Transcription Translation Research Articles

New insight into the catalytic -dependent and -independent roles of METTL3 in sustaining aberrant translation in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.

Nature-Inspired Circular-Economy Recycling for Proteins: Proof of Concept.

The billion tons of synthetic-polymer-based materials (i.e. plastics) produced yearly are a great challenge for humanity. Nature produces even more natural polymers, yet they are sustainable. Proteins are sequence-defined natural polymers that are constantly recycled when living systems feed. Digestion is the protein depolymerization into amino acids (the monomers) followed by their re-assembly into new proteins of arbitrarily different sequence and function. This breaks a common recycling paradigm where a material is recycled into itself. Organisms feed off of random protein mixtures that are "recycled" into new proteins whose identity depends on the cell's specific needs. In this study, mixtures of several peptides and/or proteins are depolymerized into their amino acid constituents, and these amino acids are used to synthesize new fluorescent, and bioactive proteins extracellularly by using an amino-acid-free, cell-free transcription-translation (TX-TL) system. Specifically, three peptides (magainin II, glucagon, and somatostatin 28) are digested using thermolysin first and then using leucine aminopeptidase. The amino acids so produced are added to a commercial TX-TL system to produce fluorescent proteins. Furthermore, proteins with high relevance in materials engineering (β-lactoglobulin films, used for water filtration, or silk fibroin solutions) are successfully recycled into biotechnologically relevant proteins (fluorescent proteins, catechol 2,3-dioxygenase).

Nucleoporin 50 mediates Kcna4 transcription to regulate cardiac electrical activity.

Emerging evidence has demonstrated that nucleoporins (Nups) play a pivotal role in cell-type-specific gene regulation, but how they control the expression and activity of ion channel genes in the heart remains unclear. Here, we show that Nup50, which is localized in the nucleus of cardiomyocytes, selectively induces an increase in the transcription and translation of Kcna4. The Kcna4 gene encodes a K+ voltage-gated channel of shaker-related subfamily member 4 and is essential for regulating the action potential in cardiac membranes. Using immunofluorescence imaging, luciferase assays and chromatin immunoprecipitation assays, we identified that the direct binding of the FG-repeat domain within Nup50 to the proximity of the Kcna4 promoter was required to activate the transcription and subsequent translation of Kcna4. Functionally, Nup50 overexpression increased the currents of KCNA4-encoded Ito,s channels, and reverse knockdown of Nup50 resulted in a remarkable decrease in the amplitude of Ito,s currents in cardiomyocytes. Moreover, a positive correlation between Nup50 and Kcna4 mRNA and protein expression was observed in heart tissues subjected to ischemic insults. These findings provide insights into the homeostatic control of cardiac electrophysiology through Nup-mediated regulation.

Multilevel Regulation of Carotenoid Synthesis by Light and Active Oxygen in Blakeslea trispora.

Although Blakeslea trispora has been used for industrial production of β-carotene, the effects of light and oxidative stress on its synthesis have not been fully clarified. The present study focuses on the effects of light and reactive oxygen species (ROS) on carotenoid synthesis and their multilevel regulation in B. trispora. Blue light significantly influenced the intracellular ROS levels, carotenoid contents, and transcription of carotenoid structural genes, while ROS levels were positively correlated with intracellular carotenoid contents and transcriptional levels of carotenoid structural genes. Blue light and ROS were both significant factors affecting carotenoid synthesis with a significant interaction between them. Irradiation by pulsed blue light and (or) addition of generating agents for active oxygen could partially compensate for the inhibition derived from the transcription inhibitor (dactinomycin) and translation inhibitor (cycloheximide) on the multilevel phenotype. Therefore, blue light and ROS act on the transcription and translation of carotenoid structural genes to promote the accumulation of carotenoid in B. trispora.

Tuning Cell-Free Composition Controls the Time Delay, Dynamics, and Productivity of TX-TL Expression.

The composition of cell-free expression systems (TX-TL) is adjusted by adding macromolecular crowding agents and salts. However, the effects of these cosolutes on the dynamics of individual gene expression processes have not been quantified. Here, we carry out kinetic mRNA and protein level measurements on libraries of genetic constructs using the common cosolutes PEG-8000, Ficoll-400, and magnesium glutamate. By combining these measurements with biophysical modeling, we show that cosolutes have differing effects on transcription initiation, translation initiation, and translation elongation rates with trade-offs between time delays, expression tunability, and maximum expression productivity. We also confirm that biophysical models can predict translation initiation rates in TX-TL using Escherichia coli lysate. We discuss how cosolute composition can be tuned to maximize performance across different cell-free applications, including biosensing, diagnostics, and biomanufacturing.

Musings from the Tribbles Research and Innovation Network.

Simple SummaryThe Tribbles Research and Innovation Network (TRAIN) was developed in the context of the medical need to understand the contribution that Tribbles (TRIB) proteins make in regulating processes governing the physiological functioning of macrophages and other immune cells, adipocytes and prostate epithelial cells in immuno-metabolic disease (such as obesity) and several cancers. To summarize, TRAIN provided a cohort of PhD students with a unique opportunity to undertake multidisciplinary research aimed at uncovering the cell-specific mechanisms by which TRIBs exert control over immuno-metabolism and impact on prostate cancer progression. The associated training programme was enhanced by contributions from the TRIB community. This review highlights the science that motivated the establishment of TRAIN, including the discovery of how the TRIB1 protein enables the marking of the transcription factor C/EBPα for degradation, and regulatory mechanisms underlying cell-specific TRIB expression. It briefly reflects on the type of TRAIN-associated tools developed for future TRIB research.This commentary integrates historical and modern findings that underpin our understanding of the cell-specific functions of the Tribbles (TRIB) proteins that bear on tumorigenesis. We touch on the initial discovery of roles played by mammalian TRIB proteins in a diverse range of cell-types and pathologies, for example, TRIB1 in regulatory T-cells, TRIB2 in acute myeloid leukaemia and TRIB3 in gliomas; the origins and diversity of TRIB1 transcripts; microRNA-mediated (miRNA) regulation of TRIB1 transcript decay and translation; the substantial conformational changes that ensue on binding of TRIB1 to the transcription factor C/EBPα; and the unique pocket formed by TRIB1 to sequester its C-terminal motif bearing a binding site for the E3 ubiquitin ligase COP1. Unashamedly, the narrative is relayed through the perspective of the Tribbles Research and Innovation Network, and its establishment, progress and future ambitions: the growth of TRIB and COP1 research to hasten discovery of their cell-specific contributions to health and obesity-related cancers.

PD62-10 THE ROLE OF MTOR IN DIFFERENTIATION OF SPERMATOZOA

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (PD62)1 Sep 2021PD62-10 THE ROLE OF MTOR IN DIFFERENTIATION OF SPERMATOZOA Karen Doersch, Kadijah Abston, Hannah Kim, Amanda Pereira, Xiaoyang Su, and Xin Zhiguo Li Karen DoerschKaren Doersch More articles by this author , Kadijah AbstonKadijah Abston More articles by this author , Hannah KimHannah Kim More articles by this author , Amanda PereiraAmanda Pereira More articles by this author , Xiaoyang SuXiaoyang Su More articles by this author , and Xin Zhiguo LiXin Zhiguo Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002099.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The mammalian target of rapamycin (mTOR) is an evolutionarily preserved protein that regulates homeostatic processes, including growth-factor response, autophagy, cytoskeletal organization, and metabolism. Rapamycin, an inhibitor of mTOR, is well-established to extend organisms’ lifespan from yeast to mice; however, its application in humans causes male infertility. Because mTOR pathway function in germ cells is not well-understood, the objective of this study was to examine the role of mTOR in germ cell development. METHODS: A testis-specific mTor-conditional knockout (CKO) mouse was created using a lox-cre system driven by Neurog3-cre, which is expressed in late spermatogenesis. Testis were examined by H&E, electron microscopy, and immunofluorescence microscopy. RNA-seq and small RNA-seq were also used. Metabolomic analysis of testis tissue with mass spectroscopy was performed. RESULTS: The CKO mutants are completely infertile with arrest of spermiogenesis at the round spermatid stage on H&E and electron microscopy. RNA-seq demonstrated decreases in markers of mature spermatozoa CKO compared to controls, corroborating the finding of arrest at the round spermatid. Furthermore, analysis of micro-RNAs (miRNAs) demonstrated aberrant miRNA expression, which may play a role in halting spermatic development. Metabolomic analysis demonstrated increased amino acids, such as threonine, glutamine, and isoleucine, in the CKO mouse compared with controls. CKO testis had altered nucleic acid levels, with elevated guanosine but decreased nucleic acid-monophosphate species. Aberrant guanosine likely represents alterations in the guanosine-triphosphate-dependent signaling via the mTORC1 pathway while decreased nucleotide monophosphates likely represent decreased energy production and translation. CONCLUSIONS: This study demonstrates a germ cell specific CKO of mTOR signaling leads to arrest of spermiogenesis at the round spermatid phase. Metabolomic analysis demonstrated decreased mRNA transcription and protein translation in the CKO. Our results demonstrate a direct role for mTOR-mediated mRNA and protein regulation in spermatozoa differentiation and metabolism. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1076-e1076 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karen Doersch More articles by this author Kadijah Abston More articles by this author Hannah Kim More articles by this author Amanda Pereira More articles by this author Xiaoyang Su More articles by this author Xin Zhiguo Li More articles by this author Expand All Advertisement Loading ...

Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome.

SUMMARYAlthough the roles of initiation factors, RNA binding proteins, and RNA elements in regulating translation are well defined, how the ribosome functionally diversifies remains poorly understood. In their human hosts, poxviruses phosphorylate serine 278 (S278) at the tip of a loop domain in the small subunit ribosomal protein RACK1, thereby mimicking negatively charged residues in the RACK1 loops of dicot plants and protists to stimulate translation of transcripts with 5′ poly(A) leaders. However, how a negatively charged RACK1 loop affects ribosome structure and its broader translational output is not known. Here, we show that although ribotoxin-induced stress signaling and stalling on poly(A) sequences are unaffected, negative charge in the RACK1 loop alters the swivel motion of the 40S head domain in a manner similar to several internal ribosome entry sites (IRESs), confers resistance to various protein synthesis inhibitors, and broadly supports noncanonical modes of translation.

YBX1 mediates translation of oncogenic transcripts to control cell competition in AML

Persistence of malignant clones is a major determinant of adverse outcome in patients with hematologic malignancies. Despite the fact that the majority of patients with acute myeloid leukemia (AML) achieve complete remission after chemotherapy, a large proportion of them relapse as a result of residual malignant cells. These persistent clones have a competitive advantage and can re-establish disease. Therefore, targeting strategies that specifically diminish cell competition of malignant cells while leaving normal cells unaffected are clearly warranted. Recently, our group identified YBX1 as a mediator of disease persistence in JAK2-mutated myeloproliferative neoplasms. The role of YBX1 in AML, however, remained so far elusive. Here, inactivation of YBX1 confirms its role as an essential driver of leukemia development and maintenance. We identify its ability to amplify the translation of oncogenic transcripts, including MYC, by recruitment to polysomal chains. Genetic inactivation of YBX1 disrupts this regulatory circuit and displaces oncogenic drivers from polysomes, with subsequent depletion of protein levels. As a consequence, leukemia cells show reduced proliferation and are out-competed in vitro and in vivo, while normal cells remain largely unaffected. Collectively, these data establish YBX1 as a specific dependency and therapeutic target in AML that is essential for oncogenic protein expression.

METTL14 facilitates global genome repair and suppresses skin tumorigenesis

Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.

The endogenous mex-3 3´UTR is required for germline repression and contributes to optimal fecundity in C. elegans.

RNA regulation is essential to successful reproduction. Messenger RNAs delivered from parent to progeny govern early embryonic development. RNA-binding proteins (RBPs) are the key effectors of this process, regulating the translation and stability of parental transcripts to control cell fate specification events prior to zygotic gene activation. The KH-domain RBP MEX-3 is conserved from nematode to human. It was first discovered in Caenorhabditis elegans, where it is essential for anterior cell fate and embryo viability. Here, we show that loss of the endogenous mex-3 3´UTR disrupts its germline expression pattern. An allelic series of 3´UTR deletion variants identify repressing regions of the UTR and demonstrate that repression is not precisely coupled to reproductive success. We also show that several RBPs regulate mex-3 mRNA through its 3´UTR to define its unique germline spatiotemporal expression pattern. Additionally, we find that both poly(A) tail length control and the translation initiation factor IFE-3 contribute to its expression pattern. Together, our results establish the importance of the mex-3 3´UTR to reproductive health and its expression in the germline. Our results suggest that additional mechanisms control MEX-3 function when 3´UTR regulation is compromised.

"Dual-Signal-On" Integrated-Type Biosensor for Portable Detection of miRNA: Cas12a-Induced Photoelectrochemistry and Fluorescence Strategy.

The abnormal expression of microRNA (miRNA) can affect the RNA transcription and protein translation, leading to tumor progression and metastasis. Currently, the accurate detection of aberrant expression of miRNA, particularly using a portable detection system, remains a great challenge. Herein, a novel dual-mode biosensor with high sensitivity and robustness for miR-21 detection was developed based on the cis-cleavage and trans-cleavage activities of Cas12a. miRNA can be combined with hairpin DNA-horseradish peroxidase anchored on a CdS/g-C3N4/B-TiO2 photoelectrode, thus the nonenzymatic amplification was triggered to form numerous HRP-modified double-stranded DNA (HRP-dsDNA). Then, HRP-dsDNA can be specifically recognized and efficiently cis-cleaved by Cas12a nucleases to detach HRP from the substrate, while the remaining HRP on HRP-dsDNA can catalyze 4-chloro-1-naphthol (4-CN) to form biocatalytic precipitation (BCP) on the surface of the photoelectrode, and thus the photocurrent can be changed. Meanwhile, the trans-cleavage ability of Cas12a was activated, and nonspecifically degrade the FQ-reporter and a significant fluorescence signal can be generated. Such two different kinds of signals with independent transmission paths can mutually support to improve the performance of the detection platform. Besides, a portable device was constructed for the point-of-care (POC) detection of miR-21. Moreover, the dual-mode detection platform can be easily expanded for the specific detection of other types of biomarkers by changing the sequence of hairpin DNA, thereby promoting the establishment of POC detection for early cancer diagnosis.

Ataluren-Promising Therapeutic Premature Termination Codon Readthrough Frontrunner.

Around 12% of hereditary disease-causing mutations are in-frame nonsense mutations. The expression of genes containing nonsense mutations potentially leads to the production of truncated proteins with residual or virtually no function. However, the translation of transcripts containing premature stop codons resulting in full-length protein expression can be achieved using readthrough agents. Among them, only ataluren was approved in several countries to treat nonsense mutation Duchenne muscular dystrophy (DMD) patients. This review summarizes ataluren’s journey from its identification, via first in vitro activity experiments, to clinical trials in DMD, cystic fibrosis, and aniridia. Additionally, data on its pharmacokinetics and mechanism of action are presented. The range of diseases with underlying nonsense mutations is described for which ataluren therapy seems to be promising. What is more, experiments in which ataluren did not show its readthrough activity are also included, and reasons for their failures are discussed.

Human oocyte meiotic maturation is associated with a specific profile of alternatively spliced transcript isoforms.

The transition from a transcriptionally active state (GV) to a transcriptionally inactive state (mature MII oocytes) is required for the acquisition of oocyte developmental competence. We hypothesize that the expression of specific genes at the in vivo matured (MII) stage could be modulated by posttranscriptional mechanisms, particularly regulation of alternative splicing (AS). In this study, we examined the transcriptional activity of GV oocytes after ovarian stimulation followed by oocyte pick-up and the landscape of alternatively spliced isoforms in human MII oocytes. Individual oocytes were processed and analyzed for transcriptional activity (GV), gene expression (GV and MII), and AS signatures (GV and MII) on HTA 2.0 microarrays. Samples were grouped according to maturation stage, and then subgrouped according to women's age and antral follicular count (AFC); array results were validated by quantitative polymerase chain reaction. Differentially expressed genes between GV and MII oocytes clustered mainly in biological processes related to mitochondrial metabolism. Interestingly, 16 genes that were related to the regulation of transcription and mitochondrial translation showed differences in alternatively spliced isoform profiles despite not being differentially expressed between groups. Altogether, our results contribute to our understanding of the role of AS in oocyte developmental competence acquisition.

The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform.

The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription–translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all-E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells.

N7-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays invitro and invivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression invivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.

IRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation.

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.

LncRNA FOXD3-AS1 Promotes the Malignant Progression of Nasopharyngeal Carcinoma Through Enhancing the Transcription of YBX1 by H3K27Ac Modification.

BackgroundLong noncoding RNAs (lncRNAs) can affect the progression of various tumors, including nasopharyngeal carcinoma (NPC). Here, lncRNA FOXD3-AS1 is highly expressed in NPC tissues through bioinformatics analysis and related to the malignant progression of NPC.MethodsBioinformatics analysis and real-time reverse transcription quantitative PCR(RT-qPCR) assay were applied to identify the expression of FOXD3-AS1 in NPC tissues and cells. Specific short hairpin RNAs (shRNAs) or overexpression plasmids were used to knockdown or upregulate FOXD3-AS1 in NPC cells. The effect of FOXD3-AS1 on proliferation and metastasis of NPC was confirmed by CCK8, colony formation, transwell assays in vitro and mouse tumor growth and metastasis models in vivo, of which the mechanism was explored by RNA pull down, mass spectrometry (MS), RNA Immunoprecipitation (RIP), chromatin immunoprecipitation (CHIP) and luciferase assays.ResultsFOXD3-AS1 was highly expressed in NPC tissues and cells. Knockdown of FOXD3-AS1 significantly inhibited proliferation, migration, and invasion of NPC cells in vitro and vivo. FOXD3-AS1 could specifically bind to YBX1 and have a positive effect on the expression of YBX1. Bioinformatics analysis showed that the promoter of YBX1 had a high enrichment of H3K27ac, which promote mRNA transcription and protein translation of YBX1. Moreover, overexpression of YBX1 could reverse the proliferation, migration and invasion arrest caused by FOXD3-AS1 knockdown.ConclusionLncRNA FOXD3-AS1 is highly expressed and promotes malignant phenotype in NPC, which may provide a new molecular mechanism for NPC.

Abstract 1832: Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy

Abstract Background: Thailanstatins are naturally occurring anti-proliferative compounds that target spliceosomes and modulate pre-mRNA splicing. Alterations in splicing machinery and mRNA splicing is common in cancer and represents a potential susceptibility that can be exploited by targeted delivery of a splicing modulator to tumors with antibody drug conjugates (ADCs). Results: PH1 payload is a novel derivative of Thailanstatin optimized for metabolic stability and anti-tumor activity. PH1 is an efficient modulator of splicing in vitro and in vivo and a poor substrate for the MDR1 transporter. Transcriptome analysis of PH1- treated NCI-N87 cells revealed multiple classes of mis-splicing events including elevated expression of transcripts with skipped exons (3364 genes/4x change) and retained introns (332 genes/2.5x change). Translation of transcripts with retained introns and novel exon-exon junctions results in generation of neoepitopes. Upon conjugation to Trastuzumab, Tras PH1 ADC exhibited nanomolar potency specific to HER2-expressing cancer cells, with durable anti-tumor response in vivo and favorable pharmacokinetic exposure in mice. Due to the potential for generation of neoepitopes, combination studies of Tras PH1 with a checkpoint inhibitor antibody was performed in a syngeneic MC38-HuHer2 model. Single agent and combination treatments induced tumor recruitment of CD11b+ positive myeloid cell subsets. While the combination treatment eradicated tumors, the treated animals also developed tumor-specific immunity. A toxicology study performed in cynomolgus monkeys confirmed favorable PK profile and a wide safety margin for Tras PH1 ADC. Conclusions: We present the development of a splicing modulator-based payload class with the ability to target tumor mRNA splicing, induce tumor neoepitopes, recruit myeloid cells and generate anti-tumor immunity. These findings support the development of ADCs using this novel class of immunostimulatory payload. Citation Format: Satyajit K. Mitra, Vasu Jammalamadaka, Jeffrey Kang, Teodora Losic, Greg Tuffy, Tony W. Liang, Kim Tipton, Adriana Lopez, Scott Savage, William Monteith, William E. Haskins, Melissa S. Jurica, Sanjeev Satyal, Mary Do. Development of a splicing modulator-based ADC payload class with immune stimulatory properties for 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 1832.