H3 on lysine 9. Self-reinforcing feedback loops enhance small RNA production and ensure robust and heritable repression. In the unicellular ciliate Paramecium tetraurelia, small-RNA-guided histone modifications lead to the elimination of transposable elements and their remnants, a definitive form of repression. In this organism, germline and somatic functions are separated within two types of nuclei with different genomes. At each sexual cycle, development of the somatic genome is accompanied by the reproducible removal of approximately a third of the germline genome. Instead of recruiting a H3K9 methyltransferase, small RNAs corresponding to eliminated sequences tether Polycomb Repressive Complex 2, which in ciliates has the unique property of catalyzing both lysine 9 and lysine 27 trimethylation of histone H3. These histone modifications that are crucial for the elimination of transposable elements are thought to guide the endonuclease complex, which triggers double-strand breaks at these specific genomic loci. The comparison between ciliates and other eukaryotes underscores the importance of investigating small-RNAs-directed chromatin silencing in a diverse range of organisms. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.

Targeting ADSS2 Enhances BH3 Mimetics Treatment Induced Mitochondrial Apoptosis in AML Cells

Recently, the BH3 mimetic-Bcl2 inhibitor venetoclax (VEN), has been approved by FDA in 2018 for the treatment of patients with AML in combination with a hypomethylating agent (HMA) such as azacytidine (AZA). This synergy is likely due to HMA-induced mitochondrial vulnerability associated with cytoplasmic RNA metabolism. Nonetheless, most AML patients treated with the combination eventually relapse, possibly due to persistence of tumor cells not dependent on antiapoptotic proteins (Bcl2, Mcl1). There is a pressing need to develop targeted therapeutics sensitizing AML cells to mitochondrial apoptosis, particularly for those patients failed from VEN associated treatment. To achieve the goal, we performed a CRISPR/Cas9 Knockout (KO) screen (using Bim peptide-based dynamic ΔBH3 profiling as read-out) targeting genes (n=534) involved in cytosolic nucleotides metabolism pathways to increase mitochondrial vulnerability in a BH3 mimetic-refractory AML cell line (HEL). That search identified ADSS2, which catalyzes the first committed/rate-limiting step of AMP biosynthesis from IMP, emerged as the top hit, exhibiting highest ΔBH3 priming. Subsequent BH3 priming assays validated that ADSS2 KO significantly enhanced sensitivity to BH3 mimetics across various AML cell lines, including MOLM13, SKM1, OCI-AML3, and HEL cells. ADSS2 KO also led to a noteworthy reduction in the IC50 values of both VEN and the Mcl1 inhibitor S63845 in these cell lines. Additionally, the synergistic effect between sgRNA targeting ADSS2 and VEN or S63845 was also confirmed in Cas9 expressing cells from two AML patient-derived-xenograft (PDX) samples. We next transplanted AML cells (form one PDX samples) with or without ADSS2-KO into NGS mice, then treated mice with VEN. Notably, VEN treatment (100 mg/kg, QDx14, PO) significantly reduced tumor burden in ADSS2-KO AML mice compared to WT AML mice. Additionally, ADSS2 KO combined with VEN treatment group displayed a survival advantage compared to single VEN treatment group. Further mechanistic investigations revealed that ADSS2 KO led to a remarkable depletion of AMP rather than ATP, and a robust repression of AMPK activity in HEL cells. To assess whether AMPK inhibition mimics the effect of ADSS2 KO, AMPKα-deficient (Mx1-Cre/Prkaa1 fl/fl/ Prkaa2 fl/fl) MLL-AF9 (MA9) transformed murine AML cells were assessed. Indeed, AMPKα depletion substantially enhanced VEN treatment efficiency ex-vivo. Notably, enforced overexpression of a constitutively active AMPKa2 (T172D) construct in a VEN sensitive cell line SKM1, promoted cell resistance to mitochondrial apoptosis by VEN or S63845. Moreover, T172D expression can also abolish ADSS2-KO mediated mitochondrial vulnerability to VEN or S63845 treatment, indicating the pivotal role of AMPK signaling in mediating ADSS2 function. There are no reported ADSS2 inhibitors. To explore ADSS inhibitor, we conducted a fragment-based virtual screen of 8,000,000 compounds (MolPort) library and requested the top-ranking hits for ADSS2 catalysis evaluation by ELISA. Notably, the top two hits AS104 and AS71 exhibited the most potent inhibitory effects (AS104 IC50=2.43µM, AS71 IC50=3.85µM). NMR-STD analysis was conducted in a cell-free system, which unequivocally confirmed the physical binding of inhibitors and the ADSS2 protein. To further investigate the interactions between AS71 and AS104 with ADSS2 in a cellular context, both compounds were subjected to a cellular thermal shift assay (CETSA). Notably, treatment with AS71 and AS104 led to substantial shifts in ADSS2 protein thermal stability, providing evidence of their interactions with ADSS2 within the cellular environment. In both HEL cells and a primary VEN-resistant AML samples, treatment with AS71 and AS104 significantly increased ΔBH3 priming. Collectively, our observation revealed that targeting ADSS2 is critical for sensitizing AML cells to BH3 mimetics. We are now conducting preclinical assessments of ADSS2 inhibitors.

Identification of the primary ciliary proteins IFT38 and IFT144 to enhance serum-mediated YAP activation and cell proliferation

Primary cilia are essential cellular antennae that transmit external signals into intracellular responses. These sensory organelles perform crucial tasks in triggering intracellular signaling pathways, including those initiated by G protein-coupled receptors (GPCRs). Given the involvement of GPCRs in serum-induced signaling, we investigated the contribution of ciliary proteins in mitogen perception and cell proliferation. We found that depletion of cilia via IFT88 silencing impaired cell growth and repressed YAP activation against serum and its mitogenic constituents, namely lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). To identify the key player of serum mitogen signaling, a mutant cell line library with 30 ablated individual ciliary proteins was established and screened based on YAP dephosphorylation and target gene induction. While 9 of them had altered signaling, ablation of IFT38 or IFT144 led to a particularly robust repression of YAP activation upon LPA and S1P. The deficiency of IFT38 and IFT144 attenuated cell proliferation, as corroborated in either 2-dimensional cultures or tumor spheroids. In subcutaneous skin melanoma patients, expression of IFT38 and IFT144 was associated with unfavorable outcomes in overall survival. In conclusion, our study demonstrates the involvement of ciliary proteins in mitogen signaling and identifies the regulatory roles of IFT38 and IFT144 in serum-mediated Hippo pathway signaling and cellular growth.

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria.

The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.

Cumulative effects of weakly repressive regulatory regions in the 3’ UTR maintain PD-1 expression homeostasis in mammals

PD-1 has become a common target for cancer treatment. However, the molecular regulation of PD-1 expression homeostasis remains unclear. Here we report the PD-1 3’ UTR can dramatically repress gene expression via promoting mRNA decay. Deletion of the PD-1 3’ UTR inhibits T cell activity and promotes T-ALL cell proliferation. Interestingly, the robust repression is attributable to cumulative effects of many weak regulatory regions, which we show together are better able to maintain PD-1 expression homeostasis. We further identify several RNA binding proteins (RBPs) that modulate PD-1 expression via the 3’ UTR, including IGF2BP2, RBM38, SRSF7, and SRSF4. Moreover, despite rapid evolution, PD-1 3’ UTRs are functionally conserved and strongly repress gene expression through many common RBP binding sites. These findings reveal a previously unrecognized mechanism of maintaining PD-1 expression homeostasis and might represent a general model for how small regulatory effects play big roles in regulation of gene expression and biology.

Pituitary Stem Cell Regulation by Zeb2 and BMP Signaling.

Epithelial to mesenchymal transition (EMT) is important for many developing organs, and for wound healing, fibrosis, and cancer. Pituitary stem cells undergo an EMT-like process as they migrate and initiate differentiation, but little is known about the input of signaling pathways or the genetic hierarchy of the transcriptional cascade. Prop1 mutant stem cells fail to undergo changes in cellular morphology, migration, and transition to the Pou1f1 lineage. We used Prop1 mutant mice to identify the changes in gene expression that are affiliated with EMT-like processes. BMP and TGF-β family gene expression was reduced in Prop1 mutants and Elf5, a transcription factor that characteristically suppresses EMT, had elevated expression. Genes involved in cell-cell contact such as cadherins and claudins were elevated in Prop1 mutants. To establish the genetic hierarchy of control, we manipulated gene expression in pituitary stem cell colonies. We determined that the EMT inducer, Zeb2, is necessary for robust BMP signaling and repression of Elf5. We demonstrated that inhibition of BMP signaling affects expression of target genes in the Id family, but it does not affect expression of other EMT genes. Zeb2 is necessary for expression of the SHH effector gene Gli2. However, knock down of Gli2 has little effect on the EMT-related genes, suggesting that it acts through a separate pathway. Thus, we have established the genetic hierarchy involved in the transition of pituitary stem cells to differentiation.

Robustness of DNA looping across multiple cell divisions in individual bacteria

DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.

Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models

BackgroundDespite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. Consequently, reactivation of the virus from latently infected cell reservoirs is a major challenge toward cure efforts. Two strategies targeting viral latency are currently under investigation: the “shock and kill” and the “block and lock.” The “Block and Lock” methodology aims to control HIV-1 latency reactivation, promoting a functional cure. We utilized the CRISPR/dCas9-KRAB platform, which was initially developed to suppress cellular genes transcription, to block drug-induced HIV-1 reactivation in latently infected T cells and myeloid cells.ResultsWe identified a set of five sgRNAs targeting the HIV-1 proviral genome (LTR1-LTR5), having the lowest nominated off-target activity, and transduced them into the latently infected lymphoid (J-Lat 10.6) and myeloid (U1) cell lines. One of the sgRNAs (LTR5), which binds specifically in the HIV-1 LTR NFκB binding site, was able to promote robust repression of HIV-1 reactivation in latently infected T cells stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a hundred times reduction of HIV-1 RNA expression levels in the latently infected myeloid cell line, U1 induced with IngB.ConclusionTaken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latently infected cells. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we have identified some useful hotspots sequences in HIV-1 LTR for the design sgRNAs.

Regulation of spatially restricted gene expression: linking RNA localization and phase separation.

Subcellular restriction of gene expression is crucial to the functioning of a wide variety of cell types. The cellular machinery driving spatially restricted gene expression has been studied for many years, but recent advances have highlighted novel mechanisms by which cells can generate subcellular microenvironments with specialized gene expression profiles. Particularly intriguing are recent findings that phase separation plays a role in certain RNA localization pathways. The burgeoning field of phase separation has revolutionized how we view cellular compartmentalization, revealing that, in addition to membrane-bound organelles, phase-separated cytoplasmic microenvironments - termed biomolecular condensates - are compositionally and functionally distinct from the surrounding cytoplasm, without the need for a lipid membrane. The coupling of phase separation and RNA localization allows for precise subcellular targeting, robust translational repression and dynamic recruitment of accessory proteins. Despite the growing interest in the intersection between RNA localization and phase separation, it remains to be seen how exactly components of the localization machinery, particularly motor proteins, are able to associate with these biomolecular condensates. Further studies of the formation, function, and transport of biomolecular condensates promise to provide a new mechanistic understanding of how cells restrict gene expression at a subcellular level.

Reawakening of Human Fetal Hemoglobin and an Epigenetic Path to the Clinic for Sickle Cell Disease and Beta-Thalassemia: Identification of an Orally-Available, Potent, and Selective Euchromatic Histone Lysine Methyltransferase 1 and 2 (EHMT1/2) Inhibitor

Expression of genes in the β-globin locus switch via epigenetic control at key stages of early gestational development from an embryonic (HbE, α2ε2) to fetal form (HbF, α2γ2) and again around birth to the adult form (HbA, α2β2). Sickle cell disease (SCD) is a multi-organ genetic disorder caused by a point mutation in the adult β-globin gene producing sickled hemoglobin (HbS, α2βs2). It is well-established that SCD patients carrying genetic variations in HbF-regulatory loci have a concomitant increase in blood levels of HbF (ranging from 5-25% of total Hb). These individuals exhibit fewer major crises or no clinical symptoms, suggesting that re-expression of the γ-gene and increasing HbF is a potential disease modifying treatment approach for SCD patients. Hydroxyurea (HU) and L-glutamine are currently the only FDA-approved drugs for the treatment of SCD, with HU working in-part, through the re-induction of HbF. Safety and efficacy responses to HU vary widely, and there remains a strong need for more robust HbF induction strategies with improved safety profiles.Previous groups have shown that inhibitors to various epigenetic modifiers are able to overcome γ-gene silencing and induce HbF; however, associated defects in hematopoiesis have been reported. Recently, histone methytransferase enzymes EHMT1 and EHMT2, responsible for the repressive chromatin mark dimethyl-lysine 9 on histone H3 (H3K9me2), have been shown to be essential for robust repression of γ-globin expression. To-date, an in vitro tool compound UNC0638 has demonstrated activation of γ-globin expression in human adult erythroid progenitor cells but no evaluation of in vivo efficacy of EHMT1/2 inhibition on HbF induction has been possible due to a lack of drug-like properties.Here we describe the generation and characterization of a potent, selective, and orally available small molecule inhibitor of EHMT1/2, EPZ035544. EHMT2 enzymatic activity in a biochemical assay, as measured by the half-maximal inhibitory concentration (IC50) is 10nM and cellular inhibition of H3K9me2 correlated well with an IC50 of 55nM. Treatment of EPZ035544 in primary human CD34+ progenitor cells led to dose-dependent decreases in H3K9me2 with concomitant induction of γ-globin relative to β-globin expression of 10-30% as measured by mass spectrometry and qPCR leading to 100% of F-positive cells. Chromatin immunoprecipitation and sequencing studies performed on EPZ035544-treated CD34+ cells demonstrated that H3K9me2 was reduced throughout the genome with an associated increase in H3K9Ac. The γ-globin gene locus displayed a reduction of H3K9me2 and an increase of H3K9Ac and RNA Polymerase II occupancy leading to significantly increased γ-globin gene expression upon EPZ035544 treatment.The drug-like properties of EPZ035544 enabled the first prolonged in vivo investigation of an EHMT1/2 inhibitor by continuous oral dosing over 90 days in C57BL/6 mice. As mice do not have HbF, we measured the levels of embryonic hemoglobin (Hbb-εy) which is normally repressed after birth. We performed twice-daily (BID) oral dosing in two dose groups: 25 and 50 mg per kilogram of body weight (mg/kg) and once-daily (QD) oral dosing in a single dose group of 75 mg/kg. Dose-dependent increases in the absolute levels of Hbb-εy mRNA in peripheral blood mononuclear cells (PBMCs) were observed with maximal induction of ~ 100 fold achieved at the 50 mg/kg BID and 75 mg/kg QD doses. Using quantitative mass spectrometry, we also observed drug-induced increases in Hbb-εy at the protein level. A corresponding reduction in H3K9me2 levels in PBMCs and bone marrow, as measured by flow cytometry and ELISA, strongly suggests that these effects were a direct consequence of EHMT1/2 inhibition. EPZ035544 was well tolerated throughout the study with no significant changes observed in body weights, spleen weights, or complete blood cell counts. In summary, we have developed an orally bioavailable EHMT1/2 inhibitor with demonstrated on-target in vivo induction of mouse Hbb-εy expression with up to 90 days of continuous dosing. To our knowledge this is the first study to test the in vivo effects of an EHMT1/2 inhibitor on hemoglobin induction. We believe this approach holds promise as a novel treatment option for β-globinopathies, and we continue to advance our program towards the clinic. DisclosuresChan-Penebre:Epizyme Inc.: Employment, Equity Ownership. Gibaja:Epizyme Inc.: Employment, Equity Ownership. Campbell:Epizyme Inc.: Employment, Equity Ownership. Olu:Epizyme Inc.: Employment, Equity Ownership. Admirand:Epizyme Inc.: Employment, Equity Ownership. Brach:Epizyme Inc.: Employment, Equity Ownership. Tang:Epizyme Inc.: Employment, Equity Ownership. Raimondi:Epizyme Inc.: Employment, Equity Ownership. Hood:Epizyme Inc.: Employment, Equity Ownership. Cocozaki:Epizyme Inc.: Employment, Equity Ownership. Karaman:Epizyme Inc.: Employment, Equity Ownership. Stickland:Epizyme Inc.: Employment, Equity Ownership. Munchhof:Michael J. Munchhof LLC: Consultancy. Armstrong:Epizyme Inc.: Employment, Equity Ownership. Riera:Epizyme Inc.: Employment, Equity Ownership. Orkin:Bioverativ: Consultancy; Epizyme Inc.: Consultancy. Boriack-Sjodin:Epizyme Inc.: Employment, Equity Ownership. Janzen:Epizyme Inc.: Employment, Equity Ownership. Blakemore:Epizyme Inc.: Employment, Equity Ownership. Ribich:Epizyme Inc.: Employment, Equity Ownership. Chesworth:Epizyme Inc.: Employment, Equity Ownership. Smith:Epizyme Inc.: Employment, Equity Ownership. Duncan:Epizyme Inc.: Employment, Equity Ownership.

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation.

The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.

A plasmid toolset for CRISPR-mediated genome editing and CRISPRi gene regulation in Escherichia coli.

SummaryCRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid‐based systems for genome editing (gene knockouts/knock‐ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR‐Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR‐Cas12 genome editing, genome integration and CRISPRi implementation. These simple‐to‐use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory.

Evaluating the therapeutic potential of ADAR1 inhibition for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of endogenous double-stranded RNAs. ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that expression of ADAR1, specifically its p150 isoform, is required for the survival of TNBC cell lines. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. Moreover, ADAR1-knockdown leads to robust translational repression. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescued the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.

MicroRNA exporter HuR clears the internalized pathogens by promoting pro‐inflammatory response in infected macrophages

HuR is a miRNA derepressor protein that can act as miRNA sponge for specific miRNAs to negate their action on target mRNAs. Here we have identified how HuR, by inducing extracellular vesicles‐mediated export of miRNAs, ensures robust derepression of miRNA‐repressed cytokines essential for strong pro‐inflammatory response in activated mammalian macrophages. Leishmania donovani, the causative agent of visceral leishmaniasis, on the contrary alters immune response of the host macrophage by a variety of complex mechanisms to promote anti‐inflammatory response essential for the survival of the parasite. We have found that during Leishmania infection, the pathogen targets HuR to promote onset of anti‐inflammatory response in mammalian macrophages. In infected macrophages, Leishmania also upregulate protein phosphatase 2A that acts on Ago2 protein to keep it in dephosphorylated and miRNA‐associated form. This causes robust repression of the miRNA‐targeted pro‐inflammatory cytokines to establish an anti‐inflammatory response in infected macrophages. HuR has an inhibitory effect on protein phosphatase 2A expression, and mathematical modelling of macrophage activation process supports antagonistic miRNA‐modulatory roles of HuR and protein phosphatase 2A which mutually balances immune response in macrophage by targeting miRNA function. Supporting this model, ectopic expression of the protein HuR and simultaneous inhibition of protein phosphatase 2A induce strong pro‐inflammatory response in the host macrophage to prevent the virulent antimonial drug‐sensitive or drug‐resistant form of L. donovani infection. Thus, HuR can act as a balancing factor of immune responses to curtail the macrophage infection process by the protozoan parasite.

Temporal Control of the TGF-β Signaling Network by Mouse ESC MicroRNA Targets of Different Affinities

SUMMARYAlthough microRNAs (miRNAs) function in the control of embryonic stem cell (ESC) pluripotency, a systems-level understanding is still being developed. Through the analysis of progressive Argonaute (Ago)-miRNA depletion and rescue, including stable Ago knockout mouse ESCs, we uncover transforming growth factor beta (TGF-β) pathway activation as a direct and early response to ESC miRNA reduction. Mechanistically, we link the derepression of weaker miRNA targets, including TGF-β receptor 1 (Tgfbr1), to the sensitive TGF-β pathway activation. In contrast, stronger miRNA targets impart a more robust repression, which dampens concurrent transcriptional activation. We verify such dampened induction for TGF-β antagonist Lefty. We find that TGF-β pathway activation contributes to the G1 cell-cycle accumulation of miRNA-deficient ESCs. We propose that miRNA target affinity is a determinant of the temporal response to miRNA changes, which enables the coordination of gene network responses.

Abstract LB-277: Characterization of the anti-cancer and immunologic activity of RGX-019, a novel pre-clinical stage humanized monoclonal antibody targeting the MERTK receptor

Abstract MERTK, a receptor tyrosine kinase of the TYRO3/AXL/MERTK (TAM) family, is expressed in innate immune cells including macrophages, dendritic cells and NK cells and is overexpressed in a wide variety of cancers, including leukemia and many solid cancers. Activation of MERTK on cancer cells via ligand binding results in activation of several tumor-promoting signaling pathways, which stimulate proliferation, migration and angiogenesis, and decrease apoptosis and chemosensitivity. Furthermore, activation of MERTK on macrophages drives immune evasion through the promotion of an immune-suppressive M2 phenotype. Pre-clinical and clinical studies have shown promising emerging evidence of anti-tumor efficacy upon modulation of TAM receptor signaling. Herein, we report the pre-clinical characterization of RGX-019, a humanized IgG1 antibody with high affinity and specificity for human MERTK. Surface Plasmon Resonance, cell-based binding assays and competition ELISA demonstrate high affinity/avidity binding for RGX-019 to human MERTK. In contrast, binding to human AXL, human TYRO3, or murine MERTK was not detected. Binding of RGX-019 to the MERTK receptor triggered its rapid internalization and degradation from the surface of human cancer cells within 4 hours and prevented Gas6 induced phosphorylation of AKT, a downstream signal transduction pathway that promotes cell growth and survival. RGX-019 treatment of SKMel5 melanoma cells effectively inhibited colony formation. RGX-019-induced degradation of MERTK in in vitro differentiated human M2 macrophages reduced AKT activation and promoted a pro-inflammatory cytokine signature. Furthermore, a related surrogate murine MERTK antibody significantly inhibited growth of MDA-MB-231 breast cancer tumors, demonstrating single agent anti-tumor efficacy in vivo. These findings demonstrate that RGX-019 is a potent and selective inhibitor of MERTK signaling with a unique mechanism-of-action that potently disrupts MERTK signaling in both cancer cells and immune-suppressive macrophages via MERTK receptor degradation. This activity results in robust repression of cancer cell growth both in vitro and in vivo. Overall, these data support further development of RGX-019 as a cancer therapeutic. Citation Format: Shugaku Takeda, Celia Andreu-Agullo, Subhasree Sridhar, Nils Halberg, Ivo C. Lorenz, Sohail Tavazoie, Isabel Kurth, Masoud Tavazoie. Characterization of the anti-cancer and immunologic activity of RGX-019, a novel pre-clinical stage humanized monoclonal antibody targeting the MERTK receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-277.

Robust repression of tRNA gene transcription during stress requires protein arginine methylation.

Protein arginine methylation is an important means by which protein function can be regulated. In the budding yeast, this modification is catalyzed by the major protein arginine methyltransferase Hmt1. Here, we provide evidence that the Hmt1-mediated methylation of Rpc31, a subunit of RNA polymerase III, plays context-dependent roles in tRNA gene transcription: under conditions optimal for growth, it positively regulates tRNA gene transcription, and in the setting of stress, it promotes robust transcriptional repression. In the context of stress, methylation of Rpc31 allows for its optimal interaction with RNA polymerase III global repressor Maf1. Interestingly, mammalian Hmt1 homologue is able to methylate one of Rpc31's human homologue, RPC32β, but not its paralogue, RPC32α. Our data led us to propose an efficient model whereby protein arginine methylation facilitates metabolic economy and coordinates protein-synthetic capacity.