RNA Polymerase I Research Articles

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the Xfm rpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Flavopiridol Mitigates the Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Targeting Cyclin-Dependent Kinase 9

PurposeTo investigate the role of cyclin-dependent kinase 9 (CDK9) and the therapeutic potential of a CDK9 inhibitor (flavopiridol) in monocrotaline (MCT)-induced pulmonary hypertension (PH).MethodsFor the in vivo experiments, rats with PH were established by a single intraperitoneal injection of MCT (60 mg/kg). After 2 weeks of MCT injection, rats were then treated with flavopiridol (5 mg/kg, i.p., twice a week) or vehicle for 2 weeks. For the in vitro experiments, human pulmonary artery smooth muscle cells (HPASMCs) were treated with flavopiridol (0.025-1 μM) or vehicle under hypoxic conditions. Hemodynamic recording, right ventricle histology, lung histology, and pulmonary arterial tissue isolation were performed. The expression levels of CDK9, RNA polymerase II, c-Myc, Mcl-1, and survivin were determined by qRT-PCR and western blotting, and the proliferation and apoptosis of rat pulmonary arterial tissues and/or HPASMCs were also assayed.ResultsCompared to the control group, CDK9 was upregulated in pulmonary arterial tissues from MCT-induced PH rats and hypoxic cultured HPASMCs. Upregulation of CDK9 was associated with enhanced phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) at serine-2 (Ser-2), promoting the expression of prosurvival and antiapoptotic proteins (c-Myc, Mcl-1, and survivin). Furthermore, treatment with flavopiridol (5 mg/kg) significantly alleviated pulmonary artery remodeling and partially reversed the progression of MCT-induced PH. Consistently, flavopiridol (0.5 μM) treatment decreased the proliferation and induced the apoptosis of cultured HPASMCs under hypoxic conditions. As a result of CDK9 inhibition and subsequent inhibition of RNA pol II CTD phosphorylation at Ser-2, flavopiridol decreased c-Myc, Mcl-1, and survivin expression in isolated pulmonary small arteries, leading to cell growth inhibition and apoptosis.ConclusionFlavopiridol mitigates the progression of MCT-induced PH in rats by targeting CDK9.

Brd4 is required for chondrocyte differentiation and endochondral ossification.

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4fl/fl: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.

The Efficiency of Global Genome-Nucleotide Excision Repair is Linked to the Fraction of Open rRNA Gene Chromatin, in Yeast.

The yeast rDNA locus is a suitable model to study nucleotide excision repair (NER) in chromatin. A portion of rRNA genes is transcribed and largely depleted of nucleosomes, the remaining genes are not transcribed and folded in nucleosomes. In G1-arrested cells, most rRNA genes do not have nucleosomes. TC-NER removes UV-induced DNA lesions from the transcribed strand of active genes. GG-NER is less efficient and removes DNA lesions from the nontranscribed strand of active genes and from the inactive genome. Different from mammalian cells, in yeast, the rRNA gene-transcribed strand is repaired by RNA polymerase-I-dependent TC-NER. The opposite nontranscribed strand is repaired faster than both strands of inactive rRNA genes. In log-phase cells, RNA polymerase-I are dislodged from the damaged transcribed strand and partially replaced by nucleosomes. Contrary to log-phase cells, in G1-phase cells few, if any, histones are deposited on the open rRNA genes during NER. In this study, we compared GG-NER efficiency in the rRNA gene coding region: without nucleosomes, partially loaded or wholly loaded with nucleosomes. The results indicate that in log-phase cells histones obstruct GG-NER, whereas in G1-phase cells GG-NER is as efficient as TC-NER.

Several Isoforms for Each Subunit Shared by RNA Polymerases are Differentially Expressed in the Cultivated Olive Tree (Olea europaea L.).

Plants contain five nuclear RNA polymerases, with RNA pols IV and V in addition to conserved eukaryotic RNA pols I, II, and III. These transcriptional complexes share five common subunits, which have been extensively analyzed only in yeasts. By taking advantage of the recently published olive tree cultivar (Olea europaea L. cv. Picual) genome, we performed a genome-wide analysis of the genomic composition corresponding to subunits common to RNA pols. The cultivated olive tree genome is quite complex and contains many genes with several copies. We also investigated, for the first time, gene expression patterns for subunits common to RNA pols using RNA-Seq under different economically and biologically relevant conditions for the cultivar “Picual”: tissues/organs, biotic and abiotic stresses, and early development from seeds. Our results demonstrated the existence of a multigene family of subunits common to RNA pols, and a variable number of paralogs for each subunit in the olive cultivar “Picual.” Furthermore, these isoforms display specific and differentiated expression profiles depending on the isoform and growth conditions, which may be relevant for their role in olive tree biology.

TRNA biogenesis and specific aminoacyl-tRNA synthetases regulate senescence stability under the control of mTOR.

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks. Using experimental models of senescence escape, we now described that the stability of this proliferative arrest relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this tumor suppressive pathway.

Nucleolar Stress Functions Upstream to Stimulate Expression of Autophagy Regulators

Simple SummaryRibosome biogenesis takes place in nucleoli and is essential for cellular survival and proliferation. In case this function is disturbed, either due to defects in regulatory factors or the structure of the nucleolus, nucleolar stress is provoked. Consequently, cells classically undergo cell cycle arrest and apoptosis. Induction of nucleolar stress is known to eliminate cells in the background of cancer therapy and paradoxically is also associated with increased cancer formation. Recent reports demonstrated that nucleolar stress triggers autophagy, a conserved pathway responsible for recycling endogenous material. Thus, it was suggested that autophagy might serve as compensatory pro-survival response. However, the mechanisms how nucleolar stress triggers autophagy are poorly understood. Here we show that induction of nucleolar stress by depleting ribosome biogenesis factors or by interfering with RNA polymerase I function, triggers expression of various key autophagy regulators. Moreover, we demonstrate that RNA pol I inhibition by CX-5461 correlates with increased ATG7 and ATGL16L1 levels, essential factors for generating autophagosomes, and stimulates autophagic flux.Ribosome biogenesis is essential for protein synthesis, cell growth and survival. The process takes places in nucleoli and is orchestrated by various proteins, among them RNA polymerases I–III as well as ribosome biogenesis factors. Perturbation of ribosome biogenesis activates the nucleolar stress response, which classically triggers cell cycle arrest and apoptosis. Nucleolar stress is utilized in modern anti-cancer therapies, however, also contributes to the development of various pathologies, including cancer. Growing evidence suggests that nucleolar stress stimulates compensatory cascades, for instance bulk autophagy. However, underlying mechanisms are poorly understood. Here we demonstrate that induction of nucleolar stress activates expression of key autophagic regulators such as ATG7 and ATG16L1, essential for generation of autophagosomes. We show that knockdown of the ribosomopathy factor SBDS, or of key ribosome biogenesis factors (PPAN, NPM, PES1) is associated with enhanced levels of ATG7 in cancer cells. The same holds true when interfering with RNA polymerase I function by either pharmacological inhibition (CX-5461) or depletion of the transcription factor UBF-1. Moreover, we demonstrate that RNA pol I inhibition by CX-5461 stimulates autophagic flux. Together, our data establish that nucleolar stress affects transcriptional regulation of autophagy. Given the contribution of both axes in propagation or cure of cancer, our data uncover a connection that might be targeted in future.

Bacteria elicit a phage tolerance response subsequent to infection of their neighbors.

Appearance of plaques on a bacterial lawn is a sign of successive rounds of bacteriophage infection. Yet, mechanisms evolved by bacteria to limit plaque spread have been hardly explored. Here, we investigated the dynamics of plaque development by lytic phages infecting the bacterium Bacillus subtilis. We report that plaque expansion is followed by a constriction phase owing to bacterial growth into the plaque zone. This phenomenon exposed an adaptive process, herein termed "phage tolerance response", elicited by non-infected bacteria upon sensing infection of their neighbors. The temporary phage tolerance is executed by the stress-response RNA polymerase sigma factor σX (SigX). Artificial expression of SigX prior to phage attack largely eliminates infection. SigX tolerance is primarily conferred by activation of the dlt operon, encoding enzymes that catalyze D-alanylation of cell wall teichoic acid polymers, the major attachment sites for phages infecting Gram-positive bacteria. D-alanylation impedes phage binding and hence infection, thus enabling the uninfected bacteria to form a protective shield opposing phage spread.

Circular RNA Foxo3 Relieves Myocardial Ischemia/Reperfusion Injury by Suppressing Autophagy via Inhibiting HMGB1 by Repressing KAT7 in Myocardial Infarction.

IntroductionMyocardial infarction is coronary artery-related heart disease, and the leading cause of mortality globally. Circular RNAs (circRNAs) are a new type of regulatory RNAs and participate in multiple pathological cardiac progression.MethodsHowever, the function of circFoxo3 in MI-induced myocardial injury remains obscure.ResultsSignificantly, we identified that circFoxo3 was downregulated in the MI rat model and the overexpression of circFoxo3 ameliorated MI-induced cardiac dysfunction and attenuated MI-induced autophagy in rat model. Meanwhile, the overexpression of circFoxo3 repressed oxygen–glucose deprivation (OGD)-induced autophagy, apoptosis, inflammation, and injury of cardiomyocyte in vitro. Mechanically, we identified that the expression of KAT7 was reduced by circFoxo3 overexpression in cardiomyocytes. Meanwhile, the expression of HMGB1 was repressed by the depletion of KAT7 in cardiomyocytes. The enrichment of histone H3 lysine 14 acetylation (H3K14ac) and RNA polymerase II (RNA pol II) on HMGB1 promoter was inhibited by the knockdown of KAT7. Moreover, the overexpression of circFoxo3 suppressed HMGB1 expression and KAT7 overexpression rescued the expression of HMGB1 in cardiomyocytes. The enrichment of KAT7, H3K14ac, and RNA poly II on HMGB1 promoter was decreased by circFoxo3 overexpression, while the overexpression of KAT7 could reverse the effect. The overexpression of KAT7 or HMGB1 could reverse circFoxo3-attenuated cardiomyocyte injury and autophagy in vitro. Thus, we conclude that circular RNA circFoxo3 relieved myocardial ischemia/reperfusion injury by suppressing autophagy via inhibiting HMGB1 by repressing KAT7 in MI.DiscussionOur finding provides new insight into the mechanism by which circFoxo3 regulates MI-related cardiac dysfunction by targeting KAT7/HMGB1 axis.

Mutation of MEDIATOR16 promotes plant biomass accumulation and root growth by modulating auxin signaling

The MEDIATOR complex influences the transcription of genes acting as a RNA pol II co-activator. The MED16 subunit has been related to low phosphate sensing in roots, but how it influences the overall plant growth and root development remains unknown. In this study, we compared the root growth of Arabidopsis wild-type (WT), and two alleles of MED16 (med16-2 and med16-3) mutants in vitro. The MED16 loss-of-function seedlings showed longer primary roots with higher cell division capacity of meristematic cells, and an increased number of lateral roots than WT plants, which correlated with improved biomass accumulation. The auxin response reported by DR5:GFP fluorescence was comparable in WT and med16-2 root tips, but strongly decreased in pericycle cells and lateral root primordia in the mutants. Dose-response analysis supplementing indole-3-acetic acid (IAA), or the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA), indicated normal responses to auxin in the med16-2 and med16-3 mutants regarding primary root growth and lateral root formation, but strong resistance to NPA in primary roots, which could be correlated with cell division and elongation. Expression analysis of pPIN1::PIN1::GFP, pPIN3::PIN3::GFP, pIAA14:GUS, pIAA28:GUS and 35S:MED16-GFP suggests that MED16 could mediate auxin signaling. Our data imply that an altered auxin response in the med16 mutants is not necessarily deleterious for overall growth and developmental patterning and may instead directly regulate basic cellular programmes.

Ketamine suppresses proliferation and induces ferroptosis and apoptosis of breast cancer cells by targeting KAT5/GPX4 axis

Breast cancer (BC) serves as a prevalent and mortal malignancy among female globally. Ferroptosis, as an oxidative cell death that characterized by abnormal iron accumulation, plays critical role in cancer development. Ketamine is a rapid-acting anesthetic agent and has presented potential anti-tumor properties. However, the effect of Ketamine on breast cancer is still obscure. Here, we aimed to explore the function of Ketamine in the modulation of proliferation and ferroptosis of breast cancer cells. The cell viability of breast cancer cells was repressed by the treatment of Ketamine, while ferroptosis inhibitor ferrostatin 1 and apoptosis inhibitor ZVAD-FMK could restore the cell viability. The treatment of Ketamine significantly decreased the Edu-positive breast cancer cells and the colony formation numbers, and the treatment of ferrostatin 1 reversed the effect of Ketamine. We observed that the levels of ferroptosis markers, such as MDA, lipid ROS, and Fe2+ were increased by the treatment of Ketamine in breast cancer cells. Regarding to the mechanism, we found that Ketamine inhibited the expression of GPX4, an anti-ferroptosis factor, by attenuating KAT5 on the promoter region of GPX4, repressing the enrichment of histone H3 lysine 27 acetylation (H3K27ac) and RNA polymerase II (RNA pol II). The treatment of Ketamine reduced the cell viability and proliferation of breast cancer cells, in which the overexpression of KAT5 or GPX4 was able to restore the phenotypes. The treatment of Ketamine induced the levels of MDA, lipid ROS, and Fe2+, while KAT5 or GPX4 overexpression could reverse this effect in breast cancer cells. Thus, we concluded that Ketamine suppressed proliferation and induced ferroptosis of breast cancer cells by targeting KAT5/GPX4 axis. Ketamine may serve as a potential therapeutic strategy for breast cancer.

AMPK Reprograms Gene Expression and Promotes Adaptation/Survival in Response to Metabolic Stress through Binding to a Chromatin-Associated Transcription Complex in Acute Lymphoblastic Leukemia

Survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL), the most common cancer in children and adolescents, remain dismal. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK) activation. AMPK has been reported to interact with chromatin-associated proteins (e.g., histone H2B) in MEF cells to epigenetically regulate gene expression in response to environmental/cellular stress (Bungard et al. Science, 2010; 329:1201). To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we first constructed Bp-ALL (NALM6, REH) and T-ALL (CCRF-CEM, KE-37) stable cell lines expressing HA-AMPKα1 or HA-AMPKα2. Next, using HA and RNA pol II antibodies, we performed ChIP-seq assays in CCRF-CEM/HA-AMPKα2 (CN2) grown in glucose-free RPMI for 24 h. ChIP-seq differentially identified 171 candidate genes in CN2 treated with no-glucose vs. 431 genes in untreated controls. Data analysis using the Encode and ChEA database identified the TATA-Box Binding Protein Associated Factor, CCAAT Enhancer Binding Protein Delta (CEBPD), the negative elongation factor complex member E (NELFE), and the Promyelocytic leukemia protein (PML) among highly ranked transcription factors (TFs) may associated with AMPKα2 on chromatin. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin gene loci regulated in response to energy/metabolic stress, we performed RNA-seq assays in CN2 cells treated with or without glucose deprivation for 24 h. RNA-seq data analysis indicated that of the 3497 genes altered by AMPK activation, two thirds were downregulated whereas the remaining were upregulated. Kyoto Encyclopedia of Genes and Genomes gene set and BioPlanet 2019 gene set analysis identified metabolic pathways, DNA replication/metabolism, and cell cycle as the main biological processes altered in CN2 cells in response to metabolic stress. Among downregulated genes in response to metabolic stress, we uncovered a cluster of histone genes. To confirm and validate our data, we used RT-qPCR and ChIP-qPCR assays on selected histone gene candidates (H1-2/ HIST1H1C, H1-3/HIST1H1D, H4C4/HIST1H4D) which exhibited both decreased recruitment of HA-AMPKα2 to chromatin and mRNA downregulation in response to metabolic stress. Further ChIP-qPCR assays using an AMPKα2 antibody confirmed these data in KASUMI-2 cells (Bp-ALL). Similar data were also observed in CN2 cells treated with AICAR, another AMPK activator. Additional experiments were conducted using the allosteric AMPK activators compound 991 and PF-06409577. Using Co-IP experiments, we uncovered that AMPKα2 interacted with putative members of an AMPK/chromatin-associated transcription factor complex which included the TATA-Box Binding Protein Associated Factor (TAF), integrator (INT), and RNA polymerase II. To investigate the role of AMPK kinase activity on AMPKα2-associated chromatin regulated gene targets, we determined the effect of genetic constructs encoding a constitutively active (CA) form of AMPKα2 on histone gene mRNA expression in CCRF-CEM and MEF AMPKα1/AMPKα2 double knockout (DKO) cells, and found that in both cell types the expression of the full-length CA-AMPKα2 (T172D) lead to decreased mRNA expression of the histone genes examined, suggesting AMPK kinase activity is required to regulate histone genes in response to energy/metabolic stress. In conclusion, our data show that in response to metabolic stress, AMPK binds directly to a multi-protein complex on chromatin to reprogram gene expression to promote cellular adaptation/survival in ALL. Further elucidation of AMPK's interactions with members of the putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions and combination strategies to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML.Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS.Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio.Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. DisclosuresLogan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Genomic analysis of Burkholderia cenocepacia isolated from a liver abscess in a patient with cystic fibrosis.

Burkholderia cenocepacia complex is associated with high transmissibility, virulence, and poor prognosis in cystic fibrosis (CF) patients. However, extrapulmonary infections are rare. We investigated the genome of a B.cenocepacia IIIA isolated from a liver abscess in a Brazilian CF patient and compared it to strain J2315. The whole genome was sequenced, and contigs were annotated by Rapid Annotation using Subsystem Technology. The Pathosystems Resource Integration Center was used to map antimicrobial and virulence genes. The genomic island (GIs) analysis was performed using two prediction methods, and the presence of putative plasmids and insertion sequences (ISs) was investigated. The isolate was confirmed as B.cenocepacia IIIA to ST-28 (ET12 lineage). A total of 64 genes for antimicrobial resistance and 47 genes related to virulence were identified. Among the virulence factors, there was a predominance of factors related to the invasion mechanism, to the flagellar biosynthesis protein, and to the RNA polymerase sigma factor for flagellar operon (cdpA). Two IS families (IS3 and IS5) and only one plasmid were found. On average 56 GIs were predicted by at least one of the methods applied. Comparative analysis showed resistance mechanisms and virulence factors revealing invasive determinants used by B. cenocepacia IIIA (ET12) in the process of disease spread to other infection sites (extrapulmonary) of highly virulent strains in CF patients.

KAT6A is associated with sorafenib resistance and contributes to progression of hepatocellular carcinoma by targeting YAP

Hepatocellular carcinoma (HCC) is a prevalent solid cancer worldwide and sorafenib is a common treatment. Nevertheless, sorafenib resistance is a severe clinical problem. In the present study, we identified that epigenetic regulator, KAT6A, was overexpressed in clinical HCC tissues and sorafenib-resistant HCC samples. The depletion of KAT6A repressed the cell viability and Edu-positive cell numbers of HCC cells. The IC50 value of sorafenib was increased in sorafenib-resistant HCC cells. In addition, the expression of KAT6A was induced in sorafenib-resistant HCC cells. The depletion of KAT6A suppressed the IC50 of sorafenib. Mechanically, YAP was decreased by the depletion of KAT6A. KAT6A was able to enrich in the promoter of YAP. The silencing of KAT6A reduced the enrichment of histone H3 lysine 23 acetylation (H3K23ac) and RNA polymerase II (RNA pol II) on the promoter of YAP in sorafenib-resistant HCC cells. KAT6A inhibitor WM-1119 repressed the cell proliferation of sorafenib-resistant HCC cells, while overexpression of KAT6A or YAP could reverse the effect in the cells. Meanwhile, the treatment of sorafenib inhibited the viability of sorafenib-resistant HCC cells, while the co-treatment of WM-1119 could improve the effect of sorafenib. Collectively, KAT6A was associated with sorafenib resistance and contributes to progression of HCC by targeting YAP. Targeting KAT6A may be served as a promising therapeutic approach for HCC.

Arabidopsis thaliana AtTFIIB1 gene displays alternative splicing under different abiotic stresses

In Arabidopsis, there are 14 TFIIB-like proteins that have been phylogenetically categorized into the TFIIB, BRF, and Rrn7/TAF1B/MEE12 subfamilies. The TFIIB transcription factor (TF) subfamily plays a key role in the regulation of gene expression in eukaryotes. To identify the expression patterns of some members of the TFIIB and BRF subfamilies in A. thaliana, different approaches were carried out to determine the possible functions of some of these transcription factors. Through an in silico analysis, we identified possible cis-acting regulatory elements in the promoter regions that drive the expression of transcription factors, as well as we evaluated their expression by means of real-time qPCR, at different growth stages and under various stress conditions. Cis-acting elements analysis showed that general transcription factors possess stress-responsive elements such as W-Box (TTGACC/T type binding WRKY TFs), ARF1 (auxin response), MYB binding site promoter (auxin response and elicitors), RAV1-A (response to dehydration and salinity), and DRE elements (dehydration response) among others. The experimental results showed differential expression of TFIIB1 and TFIIB. In addition, we demonstrate that in stress conditions a transcript of the TFIIB1 factor is generated as an alternative splicing product by retention of the third intron, where a premature termination codon is found. This is the first report of an alternative splicing event in a general transcription factor related to RNA pol II, which is synthesized when the plant is under abiotic stresses such as heat, dehydration, and salinity.

Surveillance of Pretreatment Drug Resistance Among HIV-Infected Children in Ibadan, Nigeria.

There are about 2.1 million children infected with HIV globally and about 120,000 deaths annually. Nigeria has one of the highest rates of pediatric HIV infection globally. Pretreatment HIV drug resistance data inform the choice of first- and second-line antiretroviral therapy (ART) regimens. This study investigated the prevalence of HIV drug-resistant strains among ART-naive children in Ibadan, Nigeria. A total of 20 children aged <15 years were enrolled. Demographic, clinical, and laboratory data were documented. Total nucleic acid was extracted from blood samples after which amplification of HIV-1 pol gene was done using polymerase chain reaction. Amplified gene was sequenced using big dye sequencing method. The sequenced HIV-1 pol gene was typed and analyzed for identification of mutations indicative of drug resistance across the different classes of ART. HIV-1 RNA pol gene was successfully amplified in 12/20 (60%) children. All were identified as HIV-1 and the subtypes were G and CRF 02AG, recombinant of 02_AG/G and recombinant of 02_AG/A1. Drug-resistant mutations (DRMs) were identified in 4/12 (33%). Three out of the four mutations were identified as non-nucleoside reverse transcriptase inhibitors DRM (K103N), whereas the fourth had nucleoside reverse transcriptase inhibitors DRM (M184V). Results from this preliminary study show that drug resistance among ART-naive children is a problem in Ibadan. Pretreatment drug resistance testing is desirable in children before initiation of ART to guide effective treatment.

Different Patterns of mRNA Nuclear Retention during Meiotic Prophase in Larch Microsporocytes.

Recent studies show a crucial role of post-transcriptional processes in the regulation of gene expression. Our research has shown that mRNA retention in the nucleus plays a significant role in such regulation. We studied larch microsporocytes during meiotic prophase, characterized by pulsatile transcriptional activity. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm but are retained in the cell nucleus and especially in Cajal bodies, where non-fully-spliced transcripts with retained introns are accumulated. Analysis of the transcriptome of these cells and detailed analysis of the nuclear retention and transport dynamics of several mRNAs revealed two main patterns of nuclear accumulation and transport. The majority of studied transcripts followed the first one, consisting of a more extended retention period and slow release to the cytoplasm. We have shown this in detail for the pre-mRNA and mRNA encoding RNA pol II subunit 10. In this pre-mRNA, a second (retained) intron is posttranscriptionally spliced at a precisely defined time. Fully mature mRNA is then released into the cytoplasm, where the RNA pol II complexes are produced. These proteins are necessary for transcription in the next pulse to occur.mRNAs encoding translation factors and SERRATE followed the second pattern, in which the retention period was shorter and transcripts were rapidly transferred to the cytoplasm. The presence of such a mechanism in various cell types from a diverse range of organisms suggests that it is an evolutionarily conserved mechanism of gene regulation.

Abstract 915: Preclinical evaluation of anti-CD37 antibody-targeted amanitin conjugates (ATAC) in B-cell malignancies

Abstract Background: ATACs (antibody-targeted amanitin conjugates) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of ATACs targeting CD37 (member of the tetraspanin family) are presented. CD37 is a transmembrane protein expressed exclusively on cells of the immune system and mainly on mature B-cells, as well as in many B-cell malignancies, including Richter's Syndrome (RS) or Richter´s transformation. RS is a transformation of B cell chronic lymphocytic leukemia, refractory to treatment and carries a poor prognosis, and hence is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: CD37-positive: Mec-2 (B-chronic lymphocytic leukaemia); Raji-Luc, Ramos (Burkitt´s lymphoma); CD37-negative: HL-60 (acute lymphocytic leukemia) Antibody: anti-CD37 engineered monoclonal antibody produced at Heidelberg Pharma Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody. Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega). Animal models: Disseminating Mouse xenograft tumor models (Mec-2 and Raji-Luc) and CD37-positive patient derived xenograft (PDX; Richter's Syndrome) models were performed in single-dose experiments. Tolerability was assessed in non-human primates (NHP). Results: All tested anti-CD37 ATACs showed in vitro cytotoxicity in the picomolar range on CD37-positive cell lines. No cytotoxic activity was observed on CD37-negative cells. In mouse xenograft models, 80 - 100% overall survival was achieved with two anti-CD37 ATACs at doses of 1/16 MTD (Mec-2 model) and 1/64 MTD (Raji Luc Model). Single-dose treatment caused rapid and complete tumor remission already 7 days after treatment. The tested ATACs revealed good tolerability in mice. In NHP, the anti CD37 ATACs revealed good tolerability. Hematology and clinical chemistry parameters were unaffected except liver transaminases and LDH: A mild to moderate and transient increase was observed. Conclusions: Targeted cytotoxic drug delivery to CD37 positive cell lines was achieved by using anti-CD37 ATACs. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ADCs in the therapy of B-cell lymphomas, including malignancies that underwent Richter's transformation, is a promising approach, especially by using a payload whose mode of action differs from other commonly used toxins, like ATACs. Citation Format: Stephanie Voss, Aniko Palfi, Christoph Mueller, Andreas Pahl, Torsten Hechler, Michael Kulke. Preclinical evaluation of anti-CD37 antibody-targeted amanitin conjugates (ATAC) in B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 915.

Abstract 910: Amanitin-based ADCs targeting PSMA as novel therapeutic modality for prostate cancer therapy

Abstract Background: Antibody-targeted amanitin conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of an ATAC targeting PSMA (prostate specific membrane antigen) are presented. PSMA is a type II integral membrane glycoprotein and used as tumor marker due to its predominant expression on malignant prostate cells in prostate carcinoma. As PSMA expression increases with tumor aggressiveness, metastatic and disease recurrence, it is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: PSMA+ cell lines: LNCap, 22RV1; PSMA- cell line: PC3 Antibody: cysteine engineered monoclonal anti-PSMA antibody (Albert Ludwig University Freiburg, medical center; derivatization and production at Heidelberg Pharma) Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of the anti-PSMA antibody yielding ATACs with a DAR of 2. Cell proliferation assay: Quantitative determination of cell viability was performed by BrdU-based chemiluminescent cell proliferation ELISA (Roche). Animal models: Subcutaneous Mouse xenograft tumor models with PSMA-positive cell lines 22RV1 and LNCap were performed in single-dose experiments. Tolerability was assessed in mice and non-human primates (NHP). Results: All anti-PSMA ATACs with optimized amanitin-linker variants for use in solid tumors showed favorable in vitro cytotoxicity with nano- to picomolar activity on PSMA+ cell lines and no cytotoxic activity on PSMA- cells. In mouse xenograft models, the optimized anti-PSMA ATACs caused dose-dependent tumor regression in LNCap s.c. xenografts. Complete remission was achieved after a single i.v. dose of ¼ MTD with 100% overall survival for the full duration of the studies (&amp;gt;160 days). The tolerability of the tested ATACs in mice differed between 15 and &amp;gt; 80 mg/kg Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index for all selected amanitin-linker variants. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A moderate and transient increase was observed. Low off target toxicity was confirmed by a non-binding ATAC. Conclusions: Targeted cytotoxic drug delivery to PSMA positive cell lines was achieved by using anti-PSMA ATACs optimized for the use in solid tumors. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ATACs in the therapy of PSMA positive prostate cancer is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins. Citation Format: Alexandra Braun, Aniko Palfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amanitin-based ADCs targeting PSMA as novel therapeutic modality for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 910.