Persistent Expression Research Articles

A potyvirus provides an efficient viral vector for gene expression and functional studies in Asteraceae plants.

Plant virus-derived vectors are rapid and cost-effective for protein expression and gene functional studies in plants, particularly for species that are difficult to genetically transform. However, few efficient viral vectors are available for functional studies in Asteraceae plants. Here, we identified a potyvirus named zinnia mild mottle virus (ZiMMV) from common zinnia (Zinnia elegans Jacq.) through next-generation sequencing. Using a yeast homologous recombination strategy, we established a full-length infectious cDNA clone of ZiMMV under the control of the cauliflower mosaic virus 35S promoter. Furthermore, we developed an efficient expression vector based on ZiMMV for the persistent and abundant expression of foreign proteins in the leaf, stem, root, and flower tissues with mild symptoms during viral infection in common zinnia. We showed that the ZiMMV-based vector can express ZeMYB9, which encodes a transcript factor inducing dark red speckles in leaves and flowers. Additionally, the expression of a gibberellic acid (GA) biosynthesis gene from the ZiMMV vector substantially accelerated plant height growth, offering a rapid and cost-effective method. In summary, our work provides a powerful tool for gene expression, functional studies, and genetic improvement of horticultural traits in Asteraceae plant hosts.

MDB-45. THE PTBP2 SPLICING FACTOR IS ESSENTIAL IN GROUP 3/4 MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is a common malignant pediatric brain tumor with significant heterogeneity among its four molecular subgroups: WNT, SHH, groups 3 and 4. There is a significant rate of disease recurrence with group 3/4 MB indicating a need for novel therapeutic targets. Since transcriptomic profiling of MB has not identified novel targets, we focused on a proteomic approach. We selected 157 proteins significantly over-expressed in Group 3/4 MB and performed a targeted loss-of-function CRISPR-Cas9 screen in four MB cell lines. We identified 17 essential genes shared by at least 2 of the cell lines. The PTBP2 splicing factor was essential for all cell lines. PTBP2 is an RNA-binding protein involved in splicing regulation that plays an important role during neuronal maturation. We hypothesize that persistent expression of PTBP2 results in a differentiation arrest and preservation of proliferative potential. METHODS We stably knocked-out PTBP2 expression in D556 and MB002 MB cells. We performed bulk RNA-seq and quantitative proteomics of these KO clones along with CLIP-seq to map PTBP2 targeted transcripts. We also performed phenotype assays, qPCR array and single-cell RNA-seq in KO clones to validate proliferation and neuronal differentiation. RESULTS RNA-seq in PTBP2 KO MB cells revealed 2,213 differentially expressed genes predominantly involved in neuronal differentiation. These genes were cross-referenced with quantitative proteomic and eCLIP data. Using RNAseq from clinical samples, splice inclusion analysis based upon PTBP2 bound transcripts neatly segregated Group 3/4 MB from SHH and WNT. PTBP2 KO cells exhibited reduced proliferation and, upon stimulation, increased differentiated morphology compared to controls. This latter observation was further supported by qPCR array and single-cell RNA-seq. CONCLUSIONS The results suggests that the loss of PTBP2 expression stimulates MB differentiation with a subsequent reduction of proliferation activity and nominates PTBP2 and its downstream effectors as potential therapeutic targets in group 3/4 MB.

#3029 Vitamin D-binding protein: a candidate biomarker for CKD progression

Abstract Background and Aims The toxic effects of filtered proteins on the tubules cause a more rapid progression of disease in individuals with higher levels of proteinuria, suggesting the presence of “high-risk candidate” proteins that could contribute to CKD progression. In our previous cross-sectional proteomic study, vitamin D-binding protein (VTDB) showed a significant association with kidney function. However, its persistent expression as a urinary protein in CKD patients remains unknown. Therefore, our aim was to investigate the persistent expression of VTDB in CKD patients and healthy controls during the follow-up period. Method The urinary proteomics was investigated using liquid chromatography-mass spectrometry in 18 follow-up CKD patients with stages 1-3 and 15 follow-up healthy individuals. Data analysis was performed by Mascot-SwissProt and STATA software. Parametric and nonparametric statistics were used to analyze results and the relationship between baseline characteristics and proteomic profile. Exponentially Modified Protein Abundance Index (emPAI) was used to estimate peptide count of proteins. Results The participant's baseline characteristics are presented in Table 1. The median (IQR) follow-up time for all participants was 368 (250-472) days. A statistically significant difference was found in the initial emPAI of VTDB between the HC and CKD groups. However, there was no statistically significant difference in the follow-up emPAI of VTDB between the two groups. Notably, emPAI values of VTDP decreased over the follow-up period compared to the initial values. Correlation analysis (Table 2) showed that the elevated levels of urinary VTDB emPAI are associated with decreased levels of initial and follow-up eGFR. A higher negative percentage change (a decline in eGFR over time) was also associated with elevated levels of VTDB protein over the follow-up period. Also, urinary VTDP protein has demonstrated a significant positive correlation with 24 h proteinuria. Conclusion In this proteomics study cohort, we observed persistent expression of urinary VTDB protein during the follow-up period in CKD patients compared to healthy controls. Additionally, a statistically significant negative correlation between VTDB protein and eGFR was observed. These findings need further validation using different measurement tools for urinary VTDB assessments and in a larger cohort of CKD patients. Declaration We have no conflict of interest to disclose.

Identification of Prominin-2 as a new player of cardiomyocyte senescence in the aging heart.

The aging heart is characterized by a number of structural changes leading to ventricular stiffness, impaired resistance to stress and increased risk of developing heart failure (HF). Genetic or pharmacological removal of senescent cells has recently demonstrated the possibility to relieve some cardiac aging features such as hypertrophy and fibrosis. However, the contribution of the different cell types in cardiac aging remains fragmentary due to a lack of cell-specific markers. Cardiomyocytes undergo post-mitotic senescence in response to telomere damage, characterized by persistent DNA damage response and expression of the classical senescence markers p21 and p16, which are shared by many other cell types. In the present study, we used transcriptomic approaches to discover new markers specific for cardiomyocyte senescence. We identified Prominin2 (Prom2), encoding a transmembrane glycoprotein, as the most upregulated gene in cardiomyocytes of aged mice compared to young mice. We showed that Prom2 was upregulated by a p53-dependent pathway in stress-induced premature senescence. Prom2 expression correlated with cardiomyocyte hypertrophy in the hearts of aged mice and was increased in atrial samples of patients with HF with preserved ejection fraction. Consistently, Prom2 overexpression was sufficient to drive senescence, hypertrophy and resistance to cytotoxic stress while Prom2 shRNA silencing inhibited these features in doxorubicin-treated cardiac cells. In conclusion, we identified Prom2 as a new player of cardiac aging, linking cardiomyocyte hypertrophy to senescence. These results could provide a better understanding and targeting of cell-type specific senescence in age-associated cardiac diseases.

Chronic HIV Transcription, Translation, and Persistent Inflammation.

People with HIV exhibit persistent inflammation that correlates with HIV-associated comorbidities including accelerated aging, increased risk of cardiovascular disease, and neuroinflammation. Mechanisms that perpetuate chronic inflammation in people with HIV undergoing antiretroviral treatments are poorly understood. One hypothesis is that the persistent low-level expression of HIV proviruses, including RNAs generated from defective proviral genomes, drives the immune dysfunction that is responsible for chronic HIV pathogenesis. We explore factors during HIV infection that contribute to the generation of a pool of defective proviruses as well as how HIV-1 mRNA and proteins alter immune function in people living with HIV.

Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid.

Mechanistic understanding of transient exposures that lead to adverse health outcomes will enhance our ability to recognize biological signatures of disease. Here, we measured the transcriptomic and epigenomic alterations due to exposure to the metabolic reprogramming agent, dichloroacetic acid (DCA). Previously, we showed that exposure to DCA increased liver tumor incidence in B6C3F1 mice after continuous or early life exposures significantly over background level. Using archived formalin-fixed liver samples, we utilized modern methodologies to measure gene expression and DNA methylation levels to link to previously generated phenotypic measures. Gene expression was measured by targeted RNA sequencing (TempO-seq 1500+ toxicity panel: 2754 total genes) in liver samples collected from 10-, 32-, 57-, and 78-week old mice exposed to deionized water (controls), 3.5 g/L DCA continuously in drinking water ("Direct" group), or DCA for 10-, 32-, or 57-weeks followed by deionized water until sample collection ("Stop" groups). Genome-scaled alterations in DNA methylation were measured by Reduced Representation Bisulfite Sequencing (RRBS) in 78-week liver samples for control, Direct, 10-week Stop DCA exposed mice. Transcriptomic changes were most robust with concurrent or adjacent timepoints after exposure was withdrawn. We observed a similar pattern with DNA methylation alterations where we noted attenuated differentially methylated regions (DMRs) in the 10-week Stop DCA exposure groups compared to the Direct group at 78-weeks. Gene pathway analysis indicated cellular effects linked to increased oxidative metabolism, a primary mechanism of action for DCA, closer to exposure windows especially early in life. Conversely, many gene signatures and pathways reversed patterns later in life and reflected more pro-tumorigenic patterns for both current and prior DCA exposures. DNA methylation patterns correlated to early gene pathway perturbations, such as cellular signaling, regulation and metabolism, suggesting persistence in the epigenome and possible regulatory effects. Liver metabolic reprogramming effects of DCA interacted with normal age mechanisms, increasing tumor burden with both continuous and prior DCA exposure in the male B6C3F1 rodent model.

Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity.Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Abstract 1130: Tenascin-C In Arteriovenous Fistula Failure: Unraveling Venous Remodeling Dynamics

Background: AVF are the preferred vascular access for hemodialysis, with superior patency and lower infection rates than grafts or catheters. Nevertheless, AVF patency is reduced by both early thrombosis and NIH, culminating in diminished utilization due to one-year patency rates as low as 40-50%. Tenascin-C (TnC), an ECM glycoprotein with limited adult expression, is transiently present at sites of tissue injury and inflammation. TnC has been implicated in playing a role in the development of NIH. Hypothesis: Sustained TnC expression contributes to NIH, potentially leading to AVF failure. Methods: Female and male wild-type C57Bl/6 mice, aged 9-11 weeks, had sham surgery or AVF creation by puncturing the infrarenal aorta into the inferior vena cava (IVC) with a 25-gauge needle. Doppler ultrasound confirmed AVF presence. Tissue samples were collected for IF, WB, and qPCR. Human matched vein and AVF tissue were obtained from patients undergoing planned two-stage AVF creation surgeries and analyzed with IF. In vitro analysis of human dermal microvascular endothelial cells (HDMEC) was performed to investigate the role of TnC in thrombomodulin expression. Results: TnC expression significantly increased in the AVF by day 7, returning to baseline by day 21 and reaching negligible levels by day 42. The spatial and temporal regulation of TnC expression post-AVF creation correlated with ECM remodeling and inversely with thrombomodulin. Both mice and human studies demonstrated differential regulation of TnC and thrombomodulin in patent and occluded AVF. In HDMEC, TnC knockdown suppressed NF-kB expression and increased thrombomodulin expression, while exogenous TnC had the opposite effect. Conclusion: TnC plays a pivotal role in driving pathological inflammation, with its expression being both temporally and spatially regulated during venous remodeling post-AVF creation. Persistent TnC expression is noted in occluded mouse and human AVF, leading to decreased thrombomodulin expression and heightened thrombogenicity. Sustained TnC expression regulates the shift from physiological to pathological venous remodeling, underscoring the crucial role of the immune response in this process and highlighting potential translational therapeutic strategies.

Antisense oligonucleotide therapeutic approach for Timothy syndrome

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2–6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

Nutrients, surfactants, and aeration in constructed wetlands affect bacterial persistence and metabolic activity during the remediation of crude oil-contaminated water

The use of constructed wetlands (CWs) is one of the best options to treat wastewater. In CWs, microorganisms play a major role in the degradation of organic pollutants but the concentration of nutrients, surfactant, and aeration (NSA) in oil-contaminated water is one of the factors that affect the persistence and metabolic functioning of hydrocarbon-degrading microorganisms. In the present investigation, the influence of the addition of NSA on the persistence of the augmented bacteria, copy of (alkane hydroxylase gene) alkB gene, and its expression level in the water, soil, and plants of CWs were evaluated. The CWs mesocosms were developed by the vegetation of Typha latifolia and Cyperus laevigatus and inoculated with the bacterial consortium (Pseudomonas putida TYRI39, Acinetobacter junii TYRH47, Acinetobacter sp. CYRH17, Pseudomonas sp. CYSI27, and Pseudomonas sp. TYRH42). The mesocosms were provided with nutrients (20 mg l− 1 N, 2.6 mg l− 1 P, and 16.4 mg l− 1 K) in liquid form, surfactant Tween-20 (0.2%, v/v) in liquid form, and aeration (≥ 7.0 ± 1 mg l− 1 DO) using aeration pump. The addition of NSA in CWs enhanced the persistence and metabolic functioning of the inoculated bacteria in the water, rhizospheric soil, and plants. The maximum hydrocarbon removal (97%) was observed in the water treated by CWs having C. laevigatus, bacteria, and NSA, and it is correlated with the copy numbers of alkB and its expression level. The application of NSA in CWs not only improved bacterial persistence and catabolic gene expression but also increased plant development and hydrocarbon removal.

Anaplastic lymphoma kinase-negative anaplastic large-cell lymphoma

Anaplastic large-cell lymphoma (ALCL) is a non-Hodgkin T-cell lymphoma characterized by persistent expression of CD 30 antigen that represents 2–3% of non-Hodgkin lymphoma (NHL). ALCL may present as a primary cutaneous disease or systemic disease with secondary cutaneous involvement. World Health Organization (WHO) classifies ALCL into ALK-positive and ALK-negative ALCL. ALK-negative ALCL shows poor response on treatment and has a bad prognosis. We report locally aggressive and metastasizing primary cutaneous anaplastic lymphoma kinase-negative anaplastic large-cell lymphoma in a 58-year-old female, due to its paucity of literature.

CD4+ CAR T-cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR T-cell therapy

AbstractMultiple myeloma is characterized by frequent clinical relapses after conventional therapy. Recently, chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, although >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune-intrinsic mechanisms may contribute to this resistance. Although there were no preexisting T-cell features associated with clinical outcomes, we found that patients with a durable response to CAR T-cell treatment had greater persistence of their CAR T cells than patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T-effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR T cells. These cells expand in vivo early after infusion but express exhaustion markers (hepatitis A virus cellular receptor 2 [HAVCR2] and T-cell immunoglobulin and mucin domain-containing-3 [TIGIT]) and remain polyclonal. Finally, we demonstrate that nonclassical monocytes are enriched in the myeloma niche and may induce CAR T-cell dysfunction through mechanisms that include transforming growth factor β. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR T-cell therapy.

Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2.

Significant progress has been made in enhancing recombinant adeno-associated virus (rAAV) for clinical investigation. Despite its versatility as a gene delivery platform, the inherent packaging constraint of 4.7 kb imposes restrictions on the range of diseases it can address. In this context, we present findings of an exceptionally compact and long-term promoter that facilitates the expression of larger genes compared to conventional promoters. This compact promoter originated from the genome of the alphaherpesvirus pseudorabies virus, latency-associated promoter 2 (LAP2, 404 bp). Promoter driving an mCherry reporter was packaged into single strand (ss) AAV8 and AAV9 vectors and injected into adult C57BL/6 mice at a dose of 5 × 1011 vg/mouse by single intravenous or intramuscular administration. An ssAAV8 and ssAAV9 vector with elongation factor-1α promoter (EF1α, 1264 bp) was injected side-by-side for comparison. After 400 days, we sacrificed the mice and examined mCherry expression in liver, kidney, heart, lung, spleen, pancreas, skeletal muscle, and brain. We found that LAP2 exhibited robust transgene expression across a wide range of cells and tissues comparable to the larger EF1α, which is currently recognized as a rather potent and ubiquitous promoter. The AAV8-LAP2 and AAV9-LAP2 constructs displayed strong transduction and transcription in liver, kidney, and skeletal muscle on both route of administration. However, no expression was detected in the heart, lung, spleen, pancreas, and brain. The outcomes of our investigation propose the viability of LAP2 for gene therapy applications demanding the expression of large or multiple therapeutic genes following a single viralvector administration.

Abstract 1595: Persistence of fetal gene signatures along the mesenchymal lineage trajectory defines heterogeneity and function of pancreatic cancer associated fibroblasts

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Here we aimed to investigate the origin, diversification, and function of CAFs. We constructed a dual-DNA-recombinase mouse genetic model carrying KrasG12D/+; Trp53frt/+; Pdx1Flpo/+; Isl1creER/+; R26Tomato/+ alleles, referred to as KPFIT. The DNA recombinase FlpO directs expression of an oncogene Kras (G12D mutation) and loss of a tumor suppressor p53 in pancreatic epithelial cells, while creER recombines the Tomato reporter in the splanchnic mesenchyme and its descendants. The splanchnic mesenchyme is a layer of fetal tissue surrounding the endoderm where the pancreatic epithelium arises. This approach capitalized on the independent functions of FlpO/Frt and CreER/LoxP systems, allowing us to lineage trace Tomato labeled splanchnic progenies (via IT) in a spontaneous pancreatic cancer model (via KPF) within the same mouse. Our study identified the splanchnic mesenchyme as the fetal origin of pancreatic fibroblasts during homeostasis and tumorigenesis. Notably, the other two postulated origins, bone marrow and epithelial cells, have minimal contributions to fibroblasts. Importantly, single cell transcriptomic analysis indicated persistent and dynamic gene expressions along the pancreatic mesenchymal trajectory during development, homeostasis, precancer lesion and cancer. Intriguingly, certain splanchnic factors are expressed in only subtypes of adult pancreatic fibroblasts in temporally and spatially distinct patterns. Furthermore, we constructed mouse genetic models to delete one of the splanchnic factors, Gata6, specifically in CAFs, which resulted in increased tumor burden in the pancreas. This suggests a non-cell autonomous function of GATA6 in CAFs to restrain pancreatic cancer progression. In summary, this study delineated a continuous cell trajectory of the mesenchymal lineage in the pancreas across different life stages. Moreover, persistent gene expressions along the mesenchymal trajectory contributes to pancreatic CAF heterogeneity. Importantly, such persistence may constitute an inherent mechanism to suppress pancreatic cancer. The enhancement of this mechanism could be explored further for therapeutic benefits. Citation Format: Lu Han, Tom Walter, Joseph Beaudet, Caroline Everett, Kun Fang, Michael Zimmermann, Angela Mathison, Raul Urrutia, Victor Jin, Gustavo Leone, Michael Ostrowski. Persistence of fetal gene signatures along the mesenchymal lineage trajectory defines heterogeneity and function of pancreatic cancer associated fibroblasts [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 1595.

Abstract 1446: Gastric cancer with microsatellite instability: Ex vivo generation of syngeneic mouse models

Abstract The DNA mismatch repair (MMR) system is a highly conserved pathway crucial for maintaining the genomic integrity. MMR impairment in sporadic tumors is due to MLH1 epigenetic silencing or mutations in other MMR genes (such as MSH2, MSH6, PMS2) and leads to microsatellite instability (MSI). Two independent molecular classifications reported the existence of a MSI subgroup in gastric cancer (GC), including 22-23% of all cases. GC MSI inter and intra-tumoral heterogeneity represents a significant challenge for precise patient stratification and treatment. Even if MSI status in GC is usually associated with a good prognosis, some reports described a subpopulation of MSI GC patients displaying a worse outcome. Moreover, while clinical trials testing immune checkpoint inhibitors in MSI tumors showed effectiveness across all treatment lines, the outcomes in GC patients were not as remarkable as in other cancer types. From the analysis of MSI GC patients enrolled in phase III clinical trials testing anti-PD1 monotherapy, it emerged that nearly half of the tumors are intrinsically resistant and complete and long-lasting responses are achieved in very few cases. Tumor mouse immunocompetent models allow to mimic the complex interactions between a fully functional immune system and a developing tumor. MSI GC syngeneic mouse models could thus represent a valuable tool to better understand the molecular complexity and predict the clinical behavior of this subgroup. To obtain MSI GC mouse models, we generated non-transformed organoids from normal gastric mucosa of BALB/c mice as a starting point for the induction of a neoplastic transformation ex vivo. To induce MMR deficiency, we inactivated either Mlh1 or Msh2 genes using CRISPR/Cas9 genome editing. Importantly, we did not introduce any other alteration in known oncogenes and tumor suppressor genes, thus allowing the natural emergence of driver mutations in the context of the genetic instability. Over time, MMR deficient models, but not the WT controls, exhibited MSI and increased their mutational burden. Furthermore, when cultured in stringent culture conditions (two-dimensional culture, growth factor deprivation), only MMR deficient cells generated tumor masses upon subcutaneous injection in immunodeficient NOD SCID mice. Importantly, cells retrieved from the tumor masses (n = 8) exhibited mutations in a relevant fraction of genes associated with the MSI subgroup by TCGA (21 out of 35) – including Kras, Arid1a, Erbb3 and Tpr53 – thus offering a good representation of the human MSI GC mutational landscape. Given the immunogenicity of the Cas9 protein, we finalized our models optimizing the genome editing protocol to avoid a persistent Cas9 expression. Cas9-free MMR deficient models were able to generate tumors in immunocompetent BALB/c mice, thus representing a valuable resource to investigate tumor development, progression and responses to immunotherapy in vivo. Citation Format: Daniela Conticelli, Gabriele Picco, Cristina Migliore, Claudia Orrù, Fabrizio Maina, Emanuela Boccuni, Simona Corso, Silvia Giordano. Gastric cancer with microsatellite instability: Ex vivo generation of syngeneic mouse models [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 1446.

Abstract 3402: Integration of microRNAs and transcriptome signatures identifies the persistent infection of hepatitis C virus-induced hepatocellular carcinoma

Abstract MicroRNAs (miRNAs) are short non-coding RNAs (ncRNAs) of ~22 nucleotides that mediate gene expression through various functions, including gene silencing, which can lead to translation repression and degradation of target mRNAs. Dysregulation of miRNA function has been associated with numerous diseases, particularly cancer, including hepatocellular carcinoma (HCC). During the progression of HCV-HCC, the function of miRNAs may be altered thereby altering gene expression to favor HCC. Therefore, the involved miRNA-mRNA interaction needs to be further studied. In addition, there are many clinical studies have shown that HCV patients who do not adequately control their diet which are prone to fatty liver accumulation (steatosis), it may increase the possibility of liver cirrhosis. In the current study, we aim to investigate the function and effects of persistent HCV-induced miRNAs expression in liver hepatocellular carcinoma progression and gene expression and to explore the mechanisms and regulatory genes of liver steatosis caused by HCV patients in a high-fat environment. We used the Huh751 cell line and the infected with the HCV JFH1 replicon of low-viral load S2 cells. We used an HCV miRNA array for initial identification of potential candidates. These miRNAs were analyzed for KEGG pathways via DIANA. Next, we identified miRNA-mRNA targets in 3 databases (miRDB; miRPathDB; TargetScan) and analyzed the targets for KEGG pathways using ShinyGO. The target miRNA-mRNA candidate genes were then compared with long-term JFH1 replicon infectious cell (L-HCV) NGS mRNA data to integrate miRNA-mRNA. The identified genes were analyzed in The Cancer Genome Atlas (TCGA) for survival, biological process, and expression in LIHC Big Data. Hsa-miR-215, hsa-mir-10b, and hsa-let-7a of L-HCV miRNAs group showed high significance for LIHC overall survival with a hazard ratio >1.0. Moreover, miRNAs associated with L-HCV survival were LIHC-specific but not in other cancers. Through systematic integration of these findings, we identified HMOX1 and BMF genes as being specifically associated with HCC and regulated by hsa-miR-215-5p, hsa-miR-10b-5p, and hsa-miR-7a-5p. In addition, we also found that the expression of the adipogenesis genes, FABP4 and adipogenesis gene, SREBP1, as well as the viral non-structural protein, NS3, was significantly increased in the HCV-high-fat group; and the expression of the gene IRS1, which is involved in hyperinsulinemia, showed a decreasing trend. The main cause of HCC is that HCV inhibits the expression of hsa-miR-215-5p, hsa-miR-10b-5p, and hsa-miR-7a-5p, leading to alter the expression of target genes, these have a significant effect on survival, suggesting that an essential role in virus-related liver cancer. In the future, we will further explore the regulatory mechanisms between these genes and HCV in a high-fat environment. Citation Format: Wen-Hsiu (Vivian) Su, Ciniso Sylvester Shabangu, Shu-Chi Wang. Integration of microRNAs and transcriptome signatures identifies the persistent infection of hepatitis C virus-induced hepatocellular carcinoma [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 3402.

Degradation meets development: Implications in β-cell development and diabetes.

Pancreatic development is orchestrated by timely synthesis and degradation of stage-specific transcription factors (TFs). The transition from one stage to another stage is dependent on the precise expression of the developmentally relevant TFs. Persistent expression of particular TF would impede the exit from the progenitor stage to the matured cell type. Intracellular protein degradation-mediated protein turnover contributes to a major extent to the turnover of these TFs and thereby dictates the development of different tissues. Since even subtle changes in the crucial cellular pathways would dramatically impact pancreatic β-cell performance, it is generally acknowledged that the biological activity of these pathways is tightly regulated by protein synthesis and degradation process. Intracellular protein degradation is executed majorly by the ubiquitin proteasome system (UPS) and Lysosomal degradation pathway. As more than 90% of the TFs are targeted to proteasomal degradation, this review aims to examine the crucial role of UPS in normal pancreatic β-cell development and how dysfunction of these pathways manifests in metabolic syndromes such as diabetes. Such understanding would facilitate designing a faithful approach to obtain a therapeutic quality of β-cells from stem cells.

Systematic integration of molecular and clinical approaches in HCV-induced hepatocellular carcinoma

BackgroundMicroRNAs (miRNAs) play a crucial role in gene expression and regulation, with dysregulation of miRNA function linked to various diseases, including hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). There is still a gap in understanding the regulatory relationship between miRNAs and mRNAs in HCV-HCC. This study aimed to investigate the function and effects of persistent HCV-induced miRNA expression on gene regulation in HCC.MethodsMiRNA array data were used to identify differentially expressed miRNAs and their targets, and miRNAs were analyzed via DIANA for KEGG pathways, gene ontology (GO) functional enrichment, and Ingenuity Pathways Analysis (IPA) for hepatotoxicity, canonical pathways, associated network functions, and interactive networks.ResultsSeventeen miRNAs in L-HCV and 9 miRNAs in S-HCV were differentially expressed, and 5 miRNAs in L-HCV and 5 miRNAs in S-HCV were significantly expressed in liver hepatocellular carcinoma (LIHC) tumors. Grouped miRNA survival analysis showed that L-HCV miRNAs were associated with survival in LIHC, and miRNA‒mRNA targets regulated viral carcinogenesis and cell cycle alteration through cancer pathways in LIHC. MiRNA-regulated RCN1 was suppressed through miRNA-oncogene interactions, and suppression of RCN1 inhibited invasion and migration in HCC.ConclusionPersistent HCV infection induced the expression of miRNAs that act as tumor suppressors by inhibiting oncogenes in HCC. RCN1 was suppressed while miRNAs were upregulated, demonstrating an inverse relationship. Therefore, hsa-miR-215-5p, hsa-miR-10b-5p, hsa-let-7a-5p and their target RCN1 may be ideal biomarkers for monitoring HCV-HCC progression.Graphical

Survival-Associated Cellular Response Maintained in Pancreatic Ductal Adenocarcinoma (PDAC) Switched Between Soft and Stiff 3D Microgel Culture.

Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of all pancreatic cancer cases. Five-year survival rates have remained below 12% since the 1970s, in part due to the difficulty in detection prior to metastasis (migration and invasion into neighboring organs and glands). Mechanical memory is a concept that has emerged over the past decade that may provide a path toward understanding how invading PDAC cells "remember" the mechanical properties of their diseased ("stiff", elastic modulus, E ≈ 10 kPa) microenvironment even while invading a healthy ("soft", E ≈ 1 kPa) microenvironment. Here, we investigated the role of mechanical priming by culturing a dilute suspension of PDAC (FG) cells within a 3D, rheologically tunable microgel platform from hydrogels with tunable mechanical properties. We conducted a suite of acute (short-term) priming studies where we cultured PDAC cells in either a soft (E ≈ 1 kPa) or stiff (E ≈ 10 kPa) environment for 6 h, then removed and placed them into a new soft or stiff 3D environment for another 18 h. Following these steps, we conducted RNA-seq analyses to quantify gene expression. Initial priming in the 3D culture showed persistent gene expression for the duration of the study, regardless of the subsequent environments (stiff or soft). Stiff 3D culture was associated with the downregulation of tumor suppressors (LATS1, BCAR3, CDKN2C), as well as the upregulation of cancer-associated genes (RAC3). Immunofluorescence staining (BCAR3, RAC3) further supported the persistence of this cellular response, with BCAR3 upregulated in soft culture and RAC3 upregulated in stiff-primed culture. Stiff-primed genes were stratified against patient data found in The Cancer Genome Atlas (TCGA). Upregulated genes in stiff-primed 3D culture were associated with decreased survival in patient data, suggesting a link between patient survival and mechanical priming.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.