Splice Forms Research Articles

Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5′-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Experimental Study on the Structural Performance of Reinforced Truss Concrete Composite Slabs during and after Fire

Standard fire resistance tests and post-fire tests on residual mechanical properties were carried out on two four-edge simply supported reinforced truss concrete two-way composite slabs with wide seam splicing (“integral seam”) and close seam splicing (“separated seam”), and the effects of the different splicing forms on the mechanical properties of the reinforced truss concrete composite slabs during and after exposure to high temperatures were explored. The results indicate that there are significant differences in terms of crack developments, moisture loss times on the slab surface, spalling characteristics of the bottom of the slab and cross-section temperature gradients of concrete and steel reinforcement along the thickness direction between the wide seam spliced reinforced truss concrete composite two-way slab (referred to as “S1 composite slab”) and the close seam spliced reinforced truss concrete composite two-way slab (referred to as “S2 composite slab”) in the fire test. This is mainly due to differences in the splicing forms and structural measures of the prefabricated base slabs at joints, which led to changes in the heat transfer path at the joints. As the temperature at the close seam can be released quickly, the stiffness recovery of the S2 composite slab is significantly greater than the stiffness weakening due to the thermal hysteresis of the concrete, so the deflection of the S2 composite slab recovers immediately after the fire stops. Overall, both the S1 and S2 composite slabs exhibit two-way high temperature deformation characteristics. The post-fire residual static load tests show that the static load-carrying capacity of the S1 composite slab is 12.08% higher than that of the S2 composite slab and the ultimate displacement is 8.74% lower than that of the S2 composite slab. It is appropriate to calculate the residual load capacities of the S1 and S2 composite slabs after the fire as a one-way slab.

Two splice forms of OsbZIP1, a homolog of AtHY5, function to regulate skotomorphogenesis and photomorphogenesis in rice.

Plants possess well-developed light sensing mechanisms and signal transduction systems for regulating photomorphogenesis. ELONGATED HYPOCOTYL5 (HY5), a basic leucine zipper (bZIP) transcription factor, has been extensively characterized in dicots. In this study, we show that OsbZIP1 is a functional homolog of Arabidopsis (Arabidopsis thaliana) HY5 (AtHY5) and is important for light-mediated regulation of seedling and mature plant development in rice (Oryza sativa). Ectopic expression of OsbZIP1 in rice reduced plant height and leaf length without affecting plant fertility, which contrasts with OsbZIP48, a previously characterized HY5 homolog. OsbZIP1 is alternatively spliced, and the OsbZIP1.2 isoform lacking the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-binding domain regulated seedling development in the dark. Rice seedlings overexpressing OsbZIP1 were shorter than the vector control under white and monochromatic light conditions, whereas RNAi knockdown seedlings displayed the opposite phenotype. While OsbZIP1.1 was light-regulated, OsbZIP1.2 showed a similar expression profile in both light and dark conditions. Due to its interaction with OsCOP1, OsbZIP1.1 undergoes 26S proteasome-mediated degradation under dark conditions. Also, OsbZIP1.1 interacted with and was phosphorylated by CASEIN KINASE2 (OsCK2α3). In contrast, OsbZIP1.2 did not show any interaction with OsCOP1 or OsCK2α3. We propose that OsbZIP1.1 likely regulates seedling development in the light, while OsbZIP1.2 is the dominant player under dark conditions. The data presented in this study reveal that AtHY5 homologs in rice have undergone neofunctionalization, and alternative splicing of OsbZIP1 has increased the repertoire of its functions.

Elucidating the Mesocarp Drupe Transcriptome of Açai (Euterpe oleracea Mart.): An Amazonian Tree Palm Producer of Bioactive Compounds.

Euterpe oleracea palm, endemic to the Amazon region, is well known for açai, a fruit violet beverage with nutritional and medicinal properties. During E. oleracea fruit ripening, anthocyanin accumulation is not related to sugar production, contrarily to grape and blueberry. Ripened fruits have a high content of anthocyanins, isoprenoids, fibers, and proteins, and are poor in sugars. E. oleracea is proposed as a new genetic model for metabolism partitioning in the fruit. Approximately 255 million single-end-oriented reads were generated on an Ion Proton NGS platform combining fruit cDNA libraries at four ripening stages. The de novo transcriptome assembly was tested using six assemblers and 46 different combinations of parameters, a pre-processing and a post-processing step. The multiple k-mer approach with TransABySS as an assembler and Evidential Gene as a post-processer have shown the best results, with an N50 of 959 bp, a read coverage mean of 70x, a BUSCO complete sequence recovery of 36% and an RBMT of 61%. The fruit transcriptome dataset included 22,486 transcripts representing 18 Mbp, of which a proportion of 87% had significant homology with other plant sequences. Approximately 904 new EST-SSRs were described, and were common and transferable to Phoenix dactylifera and Elaeis guineensis, two other palm trees. The global GO classification of transcripts showed similar categories to that in P. dactylifera and E. guineensis fruit transcriptomes. For an accurate annotation and functional description of metabolism genes, a bioinformatic pipeline was developed to precisely identify orthologs, such as one-to-one orthologs between species, and to infer multigenic family evolution. The phylogenetic inference confirmed an occurrence of duplication events in the Arecaceae lineage and the presence of orphan genes in E. oleracea. Anthocyanin and tocopherol pathways were annotated entirely. Interestingly, the anthocyanin pathway showed a high number of paralogs, similar to in grape, whereas the tocopherol pathway exhibited a low and conserved gene number and the prediction of several splicing forms. The release of this exhaustively annotated molecular dataset of E. oleracea constitutes a valuable tool for further studies in metabolism partitioning and opens new great perspectives to study fruit physiology with açai as a model.

SIRT3 alleviates imiquimod-induced psoriatic dermatitis through deacetylation of XBP1s and modulation of TLR7/8 inducing IL-23 production in macrophages.

Current evidence suggests that IL-23, IL-6, and TNF-α play pivotal roles in the pathogenesis of psoriasis. Although it has been established that Sirtuin 3 (SIRT3) mediates the inflammatory process, the underlying mechanisms remain largely unclear. Herein, we substantiated that the inhibition or deletion of SIRT3 increased the acetylation level of spliced form of X-box binding protein 1 (XPB1s), enhancing its transcriptional activity and IL-23a production. Pharmacologically inhibition of XBP1s with MKC8866 downregulated the expression of inflammatory cytokines in SIRT3-inhibited or Sirt3-KO BMDMs stimulated by IMQ. Inhibition or knockdown of SIRT3 could exacerbate psoriasis-like skin inflammation in an imiquimod-induced psoriasis-like mouse model. Besides, a decrease in SIRT3 expression was observed in the macrophages of psoriasis patients, which increased the expression and acetylation level of XBP1s. Overall, we provide compelling evidence of the crucial role of SIRT3 in the IL-23 axis in psoriatic inflammation and novel molecular insights into the anti-inflammatory effects of SIRT3.

Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Multi-omics analysis of the Indian ovarian cancer cohort revealed histotype-specific mutation and gene expression patterns.

Introduction: In India, OVCa is women's third most common and lethal cancer type, accounting for 6.7% of observed cancer incidences. The contribution of somatic mutations, aberrant expression of gene and splice forms in determining the cell fate, gene networks, tumour-specific variants, and the role of immune fraction infiltration have been proven essential in understanding tumorigenesis. However, their interplay in OVCa in a histotype-specific manner remains unclear in the Indian context. In the present study, we aimed to unravel the Indian population histotype-specific exome variants, differentially expressed gene modules, splice events and immune profiles of OVCa samples. Methods: We analysed 10 tumour samples across 4 ovarian cancer histotypes along with 2 normal patient samples. This included BCFtool utilities and CNVkit for exome, WGCNA and DESeq2 for obtaining differential module hub genes and dysregulated miRNA targets, CIBERSORTx for individual immune profiles and rMATS for tumour specific splice variants. Result: We identified population-specific novel mutations in Cancer Gene Census Tier1 and Tier2 genes. MUC16, MUC4, CIITA, and NCOR2 were among the most mutated genes, along with TP53. Transcriptome analysis showed significant overexpression of mutated genes MUC16, MUC4, and CIITA, whereas NCOR2 was downregulated. WGCNA revealed histotype-specific gene hubs and networks. Among the significant pathways, alteration in the immune system was one of the pathways, and immune profiling using CIBERSORTx revealed histotype-specific immune cell fraction. miRNA analysis revealed miR-200 family, miR-200a and miR-429 were upregulated in HGSOCs.Splice factor abrasion caused splicing perturbations, with the most abundant alternative splice event being exon skipping and the most spliced gene, SNHG17. Pathway analysis of spliced genes revealed translational elongation and Base excision repair as the pathways altered in OVCa. Conclusion: Integrated exome, transcriptome, and splicing patterns revealed different population-specific molecular signatures of ovarian cancer histotypes in the Indian Cohort.

Abnormalities in intron retention characterize patients with systemic lupus erythematosus

Regulation of intron retention (IR), a form of alternative splicing, is a newly recognized checkpoint in gene expression. Since there are numerous abnormalities in gene expression in the prototypic autoimmune disease systemic lupus erythematosus (SLE), we sought to determine whether IR was intact in patients with this disease. We, therefore, studied global gene expression and IR patterns of lymphocytes in SLE patients. We analyzed RNA-seq data from peripheral blood T cell samples from 14 patients suffering from systemic lupus erythematosus (SLE) and 4 healthy controls and a second, independent data set of RNA-seq data from B cells from16 SLE patients and 4 healthy controls. We identified intron retention levels from 26,372 well annotated genes as well as differential gene expression and tested for differences between cases and controls using unbiased hierarchical clustering and principal component analysis. We followed with gene-disease enrichment analysis and gene-ontology enrichment analysis. Finally, we then tested for significant differences in intron retention between cases and controls both globally and with respect to specific genes. Overall decreased IR was found in T cells from one cohort and B cells from another cohort of patients with SLE and was associated with increased expression of numerous genes, including those encoding spliceosome components. Different introns within the same gene displayed both up- and down-regulated retention profiles indicating a complex regulatory mechanism. These results indicate that decreased IR in immune cells is characteristic of patients with active SLE and may contribute to the abnormal expression of specific genes in this autoimmune disease.

Coordinated microRNA/mRNA Expression Profiles Reveal Unique Skin Color Regulatory Mechanisms in Chinese Giant Salamander (Andrias davidianus).

The Chinese giant salamander (Andrias davidianus) has been increasingly popular in the aquaculture market in China in recent years. In the breeding process of Andrias davidianus, we found that some albino individuals were extremely rare and could not be inherited stably, which severely limits their commercialization in the aquaculture market. In this study, we performed transcriptome and small RNA (sRNA) sequencing analyses in the skin samples of wild-type (WT) and albino (AL) Andrias davidianus. In total, among 5517 differentially expressed genes (DEGs), 2911 DEGs were down-regulated in AL, including almost all the key genes involved in melanin formation. A total of 25 miRNAs were differentially expressed in AL compared to WT, of which 17 were up-regulated. Through the integrated analysis, no intersection was found between the target genes of the differentially expressed miRNAs and the key genes for melanin formation. Gene Ontology (GO) and KEGG pathway analyses on DEGs showed that these genes involved multiple processes relevant to melanin synthesis and the key signal pathway MAPK. Interestingly, the transcription factors SOX10 and PAX3 and the Wnt signaling pathway that play a key role in other species were not included, while the other two transcription factors in the SOX family, SOX21 and SOX7, were included. After analyzing the key genes for melanin formation, it was interesting to note an alternative splicing form of the MITF in WT and a critical mutation of the SLC24A5 gene in AL, which might be the main reason for the skin color change of Andrias davidianus. The results contributed to understanding the molecular mechanism of skin pigmentation in Andrias davidianus and accelerating the acquisition process of individuals with specific body colors by genetic means.

Targeting XBP1 mRNA splicing sensitizes glioblastoma to chemotherapy.

Glioblastoma (GBM) is the most frequent and deadly primary brain tumor in adults. Temozolomide (TMZ) is the standard systemic therapy in GBM but has limited and restricted efficacy. Better treatments are urgently needed. The role of endoplasmic reticulum stress (ER stress) is increasingly described in GBM pathophysiology. A key molecular mediator of ER stress, the spliced form of the transcription factor x-box binding protein 1 (XBP1s) may constitute a novel therapeutic target; here we report XBP1s expression and biological activity in GBM. Tumor samples from patients with GBM (n= 85) and low-grade glioma (n= 20) were analyzed by immunohistochemistry for XBP1s with digital quantification. XBP1s expression was significantly increased in GBM compared to low-grade gliomas. XBP1s mRNA showed upregulation by qPCR analysis in a panel of patient-derived GBM cell lines. Inhibition of XBP1 splicing using the small molecular inhibitor MKC-3946 significantly reduced GBM cell viability and potentiated the effect of TMZ in GBM cells, particularly in those with methylated O6-methylguanine-DNA methyl transferase gene promoter. GBM cells resistant to TMZ were also responsive to MKC-3946 and the long-term inhibitory effect of MKC-3946 was confirmed by colony formation assay. In conclusion, this data reveals that XBP1s is overexpressed in GBM and contributes to cancer cell growth. XBP1s warrants further investigation as a clinical biomarker and therapeutic target in GBM.

Investigating the differences in metabolism, and the unfolded protein and apoptosis gene responses in two strains of the Pacific white shrimp, Penaeus vannamei Boone, 1931 (Decapoda, Penaeoidea) under ammonia stress

Abstract Ammonia is one of the major environmental pollutants in the aquatic system and poses a great threat to shrimp. In this study, we compared the differences in metabolic enzymes and the mRNA expression of unfolded protein response (UPR)-related genes between the ammonia-resistant (LV-AT) and ammonia-sensitive (LV-AS) strains of Penaeus vannamei, aiming to investigate the different tolerance mechanisms of the two extreme strains underlying ammonia stress. The results showed that elevated ammonia concentrations mainly increased glutamine synthetase (GS) content while inhibiting transglutaminase (TG) content in the muscle of both strains and the contents were significantly higher in LV-AT than in LV-AS after 48 h of ammonia stress (). As a protective or adaptive strategy, the expression of the UPR related genes, including activating transcription factors ATF6, ATF4, and the spliced form of X box binding protein 1 (XBP1) were, respectively, either significantly induced, or inhibited () after ammonia stress. The mRNA expressions of the GRP78 (Bip) gene and the apoptosis gene (caspase 3) are then activated. In hepatopancreas and gills, the associated gene expressions gradually increase in LV-AT, while several related genes in LV-AS were first induced and then gradually inhibited. The expressions of key genes (GRP78, ATF6, XBP1 and ATF4) were significantly higher in LV-AT than that in LV-AS after 48 h of ammonia stress (). These results indicate that some enzymes involved in ammonia metabolism and UPR protection genes tended to be highly expressed in LV-AT. Hence, we speculate that LV-AT has a higher ammonia excretion capacity and a more effective adaptive mechanism after ammonia stress. The experimental results will help to further understand the stress response of P. vannamei to ammonia and provide a new perspective for the underlying molecular mechanism of the ammonia adaptation strategy of at least some strains of P. vannamei.

CPSF6-mediated XBP1 3’UTR shortening attenuates cisplatin-induced ER stress and elevates chemo-resistance in lung adenocarcinoma

Alternative polyadenylation (APA) is a widespread mechanism generating RNA molecules with alternative 3' ends. Herein, we discovered that TargetScan includes a novel XBP1 transcript with a longer 3' untranslated region (UTR) (XBP1-UL) than that included in NCBI. XBP1-UL exhibited a lowered level in blood samples from lung adenocarcinoma (LUAD) patients and in those after DDP treatment. Consistently, XBP1-UL was reduced in A549 cells compared to normal BEAS-2B cells, as well as in DDP-treated/resistant A549 cells relative to controls. Moreover, due to decreased usage of the distal polyadenylation site (PAS) in 3’UTR, XBP1-UL level was lowered in A549 cells and decreased further in DDP-resistant A549 (A549/DDP) cells. Importantly, use of the distal PAS (dPAS) and XBP1-UL level were gradually reduced in A549 cells under increasing concentrations of DDP, which was attributed to DDP-induced endoplasmic reticulum (ER) stress. Furthermore, XBP1 transcripts with shorter 3’UTR (XBP1-US) were more stable and presented stronger potentiation on DDP resistance. The choice of proximal PAS (pPAS) was attributed to CPSF6 elevation, which was caused by BRCA1-distrupted R-loop accumulation in CPSF6 5’end. DDP-induced nuclear LINC00221 also facilitated CPSF6-induced pPAS choice in the pre-XBP1 3’end. Finally, we found that unlike the unspliced XBP1 protein (XBP1-u), the spliced form XBP1-s retarded p53 degradation to facilitate DNA damage repair of LUAD cells. The current study provides new insights into tumor progression and DDP resistance in LUAD, which may contribute to improved LUAD treatment.

The activation of spliced X-box binding protein 1 by isorhynchophylline therapy improves diabetic encephalopathy.

The primary symptom of diabetic encephalopathy (DE), a kind of central diabetic neuropathy caused by diabetes mellitus (DM), is cognitive impairment. In addition, the tetracyclic oxindole alkaloid isorhynchophylline (IRN) helps lessen cognitive impairment. However, it is still unclear how IRN affects DM and DE and what mechanisms are involved. The effectiveness of IRN on brain insulin resistance was carefully examined in this work, both in vitro and in vivo. We found that IRN accelerates spliced form of X-box binding protein 1 (sXBP1) translocation into the nucleus under high glucose conditions in vitro. IRN also facilitates the nuclear association of pCREB with sXBP1 and the binding of regulatory subunits of phosphatidylinositol 3-kinase (PI3K) p85α or p85β with XBP1 to restore high glucose impairment. Also, IRN treatment improves high glucose-mediated impairment of insulin signaling, endoplasmic reticulum stress, and pyroptosis/apoptosis by depending on sXBP1 in vitro. In vivo studies suggested that IRN attenuates cognitive impairment, ameliorating peripheral insulin resistance, activating insulin signaling, inactivating activating transcription factor 6 (ATF6) and C/EBP homology protein (CHOP), and mitigating pyroptosis/apoptosis by stimulation of sXBP1 nuclear translocation in the brain. In summary, these data indicate that IRN contributes to maintaining insulin homeostasis by activating sXBP1 in the brain. Thus, IRN is a potent antidiabetic agent as well as an sXBP1 activator that has promising potential for the prevention or treatment of DE.

A CRISPR/Cas9-mediated screen identifies determinants of early plasma cell differentiation.

The differentiation of B cells into antibody-secreting plasma cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood. We have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells. Among known genes whose deletion preferentially or mostly affected plasmablast formation were the transcription factors Prdm1 (BLIMP1), Irf4 and Pou2af1 (OBF-1), and the Ern1 gene encoding IRE1a, while deletion of XBP1, the transcriptional master regulator that specifies the expansion of the secretory program in plasma cells, had no effect. Defective plasmablast formation caused by Ern1 deletion could not be rescued by the active, spliced form of XBP1 whose processing is dependent on and downstream of IRE1a, suggesting that in early plasma cell differentiation IRE1a acts independently of XBP1. Moreover, we newly identified several genes involved in NF-kB signaling (Nfkbia), vesicle trafficking (Arf4, Preb) and epigenetic regulators that form part of the NuRD complex (Hdac1, Mta2, Mbd2) to be required for plasmablast formation. Deletion of ARF4, a small GTPase required for COPI vesicle formation, impaired plasmablast formation and blocked antibody secretion. After Hdac1 deletion plasmablast differentiation was consistently reduced by about 50%, while deletion of the closely related Hdac2 gene had no effect. Hdac1 knock-out led to strongly perturbed protein expression of antagonistic transcription factors that govern plasma cell versus B cell identity (by decreasing IRF4 and BLIMP1 and increasing BACH2 and PAX5). Taken together, our results highlight specific and non-redundant roles for Ern1, Arf4 and Hdac1 in the early steps of plasma cell differentiation.

Investigation of the effect of SRSF9 overexpression on HIV-1 production

Serine-arginine-rich splicing factors (SRSFs) are members of RNA processing proteins in the serine-arginine-rich (SR) family that could regulate the alternative splicing of the human immunodeficiency virus-1 (HIV-1). Whether SRSF9 has any effect on HIV-1 regulation requires elucidation. Here, we report for the first time the effects and mechanisms of SRSF9 on HIV-1 regulation. The overexpression of SRSF9 inhibits viral production and infectivity in both HEK293T and MT-4 cells. Deletion analysis of SRSF9 determined that the RNA regulation motif domain of SRSF9 is important for anti-HIV-1 effects. Furthermore, overexpression of SRSF9 increases multiple spliced forms of viral mRNA, such as Vpr mRNA. These data suggest that SRSF9 overexpression inhibits HIV-1 production by inducing the imbalanced HIV-1 mRNA splicing that could be exploited further for a novel HIV-1 therapeutic molecule. [BMB Reports 2022; 55(12): 639-644].

A clinically relevant mouse model of the Trem2*R47H Alzheimer’s disease risk variant

AbstractBackgroundThe TREM2 (triggering receptor expressed on myeloid cells 2) locus harbors numerous relatively rare variants associated with Alzheimer’s disease risk. The TREM2*R47H allele is one of the strongest genetic risk factors for developing late‐onset Alzheimer’s disease; patients exhibit both earlier onset of symptoms and rapid cognitive decline. TREM2 is expressed by microglia in the brain and plays a key role in modulating microglial activity. TREM2 expression is increased in AD and is associated with recruitment of microglia to amyloid plaques. An experimental model is therefore critical to study mechanisms of late‐onset AD, and the MODEL‐AD set out to create a clinically relevant mouse model.MethodWe used CRISPR to engineer the R47H variant into the mouse Trem2 locus; this model is available as JAX #27918. This model, as well as two other CRISPR‐generated Trem2*R47H models, display a splicing event seen in mice, but not humans, that leads to a novel splice form with a deletion of 119bp at the 5’ end of exon 2 (Xiang et al, Molecular Neurodegeneration, 2018). This leads to a decrease in ∼50% of both transcript and protein levels in brain homogenate in the mouse, indicating that the model expresses mutant Trem2 protein, but is also a hypomorph. To generate a more clinically relevant model, we have modified the existing Trem2*R47H mouse allele by using CRISPR to humanize the exon 2 splice site. Expression in these models was compared to normal Trem2 levels using both RNA‐seq and Western blot.ResultThe new model, which we are calling Trem2*R47HHSS for humanized splice site (JAX #33781), showed normal levels of expression. Relative to B6J controls, this new model expresses normal levels of each of the four transcripts. No truncated transcript was detected. Protein levels were restored to wild‐type levels in the model with the humanized splice site.ConclusionWe expect that this model Trem2*R47H model will facilitate research into the mechanisms and pathways related to TREM2 genetic risk in late‐onset AD. For more information see www.model-ad.org.

Preclinical Translational Research Suggests a Clinical Trial Strategy for a Novel MALT1 Inhibitor ONO-7018/CTX-177 Against Malignant Lymphomas

Introduction Malignant lymphomas, relapsed or refractory B Cell Non-Hodgkin Lymphoma like B-cell diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), are still clinically intractable as patients with these lymphomas carry a dismal prognosis. Genome sequence analysis data on these lymphomas suggest that the gene mutations of mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) -upstream molecules, such as CD79A/B and CARD11, eventually promotes the activation of MALT1's protease activity; this protease activity of MALT1 is known as a key growth or survival regulator for intractable lymphomas that carry these mutations. Previously, we reported the generation of a novel small molecule inhibitor, CTX-177 (currently renamed to ONO-7018 by Ono Pharmaceutical Co., Ltd.), which acts on MALT1 with high potency and specificity. In addition, we showed that ONO-7018 demonstrated preclinical efficacy along with target engagement in several lymphoma models (Morishita D et al., Blood (2020) 136 (Supplement 1): 3-4.). Results To identify potential strategies for a clinical trial of ONO-7018, we conducted translational research to elucidate (1) combination strategies with other key medications, (2) patient selection biomarkers to illuminate sensitivity to ONO-7018 and (3) pharmacodynamics (PD) markers to monitor the suppression of MALT1. After searching for combination strategies (1), ONO-7018 exhibited synergistic combination effect with a BTK inhibitor in vitro along with target inhibition. In addition, the MALT1 inhibitor showed an anti-tumor effect against an ABC-DLBCL cell line derived xenograft (CDX) model. Thus, combination treatment with BTK inhibitor is expected to be the treatment strategy of ONO-7018. Next, regarding biomarkers of sensitivity (2), we found that ONO-7018 selectively inhibits the in vitro growth of cell lines expressing cleaved forms of MALT1 substrates. Additionally, in vivo data from CDX and patient derived xenograft model were consistent with in vitro data. Interestingly, CRISPR-Cas9 mediated A20 knockout attenuates the sensitivity of cell line model toward ONO-7018 treatment. Given these findings, these cleaved forms of MALT1 substrates and the loss of function gene mutation on A20 could become biomarkers to illuminate sensitivity to ONO-7018. Finally, to identify pharmacodynamic markers of ONO-7018 (3), panel analysis of cytokine production following CD3/CD28 stimulation was conducted. Before starting this experiment, we confirmed that ONO-7018 could inhibit the cellular activity of the two T-cell receptor signaling stimulated forms of MALT1 splice variants. Consistent with the inhibitory mode of ONO-7018 on these MALT1 splicing forms, some cytokines’ production was clearly abrogated; these cytokines could be potential PD markers in a clinical trial. Conclusion The results from these translational research studies support the preparation of a clinical trial by clearly defining patient stratification biomarkers, combination strategies, and PD monitoring. These data underscore the potential therapeutic impact of ONO-7018 as a single-agent or combination partner with other inhibitors like BTK inhibitor and elucidate the sensitivity and response of ONO-7018 for the treatment of malignant lymphomas.

LBODP078 Role Of Ire1a-xbp1 Pathway In Rodent Functional Pituitary Tumor Cells.

Abstract Introduction Pituitary tumors (PTs) occur in almost 17% of the population and they represent about 10% of all intracranial tumors. At present, many patients with pituitary tumors suffer from symptoms and complications caused by abnormal hormone production and/or mass effects due to limited treatment options. Therefore, the development of novel therapies is an urgent issue. The inositol requiring enzyme 1a (IRE1a)-X-box binding protein 1 (XBP1) pathway is an important component for the endoplasmic reticulum (ER) stress response. This pathway is activated by immature protein overload in the ER and elimination or repair of such protein can reduce ER stress. This pathway has been reported to regulate cancer cell growth and secretory protein production. The detection of potential XBP1 binding sites in the promoter region of rat GH and mouse POMC similar to those of human genes suggests the involvement of the IRE1a-XBP1 pathway in the production of these hormones and pituitary tumor cell growth. In this study, we aimed to clarify the role of the IRE1a-XBP1 pathway in functional pituitary tumor cells. Methods and Results We conducted in vitro experiments by using rat GH-producing pituitary GH4 cells and mouse ACTH-producing AtT-20 cells as the models of GH- and ACTH-producing pituitary adenomas, respectively. Cell proliferation was measured by WST-8 assay, gene expression levels by qPCR, and protein expression by western blotting, respectively. In GH4 cells, STF08310 (40µM), an IRE1a inhibitor, reduced levels of GH and XBP1 spliced form (XBP1s) mRNA and GH protein with suppressed cell proliferation. In AtT-20 cells, STF08310 (40µM) also suppressed protein expression of proopiomelanocortin (POMC) and cell proliferation. Gene suppression of IRE1a by siRNA and over-expression of XBP1s showed marked reduction and increase of POMC protein, respectively. Conclusion Our findings suggest that the IRE1a-XBP1s pathway would be a possible therapeutic target that has both anti-tumoral and hormone control effects against functional pituitary tumor. Presentation: No date and time listed

Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

Oncogenic transformation leads to changes in glutamine metabolism that make transformed cells highly dependent on glutamine for anabolic growth and survival. Herein, we investigated the cell death mechanism activated in glutamine-addicted tumor cells in response to the limitation of glutamine metabolism. We show that glutamine starvation triggers a FADD and caspase-8-dependent and mitochondria-operated apoptotic program in tumor cells that involves the pro-apoptotic TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2), but is independent of its cognate ligand TRAIL. In glutamine-depleted tumor cells, activation of the amino acid-sensing general control nonderepressible-2 kinase (GCN2) is responsible for TRAIL-R2 upregulation, caspase-8 activation, and apoptotic cell death. Interestingly, GCN2-dependent ISR signaling induced by methionine starvation also leads to TRAIL-R2 upregulation and apoptosis. Moreover, pharmacological inhibition of transaminases activates a GCN2 and TRAIL-R2-dependent apoptotic mechanism that is inhibited by non-essential amino acids (NEAA). In addition, metabolic stress upon glutamine deprivation also results in GCN2-independent FLICE-inhibitory protein (FLIP) downregulation facilitating caspase-8 activation and apoptosis. Importantly, downregulation of the long FLIP splice form (FLIPL) and apoptosis upon glutamine deprivation are inhibited in the presence of a membrane-permeable α-ketoglutarate. Collectively, our data support a model in which limiting glutamine utilization in glutamine-addicted tumor cells triggers a previously unknown cell death mechanism regulated by GCN2 that involves the TRAIL-R2-mediated activation of the extrinsic apoptotic pathway.

Exploring Transcriptomic Landscapes in Red Blood Cells, in Their Extracellular Vesicles and on a Single-Cell Level.

Being enucleated, RBCs lack typical transcriptomes, but are known to contain small amounts of diverse long transcripts and microRNAs. However, the exact role and importance of these RNAs are lacking. Shedding of extracellular vesicles (EVs) from the plasma membrane constitutes an integral mechanism of RBC homeostasis, by which RBCs remove unnecessary cytoplasmic content and cell membrane. To study this further, we explored the transcriptomes of RBCs and extracellular vesicles (EVs) of RBCs using next-generation sequencing. Furthermore, we performed single-cell RNA sequencing on RBCs, which revealed that approximately 10% of the cells contained detectable levels of mRNA and cells formed three subpopulations based on their transcriptomes. A decrease in the mRNA quantity was observed across the populations. Qualitative changes included the differences in the globin transcripts and changes in the expression of ribosomal genes. A specific splice form of a long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), was the most enriched marker in one subpopulation of RBCs, co-expressing with ribosomal structural transcripts. MALAT1 expression was confirmed by qPCR in CD71-enriched reticulocytes, which were also characterized with imaging flow cytometry at the single cell level. Analysis of the RBC transcriptome shows enrichment of pathways and functional categories required for the maturation of reticulocytes and erythrocyte functions. The RBC transcriptome was detected in their EVs, making these transcripts available for intercellular communication in blood.