Intronic Polyadenylation Sites Research Articles

Pre-eclampsia intronic polyadenylation enriched in VEGFA-VEGFR2 signaling pathway

AimsThis study aims to reveal transcriptome-wide intronic polyadenylation (IPA) events associated with Pre-eclampsia (PE). BackgroundPre-eclampsia (PE) is a potentially life-threatening complication of pregnancy, affecting both maternal and fetal health. However, our understanding of the underlying molecular mechanisms of PE remains limited. ObjectiveIn this study, we conducted a transcriptome-wide analysis of gene expression levels and intronic polyadenylation (IPA) events in samples of patients with PE. We also conducted motif analysis and scanned the microRNA targeting IPA network. MethodWe collected 90 PE-related samples from GEO database. IPA events were analyzed using IPAfinder software from hg38 alignment files from STAR. Miranda software was used to perform miRNA target gene prediction. Differentially expressed genes (DEG) were evaluated using edgeR with log2 fold change >1 and adjusted p-value <0.05. Function enrichment was performed through Clusterprofiler. ResultOur analysis revealed that genes in the VEGFA-VEGFR2 signaling pathway were functionally enriched with IPA events related to PE. We observed a negative correlation between gene expression levels and IPA events in VEGFA-VEGFR2 pathway. We identified LIN28B and AGO2 as the most significantly binding motifs to IPA sites. Furthermore, our analysis of miRNA binding sites associated with these IPA events revealed the central regulatory roles of miR-193b and miR-365a in IPA genes. ConclusionOur findings suggest that transcriptome data-mining might be a useful approach in future studies aimed at identifying potential biomarkers for PE.

Abstract 5646: Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer

Abstract Introduction: Changes in alternative pre-mRNA processing have been found in many cancers. Aberrant intronic polyadenylation (IPA) causes a stop of normal transcription resulting in the production of truncated mRNA isoform or lncRNA. We sought to examine the role of mutant p53 in pre-mRNA processing, especially in IPA regulation in lung cancer. Methods: We established stable cell lines transfected with mutant p53 (R273H, R175H, H179Q, C238Y, and C242F) or vector control using the NSCLC cell line H1299 with a homozygous deletion of the endogenous p53. Total RNA was used to build libraries enriched in sequences near polyadenylation sites (PASs) using the 3’mRNA-Seq REV library kit (Lexogen; Greenland, NH). Sequencing reads were mapped to the human genome (GRCh37), and PASs with ≥2 supporting reads and ≥0.05 usage in any experiment were identified. Counts for each Intronic Polyadenylation (IPA) and total reads in the terminal exon were compiled into matrices for each condition. DEXseq was applied to detect changes in the relative abundance of each exon part normalized to all exons within the gene. Gene Ontology (GO) analysis was implemented using the enrichR package. Results: We identified 32,752 IPA sites in 5,718 genes, and 26,416 distal PASs (dPASs) in the 3’UTRs of 10,298 genes. Across all five cell lines, when compared individually to the vector control, a comprehensive shift in IPA events was observed, with a concurrent decrease in dPASs. Many of IPA isoforms exhibited consistent patterns of increased or decreased abundance changes. Upon closer examination of each individual cell line, such as the p53-R273H cell line (vs. vector), significant usage changes were identified for 2,389 IPA sites, of which 1,210 were increased and 1,128 terminal exons were altered, with 650 being suppressed. At the gene level, a total of 1,183 genes had at least one significantly increased IPA isoform, a significantly decreased distal isoform, or both. GO analysis showed that these genes were highly enriched in DNA repair-related functional terms such as DNA repair, double-strand break repair, and nucleotide excision repair. The observed IPA changes in DNA-repair genes were consistently observed across the other 4 mutant lines (vs. vector). This observation was further supported by analyzing data from patients with lung adenocarcinoma (LUAD) in the TCGA cohort, where we selected samples carrying nonsense/frameshift mutations, and/or a deep deletion as the p53-null control group, and those carrying missense mutations as the p53-mutant group. Conclusions: Our study illustrated a novel role of p53 mutants in the regulation of intronic polyadenylation processing that contributes to deficient DNA repair. These observations offer a fresh perspective on the multifaceted roles of p53 in cellular homeostasis. Acknowledgment: This work was supported by NCI Cancer Center Support Grant P30CA012197 and NIH/NCI grant R03CA256100. Citation Format: Liang Liu, Elizabeth Forbes, Wei Zhang. Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5646.

Abstract 3301: Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy

Abstract Gene dysregulation is a hallmark of cancer, arising from genetic alterations that invariably lead to dysregulated transcriptional programs. This dysregulation renders cancer cells highly dependent on certain gene expression regulators, leading to a potential for novel therapeutic strategies. Consequently, ongoing research aims to develop small molecules targeting RNA transcriptional processes. Cyclin-dependent kinase 12/13 (CDK12/13) is a member of the cyclin-dependent kinase family of serine/threonine protein kinases. CDK12/13 regulates the RNA transcription elongation by phosphorylating RNA polymerase II and promotes the elongation of transcripts, specifically those involved in DNA damage responses, such as BRCA1/2. The inhibition of CDK12/13 is expected to exert a synergistic effect when combined with PARP inhibitors and chemotherapeutic reagents. Therefore, we developed a novel small molecule inhibitor, CTX-439 (former name: CRD-1835439). Additionally, we demonstrated that CTX-439 exhibits a preclinical efficacy in vitro and in vivo [Cancer Res. (2022) 82 (12_Supplement): 5485]. To identify potential strategies for a clinical trial of CTX-439, in the current study, we conducted translational research to elucidate (1) pharmacodynamics (PD) markers to monitor the suppression of CDK12/13 in humans, (2) patient selection biomarkers to determine sensitivity to CTX-439, and (3) combination strategies with other key medications. Firstly, to identify PD markers of CTX-439, we performed a comprehensive analysis employing RNA-seq, Pol II-ChIP-seq, and Poly(A)-seq. We confirmed that CTX-439 induced the usage of intronic polyadenylation sites by inhibiting CDK12, resulting in the production of shortened mRNA. Of these transcripts, PCF11 emerged as a potential PD marker for clinical trials, which was confirmed using qPCR analysis of human blood. Secondly, to clarify the indication of CTX-439, we performed an in vitro survey involving 400 cell lines and 100 PDX-derived organoids (PDXOs) to identify biomarkers displaying sensitivity and insensitivity to CTX-439. Furthermore, a significant anti-tumor effect of CTX-439 was revealed in PDX models that showed sensitivity in the survey. CTX-439 exhibited a synergistic effect in combination with PARP inhibitors both in vitro and in vivo, highlighting its potential for use in combination strategies. Additionally, CTX-439 showed an anti-tumor effect when combined with other chemotherapy and molecular-targeted drugs. Collectively, the results from these translational research studies provide a foundation for developing a clinical trial by clearly defining PD monitoring, patient stratification biomarkers, and combination strategies. These findings highlight the therapeutic potential of CTX-439 as a single-agent and in combination with inhibitors, such as PARP, for the treatment of cancers characterized by transcriptional addiction. Citation Format: Hiroko Yamakawa, Shunsuke Ebara, Akio Mizutani, Misaki Yoshida, Midori Sugiyama, Koji Yamamoto, Daisuke Komura, Miwako Kakiuchi, Shumpei Ishikawa, Kosuke Yusa, Daisuke Morishita. Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3301.

The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs

Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both invitro and invivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.

Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation.

Alternative splicing (AS) and alternative polyadenylation (APA) are two crucial steps in the post-transcriptional regulation of eukaryotic gene expression. Protocols capturing and sequencing RNA 3'-ends have uncovered widespread intronic polyadenylation (IPA) in normal and disease conditions, where it is currently attributed to stochastic variations in the pre-mRNA processing. Here, we took advantage of the massive amount of RNA-seq data generated by the Genotype Tissue Expression project (GTEx) to simultaneously identify and match tissue-specific expression of intronic polyadenylation sites with tissue-specific splicing. A combination of computational methods including the analysis of short reads with non-templated adenines revealed that APA events are more abundant in introns than in exons. While the rate of IPA in composite terminal exons and skipped terminal exons expectedly correlates with splicing, we observed a considerable fraction of IPA events that lack AS support and attributed them to spliced polyadenylated introns (SPI). We hypothesize that SPIs represent transient byproducts of a dynamic coupling between APA and AS, in which the spliceosome removes the intron while it is being cleaved and polyadenylated. These findings indicate that cotranscriptional pre-mRNA splicing could serve as a rescue mechanism to suppress premature transcription termination at intronic polyadenylation sites.

CRISPR-iPAS: a novel dCAS13-based method for alternative polyadenylation interference.

Alternative polyadenylation (APA) plays an important role in gene regulation. With the recent application of novel sequencing technology in APA profiling, an ever-increasing number of APA genes/sites have been identified. However, the phenotypic relevance of most of these APA isoforms remains elusive, which is largely due to the lack of a convenient genetics tool for APA interference. To address this issue, herein, an efficient method is developed based on the CRISPR-dCas13 system, termed as CRISPR-iPAS. Out of eight different dCas13 proteins, Porphyromonas gulae (Pgu) dCas13b, is identified as the most effective one in blocking the usage of the polyadenylation site (PAS). With guide RNAs targeting at core regulatory elements, dPguCas13b enabled APA regulation of endogenous genes with different APA types, including tandem 3′UTR, alternative terminal exon, as well as intronic PAS. Finally, we demonstrated that the proposed APA perturbation tool could be used to investigate the functional relevance of APA isoforms.

Heat shock induces premature transcript termination and reconfigures the human transcriptome

SummaryThe heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5′-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.

Androgen signaling connects short isoform production to breakpoint formation at Ewing sarcoma breakpoint region 1.

Ewing sarcoma breakpoint region 1 (EWSR1) encodes a multifunctional protein that can cooperate with the transcription factor ERG to promote prostate cancer. The EWSR1 gene is also commonly involved in oncogenic gene rearrangements in Ewing sarcoma. Despite the cancer relevance of EWSR1, its regulation is poorly understood. Here we find that in prostate cancer, androgen signaling upregulates a 5′ EWSR1 isoform by promoting usage of an intronic polyadenylation site. This isoform encodes a cytoplasmic protein that can strongly promote cell migration and clonogenic growth. Deletion of an Androgen Receptor (AR) binding site near the 5′ EWSR1 polyadenylation site abolished androgen-dependent upregulation. This polyadenylation site is also near the Ewing sarcoma breakpoint hotspot, and androgen signaling promoted R-loop and breakpoint formation. RNase H overexpression reduced breakage and 5′ EWSR1 isoform expression suggesting an R-loop dependent mechanism. These data suggest that androgen signaling can promote R-loops internal to the EWSR1 gene leading to either early transcription termination, or breakpoint formation.

Epidermal progenitors suppress GRHL3-mediated differentiation through intronic polyadenylation promoted by CPSF-HNRNPA3 collaboration

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.

MeCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation.

The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2′-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner. Here we describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, we have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide–alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N6-methyladenosine (m6A), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we show that METTL16 deposits m6A to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation.

Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer

Epigenetic regulation enables tumors to respond to changing environments during tumor progression and metastases and facilitates treatment resistance. Targeting chromatin modifiers or catalytic effectors of transcription is an emerging anti-cancer strategy. The cyclin-dependent kinases (CDKs) 12 and 13 phosphorylate the C-terminal domain of RNA polymerase II, regulating transcription and co-transcriptional processes. Here we report the development of SR-4835, a highly selective dual inhibitor of CDK12 and CDK13, which disables triple-negative breast cancer (TNBC) cells. Mechanistically, inhibition or loss of CDK12/CDK13 triggers intronic polyadenylation site cleavage that suppresses the expression of core DNA damage response proteins. This provokes a "BRCAness" phenotype that results in deficiencies in DNA damage repair, promoting synergy with DNA-damaging chemotherapy and PARP inhibitors.

Suboptimal RNA–RNA interaction limits U1 snRNP inhibition of canonical mRNA 3’ processing

ABSTRACT It is increasingly appreciated that U1 snRNP transcriptomically suppresses the usage of intronic polyadenylation site (PAS) of mRNAs, an outstanding question is why frequently used PASs are not suppressed. Here we found that U1 snRNP could be transiently associated with sequences upstream of actionable PASs in human cells, and RNA–RNA interaction might contribute to the association. By focusing on individual PAS, we showed that the stable assembly of U1 snRNP near PAS might be generally required for U1 inhibition of mRNA 3ʹ processing. Therefore, actionable PASs that often lack optimal U1 snRNP docking site nearby is free from U1 inhibitory effect. Consistently, natural 5ʹ splicing site (5ʹ-SS) is moderately enriched ~250 nt upstream of intronic PASs whose usage is sensitive to functional knockdown of U1 snRNA. Collectively, our results provided an insight into how U1 snRNP selectively inhibits the usage of PASs in a cellular context, and supported a prevailing model that U1 snRNP scans pre-mRNA through RNA–RNA interaction to find a stable interaction site to exercise its function in pre-mRNA processing, including repressing the usage of cryptic PASs.

Abstract 4780: An antisense-based approach to induce anti-tumorigenic variants of MET receptor

Abstract Rational: Aberrant activity of MET receptor, a receptor tyrosine kinase (RTK), is implicated in various forms of cancer. Moreover, functional crosstalk of MET with other RTKs has been reported in tumors and MET has emerged both as a driver and as a primary mechanism of resistance, thus, making MET an attractive target for cancer therapeutics. Aim: Recently, we have described natural soluble decoy variants for multiple RTKs (sdRKTs), including MET, which can antagonize RTK signaling by ligand sequestration and non-functional dimerization. These isoforms can be generated via intronic polyadenylation (IPA) of RTK’s pre-mRNA in a U1-snRNP dependent manner. Our aim is to activate IPA in MET pre-mRNA using antisense compounds and to increase mRNA variants that express dominant-negative soluble decoy MET (sdMET). Methods: Identify actionable IPA sites within introns upstream of the transmembrane-coding exon. Express potential sdMET isoforms and characterize their biological properties. For sdMET isoforms with inhibitory functions, anti-sense compounds are utilized to block the upstream U1 binding site, activate IPA sites, and thus increase expression of sdMET variants in MET-dependent cancer cells. Results: Ectopically expressed sdMET isoforms are secreted into the culture medium. When then added to MET-expressing cells, sdMET variants functioned in a dominant-negative manner over full length MET (FL-MET) and suppressed activation of AKT and MAPK pathways. Moreover, antisense compounds that induce IPA effectively convert oncogenic FL-MET into sdMET variants in multiple cell lines, leading to suppression of downstream pathways and dramatic reduction in cell viability of MET-dependent cells. Conclusion: Most sdMET isoforms are secreted into extracellular space and have inhibitory function. Our anti-sense approach induces IPA, generates truncated sdMET variants at the expense of FL-MET and efficiently blocks FL-MET’s biological activity. Citation Format: Trushar Rathod, Luca Cartegni. An antisense-based approach to induce anti-tumorigenic variants of MET 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 4780.

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes

SUMMARYRegulation of cleavage and polyadenylation (CPA) affects gene expression and polyadenylation site (PAS) choice. Here, we report that the CPA and termination factor PCF11 modulates gene expression on the basis of gene size. Although downregulation of PCF11 leads to inhibition of short gene expression, long genes are upregulated because of suppressed intronic polyadenylation (IPA) enriched in large introns. We show that this regulatory scheme, named PCF11-mediated expression regulation through IPA (PEIPA), takes place in cell differentiation, during which downregulation of PCF11 is coupled with upregulation of long genes with functions in cell morphology, adhesion, and migration. PEIPA targets distinct gene sets in different cell contexts with similar rules. Furthermore, PCF11 is autoregulated through a conserved IPA site, the removal of which leads to global activation of PASs close to gene promotors. Therefore, PCF11 uses distinct mechanisms to regulate genes of different sizes, and its autoregulation maintains homeostasis of PAS usage in the cell.

CDK12 regulates DNA repair genes by suppressing intronic polyadenylation.

Mutations that attenuate homologous recombination (HR) repair promote tumorigenesis and sensitize cells to chemotherapeutics that cause replication fork collapse, a phenotype known as “BRCAness.”1 BRCAness tumors arise from loss-of-function mutations in 22 genes.1 Of these genes, all but one (Cdk12) directly function in the HR repair pathway.1 Cdk12 phosphorylates Serine 2 of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeat,2–7 a modification that regulates transcription elongation, splicing, and cleavage/polyadenylation.8,9 Genome-wide expression studies suggest that Cdk12 depletion abrogates the expression of several HR genes relatively specifically, blunting HR repair.3–7,10,11 This observation suggests that Cdk12 mutational status may predict sensitivity to targeted treatments against BRCAness, such as Parp1 inhibitors, and that Cdk12 inhibitors may induce sensitization of HR-competent tumors to these treatments.6,7,10,11 Despite growing clinical interest, the mechanism by which Cdk12 regulates HR genes remains unknown. Here we find that Cdk12 globally suppresses intronic polyadenylation events, enabling the production of full-length gene products. Many HR genes harbor more intronic polyadenylation sites than other expressed genes, and these sites are particularly sensitive to Cdk12 loss. The cumulative effect of these sites accounts for the enhanced sensitivity of HR gene expression to Cdk12 loss, and we find that this mechanism is conserved in human tumors harboring Cdk12 loss-of-function mutations. This work clarifies the function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumor biomarker.

Abstract 1836: Induction of secreted soluble decoy EGFR variants by splicing interference overcomes drug resistance in human non-small cell lung cancer

Abstract Activation of the epidermal growth factor receptor (EGFR) is involved in the oncogenesis of multiple cancers, particularly lung cancer. Current treatments are limited by the appearance of TKI resistance from secondary somatic mutations in the EGFR gene, such as the T790M mutation (&amp;gt;50% of biopsies) in the EGFR kinase domain following erlotinib or gefitinib treatment. As these tumors are still EGFR-dependent, EGFR remains a prime therapeutic target in NSCLC. We have recently described multiple natural soluble decoy isoforms for multiple RTKs, including EGFR, which depend on the differential inclusion of functional domains by alternative splicing. We employ antisense oligo nucleotide (ASO) to reprogram EGFR pre-mRNA processing in order to increase expression of mRNA variants that encode for dominant-negative, soluble decoys EGFR (sdEGFR), at the expense of the oncogenic full-length EGFR receptor. These alternative splicing isoforms are generated via intronic polyadenylation (IPA) of pre-mRNA in a U1-snRNP (U1)-dependent manner. More specifically, we are able to induce the sdEGFR variants by using specific ASOs, designed to block the upstream U1 binding site and thus activate the appropriate IPA sites. Here, we show that our novel strategy effectively induces the expression of potent natural inhibitors of EGFR signaling in treatment-refractory lung cancer models in vitro and in vivo, leading to suppression of downstream pathways and tumor growth inhibition. The ASO-induced natural sdEGFR compounds function in a dominant-negative manner and induce dramatic cell death in NSCLC cells harboring multiple activating and resistance EGFR mutations, and thus provides a novel alternative strategy for treatment of refractory NSCLC, to overcome resistance mediated by the EGFR secondary mutations (including T790M, C797S and others). Citation Format: Jeong Eun Park, Lee Spraggon, Elisa de Stanchina, Luca Cartegni. Induction of secreted soluble decoy EGFR variants by splicing interference overcomes drug resistance in human non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1836.

Alternative polyadenylation expands the mRNA isoform repertoire of human CD46

Alternative polyadenylation is a prevalent mechanism regulating mammalian gene expression. While tandem 3′-Untranslated-Region (3′UTR) polyadenylation changes expression levels, Intronic PolyAdenylation generates shorter transcripts encoding truncated proteins. Intronic PolyAdenylation regulates 20% of genes and is especially common in receptor tyrosine-kinase transcripts, generating soluble repressors. Here we report that human CD46, encoding a TransMembrane repressor of complement and T-cell co-stimulator, expresses multiple isoforms by alternative polyadenylation. We provide evidence for polyadenylation at several introns by RT-PCR of 5′ intronic fragments, and by increase in such isoforms via functional U1 knockdown. We mapped various Intronic PolyAdenylation Sites by 3′ Rapid Amplification of cDNA Ends (3′RACE), which could generate soluble or membrane-bound but tail-less CD46. Intronic PolyAdenylation could add to the source of soluble CD46 isoforms in fluids and tissues, which increase in cancers and autoimmune syndromes. Furthermore, 3′RACE identified three PolyAdenylation Sites within the last intron and exon, whose transcripts with shortened 3′UTRs could support higher CD46 expression. Finally, 3′RACE revealed that the CD46 Pseudogene only expresses short transcripts by early polyadenylation in intron 2. Overall, we report a wide variety of CD46 mRNA isoforms which could generate new protein isoforms, adding to the diverse physiological and pathological roles of CD46.

An alanine expanded PABPN1 causes increased utilization of intronic polyadenylation sites

In eukaryote genomes, the polyadenylation site marks termination of mature RNA transcripts by a poly-adenine tail. The polyadenylation site is recognized by a dynamic protein complex, among which the poly-adenine-binding protein nuclear1 plays a key role. Reduced poly-adenine-binding protein nuclear1 levels are found in aged muscles and are even lower in oculopharyngeal muscular dystrophy patients. Oculopharyngeal muscular dystrophy is a rare, late onset autosomal dominant myopathy, and is caused by an alanine expansion mutation in poly-adenine-binding protein nuclear1. Mutant poly-adenine-binding protein nuclear1 forms insoluble nuclear aggregates leading to depletion of functional poly-adenine-binding protein nuclear1 levels. In oculopharyngeal muscular dystrophy models, increased utilization of proximal polyadenylation sites has been observed in tandem 3′-untranslated regions, and most often cause gene upregulation. However, global alterations in expression profiles canonly partly be explained by polyadenylation site switches within the most distal 3′-untranslated region. Most poly-adenine signals are found at the distal 3′-untranslated region, but a significant part is also found in internal gene regions, like introns, exons, and internal 3′-untranslated regions. Here, we investigated poly-adenine-binding protein nuclear1’s role in polyadenylation site utilization in internal gene regions. In the quadriceps muscle of oculopharyngeal muscular dystrophy mice expressing expPABPN1 we found significant polyadenylation site switches between gene regions in 17% of genes with polyadenylation site in multiple regions (N = 574; 5% False Discovery Rate). Polyadenylation site switches between gene regions were associated with differences in transcript expression levels and alterations in open reading frames. Transcripts ending at internal polyadenylation site were confirmed in tibialis anterior muscles from the same mice and in mouse muscle cell cultures overexpressing expPABPN1. The polyadenylation site switches were associated with nuclear accumulation of full-length transcripts. Our results provide further insights into the diverse roles of poly-adenine-binding protein nuclear1 in the post-transcriptional control of muscle gene expression and its relevance for oculopharyngeal muscular dystrophy pathology and muscle aging.

Abstract IA29: Role of eIF4F in anticancer drug resistance

Abstract Recent work, including large-scale genetic and molecular analyses, identified RNA-binding proteins (RBPs) as major players in the hallmarks of cancer. Specific RBPs allow the selective regulation of cancer genes at multiple post-transcriptional levels from pre-mRNA splicing/polyadenylation to mRNA stability/translation. These multiple activities are mediated by RBP binding to mRNAs. In recent years, we have studied how the eIF4F translation initiation factor bound to the 7-methylguanosine cap structure present at the 5'-end of all cellular mRNAs contributes to tumorigenesis with a focus on its role in chemoresistance as well as the promising use of new small molecule inhibitors of the complex, including flavaglines/rocaglates, hippuristanol and pateamine A. We have specifically shown that, due to its location downstream of the the PI(3)K/AKT/mTOR pathway and the RAS-RAF-MEK-ERK-MNK mitogen-activated protein kinase (MAPK) signal transduction pathways, eIF4F is a nexus of resistance to anti-BRAF and anti-MEK therapies in both BRAF-mutated (melanoma, colon, thyroid) and NRAS-mutated (melanoma) cancer cells. Furthermore, inhibiting the eIF4A component of the eIF4F complex, with a novel flavagline (FL3) that is insensitive to P-glycoprotein-mediated multidrug resistance, synergizes with inhibiting BRAF and MEK to kill BRAF-mutant cancer cells and synergizes with inhibiting MEK to kill NRAS-mutant cancer cells in melanoma. In parallel, while investigating a set of data based on high-throughput sequencing of polyadenylated transcripts 3'-ends (3'-Seq), we have found that a short eIF4E mRNA isoform is generated through alternative use on an intronic polyadenylation (IPA) site. The global generation of transcripts with shorter 3' untranslated region (3'UTR) is known to occur during enhanced cell proliferation and transformation into cancer cells. However, the involvement of alternative polyadenylation in the response to targeted therapies and in the spreading of cancer cells to metastatic sites is unknown. IPA site usage is known to be widely inhibited by the U1 small ribonucleoprotein particle (U1) bound to an adjacent 5' splice site (5'ss). We have found that targeting U1 with an antisense oligonucleotide (U1-ASO) leads to activation of the use of IPA sites in the eIF4E gene but also in several genes with functions enriched in cell movement and involved in the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) and PI(3)K-AKT-mTOR signal transduction pathways. We have also demonstrated that the specificity of IPA regulation by U1 during tumor cell spreading is provided by the regulation of its binding to specific 5'ss by the RBP and U1-interacting splicing factor TIA1. The significance of these findings in tumor cell spreading and in the response to targeted therapies will be discussed. Citation Format: Galina Boldina, Maricarmen Vallejos, Delphine Allard, Isabelle Girault, Hélène Malka-Mahieu, Laurent Désaubry, Martin Dutertre, Caroline Robert, Stéphan Vagner. Role of eIF4F in anticancer drug resistance. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA29.