Correlations of Long Noncoding RNA HNF4A-AS1 Alternative Transcripts with Liver Diseases and Drug Metabolism.

Hepatocyte nuclear factor 4 alpha antisense 1 (HNF4A-AS1) is a long noncoding RNA (lncRNA) gene physically located next to the transcription factor HNF4A gene in the human genome. Its transcription products have been reported to inhibit the progression of hepatocellular carcinoma (HCC) and negatively regulate the expression of cytochrome P450s (CYPs), including CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4. By altering CYP expression, lncRNA HNF4A-AS1 also contributes to the susceptibility of drug-induced liver injury. Thus, HNF4A-AS1 lncRNA is a promising target for controlling HCC and modulating drug metabolism. However, HNF4A-AS1 has four annotated alternative transcripts in the human genome browsers, and it is unclear which transcripts the small interfering RNAs or small hairpin RNAs used in the previous studies are silenced and which transcripts should be used as the target. In this study, four annotated and two newly identified transcripts were confirmed. These six transcripts showed different expression levels in different liver disease conditions, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and obesity. The expression patterns of all HNF4A-AS1 transcripts were further investigated in liver cell growth from human embryonic stem cells to matured hepatocyte-like cells, HepaRG differentiation, and exposure to rifampicin treatment. Several HNF4A-AS1 transcripts highly displayed correlations with these situations. In addition, some of the HNF4A-AS1 transcripts also showed a strong correlation with CYP3A4 during HepaRG maturation and rifampicin exposure. Our findings provide valuable insights into the specific roles of HNF4A-AS1 transcripts, paving the way for more targeted therapeutic strategies for liver diseases and drug metabolism. SIGNIFICANCE STATEMENT: This study explores the alternative transcripts of HNF4A-AS1, showing how their expression changes in different biological conditions, from various liver diseases to the growth and differentiation of hepatocytes and drug metabolism. The generated knowledge is essential for understanding the independent roles of different transcripts from the same lncRNA in different liver diseases and drug metabolism situations.

Ide-cel vs standard regimens in triple-class-exposed relapsed and refractory multiple myeloma: updated KarMMa-3 analyses.

Outcomes are poor in triple-class-exposed (TCE) relapsed/refractory multiple myeloma (RRMM). In the phase 3 KarMMa-3 (clinicaltrials.gov; NCT03651128) trial, patients with TCE RRMM and 2-4 prior regimens were randomized 2:1 to idecabtagene vicleucel (ide-cel) or standard regimens (SRs). An interim analysis (IA) demonstrated significantly longer median progression-free survival (PFS; primary endpoint; 13.3 vs 4.4 months; P<.0001) and higher overall response rate (ORR) with ide-cel vs SRs. At final PFS analysis (median follow-up, 30.9 months), ide-cel further improved median PFS vs SRs (13.8 vs 4.4 months; hazard ratio (HR), 0.49; 95% confidence interval (CI), 0.38-0.63). PFS benefit with ide-cel vs SRs was observed regardless of number of prior lines of therapy, with greatest benefit after 2 prior lines (16.2 vs 4.8 months, respectively). ORR benefit was maintained with ide-cel vs SRs (71% vs 42%; complete response, 44% vs 5%). Patient-centric design allowed crossover from SRs (56%) to ide-cel upon progressive disease, confounding overall survival (OS) interpretation. At IA of OS, median (95% CI) was 41.4 (30.9-not reached [NR]) vs 37.9 (23.4-NR) months with ide-cel and SRs, respectively (HR, 1.01; 95% CI 0.73-1.40); median OS in both arms was longer than historical data (9-22 months). Two prespecified analyses adjusting for crossover showed OS favoring ide-cel. This trial highlighted the importance of individualized bridging therapy to ensure adequate disease control during ide-cel manufacturing. Ide-cel improved patient-reported outcomes vs SRs. No new safety signals were reported. These results demonstrate the continued favorable benefit-risk profile of ide-cel in early-line and TCE RRMM. NCT03651128.

Mechanisms of Action of the US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

Antisense oligonucleotides (ASOs) are single stranded nucleic acids that target RNA. The US Food and Drug Administration has approved ASOs for several diseases. ASOs utilize three principal modes of action (MOA). The first MOA is initiated by base-pairing between the ASO and its target mRNA, followed by RNase H-dependent mRNA degradation. The second MOA is triggered by ASOs that occlude splice acceptor sites in pre-mRNAs leading to skipping of a mutation-bearing exon. The third MOA involves ASOs that sterically hinder mRNA function, often inhibiting translation. ASOs contain a variety of modifications to the sugar-phosphate backbone and bases that stabilize the ASO or render them resistant to RNase activity. RNase H-dependent ASOs include inotersen and eplontersen (for hereditary transthyretin amyloidosis), fomiversen (for opportunistic cytomegalovirus infection), mipomersen (for familial hypercholesterolemia), and tofersen [for amyotrophic lateral sclerosis (ALS)]. Splice modulating ASOs include nursinersen (for spinal muscular atrophy) and eteplirsen, golodirsen, viltolarsen, and casimersen (all for the treatment of Duchenne muscular dystrophy). In addition, a designer ASO, milasen, was used to treat a single individual afflicted with Batten disease. Since ASO design relies principally upon knowledge of mRNA sequence, the bench to bedside pipeline for ASOs is expedient compared with protein-directed drugs. [Graphical abstract available.].

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Identification and Functional Characterization of Alternative Transcripts of LncRNA HNF1A-AS1 and Their Impacts on Cell Growth, Differentiation, Liver Diseases, and in Response to Drug Induction.

The long non-coding RNA (lncRNA) hepatocyte nuclear factor-1 alpha (HNF1A) antisense RNA 1 (HNF1A-AS1) is an important lncRNA for liver growth, development, cell differentiation, and drug metabolism. Like many lncRNAs, HNF1A-AS1 has multiple annotated alternative transcripts in the human genome. Several fundamental biological questions are still not solved: (1) How many transcripts really exist in biological samples, such as liver samples and liver cell lines? (2) What are the expression patterns of different alternative HNF1A-AS1 transcripts at different conditions, including during cell growth and development, after exposure to xenobiotics (such as drugs), and in disease conditions, such as metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD) cirrhosis, and obesity? (3) Does the siRNA used in previous studies knock down one or multiple transcripts? (4) Do different transcripts have the same or different functions for gene regulation? The presented data confirm the existence of several annotated HNF1A-AS1 transcripts in liver samples and cell lines, but also identify some new transcripts, which are not annotated in the Ensembl genome database. Expression patterns of the identified HNF1A-AS1 transcripts are highly correlated with the cell differentiation of matured hepatocyte-like cells from human embryonic stem cells (hESC), growth and differentiation of HepaRG cells, in response to rifampicin induction, and in various liver disease conditions. The expression levels of the HNF1A-AS1 transcripts are also highly correlated to the expression of cytochrome P450 enzymes, such as CYP3A4, during HepaRG growth, differentiation, and in response to rifampicin induction.

The long noncoding RNA HNF1A-AS1 with dual functions in the regulation of cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) is the most important and abundant drug-metabolizing enzyme in the human liver. Inter-individual differences in the expression and activity of CYP3A4 affect clinical and precision medicine. Increasing evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the regulation of CYP3A4 expression. Here, we showed that lncRNA hepatocyte nuclear factor 1 alpha-antisense 1 (HNF1A-AS1) exerted dual functions in regulating CYP3A4 expression in Huh7 and HepG2 cells. Mechanistically, HNF1A-AS1 served as an RNA scaffold to interact with both protein arginine methyltransferase 1 and pregnane X receptor (PXR), thereby facilitating their protein interactions and resulting in the transactivation of PXR and transcriptional alteration of CYP3A4 via histone modifications. Furthermore, HNF1A-AS1 bound to the HNF1A protein, a liver-specific transcription factor, thereby blocking its interaction with the E3 ubiquitin ligase tripartite motif containing 25, ultimately preventing HNF1A ubiquitination and protein degradation, further regulating the expression of CYP3A4. In summary, these results reveal the novel functions of HNF1A-AS1 as the transcriptional and post-translational regulator of CYP3A4; thus, HNF1A-AS1 may serve as a new indicator for establishing or predicting individual differences in CYP3A4 expression.

Targeting the Liver with Nucleic Acid Therapeutics for the Treatment of Systemic Diseases of Liver Origin.

Systemic diseases of liver origin (SDLO) are complex diseases in multiple organ systems, such as cardiovascular, musculoskeletal, endocrine, renal, respiratory, and sensory organ systems, caused by irregular liver metabolism and production of functional factors. Examples of such diseases discussed in this article include primary hyperoxaluria, familial hypercholesterolemia, acute hepatic porphyria, hereditary transthyretin amyloidosis, hemophilia, atherosclerotic cardiovascular diseases, α-1 antitrypsin deficiency-associated liver disease, and complement-mediated diseases. Nucleic acid therapeutics use nucleic acids and related compounds as therapeutic agents to alter gene expression for therapeutic purposes. The two most promising, fastest-growing classes of nucleic acid therapeutics are antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). For each listed SDLO disease, this article discusses epidemiology, symptoms, genetic causes, current treatment options, and advantages and disadvantages of nucleic acid therapeutics by either ASO or siRNA drugs approved or under development. Furthermore, challenges and future perspectives on adverse drug reactions and toxicity of ASO and siRNA drugs for the treatment of SDLO diseases are also discussed. In summary, this review article will highlight the clinical advantages of nucleic acid therapeutics in targeting the liver for the treatment of SDLO diseases. SIGNIFICANCE STATEMENT: Systemic diseases of liver origin (SDLO) contain rare and common complex diseases caused by irregular functions of the liver. Nucleic acid therapeutics have shown promising clinical advantages to treat SDLO. This article aims to provide the most updated information on targeting the liver with antisense oligonucleotides and small interfering RNA drugs. The generated knowledge may stimulate further investigations in this growing field of new therapeutic entities for the treatment of SDLO, which currently have no or limited options for treatment.

A holistic analysis of the intrinsic and delivery-mediated toxicity of siRNA therapeutics.

Small interfering RNAs (siRNAs) are among the most promising therapeutic platforms in many life-threatening diseases. Owing to the significant advances in siRNA design, many challenges in the stability, specificity and delivery of siRNA have been addressed. However, safety concerns and dose-limiting toxicities still stand among the reasons for the failure of clinical trials of potent siRNA therapies, calling for a need of more comprehensive understanding of their potential mechanisms of toxicity. This review delves into the intrinsic and delivery related toxicity mechanisms of siRNA drugs and takes a holistic look at the safety failure of the clinical trials to identify the underlying causes of toxicity. In the end, the current challenges, and potential solutions for the safety assessment and high throughput screening of investigational siRNA and delivery systems as well as considerations for design strategies of safer siRNA therapeutics are outlined.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-12.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently [...].

Epigenetic Mechanisms Contribute to Intraindividual Variations of Drug Metabolism Mediated by Cytochrome P450 Enzymes.

Significant interindividual and intraindividual variations on cytochrome P450 (CYP)-mediated drug metabolism exist in the general population globally. Genetic polymorphisms are one of the major contribution factors for interindividual variations, but epigenetic mechanisms mainly contribute to intraindividual variations, including DNA methylation, histone modifications, microRNAs, and long non-coding RNAs. The current review provides analysis of advanced knowledge in the last decade on contributions of epigenetic mechanisms to intraindividual variations on CYP-mediated drug metabolism in several situations, including (1) ontogeny, the developmental changes of CYP expression in individuals from neonates to adults; (2) increased activities of CYP enzymes induced by drug treatment; (3) increased activities of CYP enzymes in adult ages induced by drug treatment at neonate ages; and (4) decreased activities of CYP enzymes in individuals with drug-induced liver injury (DILI). Furthermore, current challenges, knowledge gaps, and future perspective of the epigenetic mechanisms in development of CYP pharmacoepigenetics are discussed. In conclusion, epigenetic mechanisms have been proven to contribute to intraindividual variations of drug metabolism mediated by CYP enzymes in age development, drug induction, and DILI conditions. The knowledge has helped understanding how intraindividual variation are generated. Future studies are needed to develop CYP-based pharmacoepigenetics to guide clinical applications for precision medicine with improved therapeutic efficacy and reduced risk of adverse drug reactions and toxicity. SIGNIFICANCE STATEMENT: Understanding epigenetic mechanisms in contribution to intraindividual variations of CYP-mediated drug metabolism may help to develop CYP-based pharmacoepigenetics for precision medicine to improve therapeutic efficacy and reduce adverse drug reactions and toxicity for drugs metabolized by CYP enzymes.

Effect of concurrent beta-blocker use in patients receiving immune checkpoint inhibitors for advanced solid tumors.

PURPOSE: Stress-induced adrenergic signaling can suppress the immune system. In animal models, pharmacological beta-blockade stimulates CD8 + T-cell activity and improves clinical activity of immune checkpoint blockade (ICB) in inhibiting tumor growth. Herein, we investigated the effect of BB on clinical outcomes of patients receiving ICB in advanced solid tumors. METHODS: We retrospectively evaluated patients with solid tumors treated with ICB at our institution from January 1, 2011 to April 28, 2017. The primary clinical outcome was disease control. Secondary clinical outcomes were overall survival (OS), and duration of therapy (DoT). The primary predictor was use of BB. Association between disease control status and BB use was assessed in univariable and multivariable logistic regression. OS was calculated using hazard ratios of BB-recipient patients vs. BB non-recipient patients via Cox proportional hazards regression models. All tests were two-sided at a significance level of 0.05. RESULTS: Of 339 identified patients receiving ICB, 109 (32%) also received BB. In covariate-adjusted analysis, odds of disease control were significantly higher among BB recipients compared to BB-non-recipients (2.79; [1.54-5.03]; P = 0.001). While we did not observe significant association of OS with the use of BB overall, significant association with better OS was observed for the urothelial carcinoma cohort (HR: 0.24; [0.09, 0.62]; P = 0.0031). CONCLUSIONS: Concurrent use of BB may enhance the clinical activity of ICB and influence overall survival, particularly in patients with urothelial carcinoma. Our findings warrant further investigation to understand the interaction of beta adrenergic signaling and antitumor immune activity and explore a combination strategy.