Rituximab induces ferroptosis and RSL3 overcomes rituximab resistance in diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common malignant lymphoma in adults, and the use of rituximab has greatly improved the survival of DLBCL patients. Currently, the first-line treatment regimen for DLBCL is still rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP), which significantly improves outcomes for DLBCL patients. However, a percentage of patients still experience refractory or relapsed disease. Since Dr. Brent R Stockwell proposed ferroptosis in 2012, Roudkenar, M. H. Roushandeh, A. M. Valashedi, M. R. and others proved the importance of ferroptosis in cancer drug resistance. The purpose of this study was to elucidate whether rituximab could exert anticancer effects on DLBCL cells by promoting ferroptosis. Cell viability was assessed using the Cell Counting Kit-8. The results showed that rituximab exposure induced ferroptosis in OCI-LY1 cells. However, combination with ferroptosis inhibitor ferrostatin (Fer-1) rescued ferroptosis-induced injury, indicating that ferroptosis plays a key role in rituximab-induced cell death. Western blotting was performed to detect the levels of specific ferroptosis-associated proteins in DLBCL. Moreover, GSH depletion and MDA upregulation was assessed using GSH assays and MDA assay kits in rituximab-treated OCI-LY1 cells. In addition, rituximab failed to induce ferroptosis in rituximab-resistant cell lines. Treatment with RSL3 enhanced the effects of rituximab on DLBCL cells by inhibiting cell viability. In conclusion, we report for the first time that rituximab induces ferroptosis in lymphoma cells, at least partially through the SLC7A11/GPX4 axis. We also identify targeting ferroptosis as a promising therapeutic option for both sensitive cells and resistant cells in the treatment of DLBCL.

Application and progress of CRISPR/Cas9 gene editing in B-cell lymphoma: a narrative review.

Background and Objective: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) gene editing and CRISPR/Cas9 screening libraries are hot topics, and have high application values in the diagnosis and treatment of genetic diseases, and the improvement of prognosis. The major treatment of B-cell lymphoma is chemotherapy combined with biological therapy. Due to the individual specificity and the emergence of drug resistance, the therapeutic efficacy varies. The objective of this article is to explore potential targets to enhance therapeutic effects, optimize treatment plans, and improve the prognosis of patients with B-cell lymphoma. Methods: We undertook a comprehensive, narrative review of the latest literature to define the current application and progress of CRISPR/Cas9 in B-cell lymphoma. Key Content and Findings: The concepts of CRISPR/Cas9, the mechanism of gene editing, and the procedures of CRISPR/Cas9 screening libraries are investigated for candidate genes. We mainly focus on application and progress of CRISPR/Cas9 in B-cell lymphoma and screen out some genes, signaling pathways, and cytokines, which may become potential targets for clinical treatment. Conclusions: CRISPR/Cas9 gene editing has great promise in the treatment of B-cell lymphoma. This article reviews some genes, signaling pathways, and cytokines related to the progression and prognosis of B-cell lymphoma to provide a strong theoretical basis.

CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat.

Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER-LIKE 4 (DCL4), or DCL5 to produce 21- or 24 nucleotide (nt) double-strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21- and 24-nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5-A/B becoming non-functioning and TaRDR6-A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub-genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding.

Bortezomib-based chemotherapy for patients with Waldenström macroglobulinemia: a single-center experience.

Waldenström Macroglobulinemia (WM) is a rare type of non-Hodgkin lymphoma with no standard first-line treatment, and the disease is still incurable. This study evaluated the clinical efficacy, safety, and prognostic factors of bortezomib-based chemotherapy as initial treatment in WM patients. We retrospectively analyzed the clinical data collected from 44 newly diagnosed WM patients treated with bortezomib-based regimens at the Affiliated Hospital of Nantong University from December 2011 to June 2021. Univariate and multivariate analyses were used to assess prognostic factors for overall survival (OS) and progression-free survival (PFS). The median age was 67 years old, with an overall response rate (ORR) of 93.2%, complete response (CR) rate of 6.8%, and very good partial response (VGPR) rate of 29.5%. With a median follow-up of 39 months, the 2-year overall survival (OS) and 2-year PFS rates were 88.0% and 59.0%, respectively. By the last follow-up, eight patients (18.2%) had died. Univariate analysis showed patients with B symptoms, elevated LDH, international prognostic stage system of WM (IPSSWM) stage III, high Revised IPSSWM (R-IPSSWM) score, and those who did not achieve VGPR were associated with shorter PFS. And patients with B symptoms, with high R-IPSSWM score, and who do not achieve VGPR also had shorter OS than their counterparts. Multivariate analysis confirmed that failure to achieve VGPR was an independent adverse prognostic factor for OS and PFS. In conclusion, we showed that bortezomib-based chemotherapy effectively treated newly diagnosed patients with WM. However, combinations of drugs with different mechanisms are recommended for patients with a high tumor burden. In addition, deep remission can improve patients' survival.

RUNX1T1 function in cell fate.

RUNX1T1 (Runt-related transcription factor 1, translocated to 1), a myeloid translocation gene (MTG) family member, is usually investigated as part of the fusion protein RUNX1-RUNX1T1 for its role in acute myeloid leukemia. In the main, by recruiting histone deacetylases, RUNX1T1 negatively influences transcription, enabling it to regulate the proliferation and differentiation of hematopoietic progenitors. Moreover, the formation of blood vessels, neuronal differentiation, microglial activation following injury, and intestinal development all relate closely to the expression of RUNX1T1. Furthermore, through alternative splicing of RUNX1T1, short and long isoforms have been noted to mediate adipogenesis by balancing the differentiation and proliferation of adipocytes. In addition, RUNX1T1 plays wide-ranging and diverse roles in carcinoma as a biomarker, suppressor, or positive regulator of carcinogenesis, closely correlated to specific organs and dominant signaling pathways. The aim of this work was to investigate the structure of RUNX1T1, which contains four conserved nervy homolog domains, and to demonstrate crosstalk with the Notch signaling pathway. Moreover, we endeavored to illustrate the effects of RUNX1T1 on cell fate from multiple aspects, including its influence on hematopoiesis, neuronal differentiation, microglial activation, intestinal development, adipogenesis, angiogenesis, and carcinogenesis.

Runx1t1 promotes the neuronal differentiation in rat hippocampus.

BACKGROUND: Runt-related transcription factor 1 translocated to 1 (Runx1t1) is one of the members of the myeloid translocation gene family. Our previous work showed that Runx1t1 induced the neuronal differentiation of radial glia cells in vitro. METHODS: To better uncover the role of Runx1t1 in hippocampal neurogenesis, in this study, we further explore its localization and function during the hippocampal neurogenesis. RESULTS: Our results showed that insufficient expression of Runx1t1 reduced the neuronal differentiation, and overexpression of Runx1t1 promoted the neuronal differentiation in vitro. We also found that Runx1t1 localized in neurons but not astrocytes both in vivo and in vitro. Furthermore, we found that Runx1t1 overexpression elevated the number of newborn neurons in the hippocampal dentate gyrus. CONCLUSIONS: Taken together, our results further proved that Runx1t1 could be worked as a regulator in the process of hippocampal neurogenesis.

Evolution of PHAS loci in the young spike of Allohexaploid wheat.

BACKGROUND: PhasiRNAs (phased secondary siRNAs) play important regulatory roles in the development processes and biotic or abiotic stresses in plants. Some of phasiRNAs involve in the reproductive development in grasses, which include two categories, 21-nt (nucleotide) and 24-nt phasiRNAs. They are triggered by miR2118 and miR2275 respectively, in premeiotic and meiotic anthers of rice, maize and other grass species. Wheat (Triticum aestivum) with three closely related subgenomes (subA, subB and subD), is a model of allopolyploid in plants. Knowledge about the role of phasiRNAs in the inflorescence development of wheat is absent until now, and the evolution of PHAS loci in polyploid plants is also unavailable. RESULTS: Using 261 small RNA expression datasets from various tissues, a batch of PHAS (phasiRNA precursors) loci were identified in the young spike of wheat, most of which were regulated by miR2118 and miR2275 in their target site regions. Dissection of PHAS and their trigger miRNAs among the diploid (AA and DD), tetraploid (AABB) and hexaploid (AABBDD) genomes of Triticum indicated that distribution of PHAS loci were dominant randomly in local chromosomes, while miR2118 was dominant only in the subB genome. The diversity of PHAS loci in the three subgenomes of wheat and their progenitor genomes (AA, DD and AABB) suggested that they originated or diverged at least before the occurrence of the tetraploid AABB genome. The positive correlation between the PHAS loci or the trigger miRNAs and the ploidy of genome indicated the expansion of genome was the major drive force for the increase of PHAS loci and their trigger miRNAs in Triticum. In addition, the expression profiles of the PHAS transcripts suggested they responded to abiotic stresses such as cold stress in wheat. CONCLUSIONS: Altogether, non-coding phasiRNAs are conserved transcriptional regulators that display quick plasticity in Triticum genome. They may be involved in reproductive development and abiotic stress in wheat. It could be referred to molecular research on male reproductive development in Triticum.

Promising RNA-based cancer gene therapy using extracellular vesicles for drug delivery.

INTRODUCTION: RNA-based cancer gene therapy shows potential in cancer treatment. However, the safe and efficient transfer of therapeutic RNA to target cells has always been a challenge. The ideal drug delivery system should be effective with low immunogenicity and toxicity. Besides, a high specificity of drug delivery is necessary to improve efficacy and avoid the side effects associated with tumor heterogeneity. As endogenous RNA vehicles, extracellular vesicles (EVs) have shown their advantages and potential as drug delivery systems in gene therapy. AREAS COVERED: We summarize the performance of EVs as a drug delivery system in RNA-based cancer gene therapy and discuss the advantages, limitations, and potentials of this translational medicine. In addition, we compare the characteristics and differences of current drug delivery systems and expound the principles of selecting a drug delivery system suitable for cancer gene therapy. EXPERT OPINION: EVs are highly biocompatible membrane structures with low cytotoxicity which provide a new choice for drug delivery in RNA-based cancer gene therapy. The specificity of engineered EVs and artificial EV-mimetics can be improved through peptide or polymer decoration. However, apart from therapeutic RNA, EVs naturally carry many molecules. This may lead to unpredictable effects and thus should be applied with caution.

Highly Efficient and Heritable Targeted Mutagenesis in Wheat via the Agrobacterium tumefaciens-Mediated CRISPR/Cas9 System.

The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering.

Lineage-Specific Evolved MicroRNAs Regulating NB-LRR Defense Genes in Triticeae.

Disease resistance genes encoding proteins with nucleotide binding sites and Leucine-Rich Repeat (NB-LRR) domains include many members involved in the effector-triggered immunity pathway in plants. The transcript levels of these defense genes are negatively regulated by diverse microRNAs (miRNAs) in angiosperms and gymnosperms. In wheat, using small RNA expression datasets and degradome datasets, we identified five miRNA families targeting NB-LRR defense genes in monocots, some of which arose in the Triticeae species era. These miRNAs regulate different types of NB-LRR genes, most of them with coil-coiled domains, and trigger the generation of secondary small interfering RNAs (siRNA) as a phased pattern in the target site regions. In addition to acting in response to biotic stresses, they are also responsive to abiotic stresses such as heat, drought, salt, and light stress. Their copy number and expression variation in Triticeae suggest a rapid birth and death frequency. Altogether, non-conserved miRNAs as conserved transcriptional regulators in gymnosperms and angiosperms regulating the disease resistance genes displayed quick plasticity including the variations of sequences, gene copy number, functions, and expression level, which accompanied with NB-LRR genes may be tune-regulated to plants in natural environments with various biotic and abiotic stresses.

Mechanism and Treatment of Rituximab Resistance in Diffuse Large Bcell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype B non-Hodgkin lymphoma in adults. After rituximab being introduced to treat DLBCL, the current first-line treatment is R-CHOP regimen. This regimen greatly improves patient's prognosis, however, relapsed or refractory cases are commonly seen, mainly due to the resistance to rituximab. Although a large number of experiments have been conducted to investigate rituximab resistance, the exac mechanisms and solutions are still unclear. This review mainly explores the possible mechanisms oft rituximab resistance and current new effective treatments for rituximab resistance in DLBCL.

The genome-wide transcriptional consequences of the nullisomic-tetrasomic stocks for homoeologous group 7 in bread wheat.

BACKGROUND: Hexaploid bread wheat (Triticum aestivum L) arose by two polyploidisation events from three diploid species with homoeologous genomes. Nullisomic-tetrasomic (nulli-tetra or NT) lines are aneuploid wheat plants lacking two and adding two of six homoeologous chromosomes. These plants can grow normally, but with significantly morphological variations because the adding two chromosomes or the remaining four chromosomes compensate for those absent. Despite these interesting phenomena, detailed molecular mechanisms underlying dosage deletion and compensation in these useful genetic materials have not been determined. RESULTS: By sequencing the transcriptomes of leaves in two-week-old seedlings, we showed that the profiles of differentially expressed genes between NT stocks for homoeologous group 7 and the parent hexaploid Chinese Spring (CS) occurred throughout the whole genome with a subgenome and chromosome preference. The deletion effect of nulli-chromosomes was compensated partly by the tetra-chromosomes via the dose level of expressed genes, according to the types of homoeologous genes. The functions of differentially regulated genes primarily focused on carbon metabolic process, photosynthesis process, hormone metabolism, and responding to stimulus, and etc., which might be related to the defective phenotypes that included reductions in plant height, flag leaf length, spikelet number, and kernels per spike. CONCLUSIONS: The perturbation of the expression levels of transcriptional genes among the NT stocks for homoeologous group 7 demonstrated the gene dosage effect of the subgenome at the genome-wide level. The gene dosage deletion and compensation can be used as a model to elucidate the functions of the subgenomes in modern polyploid plants.

Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat.

BACKGROUND: Recently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation. RESULTS: In the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Seven target sites in three genes (Pinb, waxy and DA1) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens-mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences. CONCLUSIONS: Our results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.

Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat.

BACKGROUND: This study aimed to validate the function of CKX gene on grain numbers in wheat. METHODS: we constructed and transformed a RNA interference expression vector of TaCKX2.4 in bread wheat line NB1. Southern blotting analysis was used to select transgenic plants with single copy. The expression of TaCKX2.4 gene was estimated by Quantitative real-time PCR (qRT-PCR) analysis. Finally, the relation between expression of TaCKX2.4 gene and grain numbers was validated. RESULTS: Totally, 20 positive independent events were obtained. Homozygous lines from 5 events with a single copy of transformed gene each were selected to evaluate the expression of TaCKX2.4 and grain numbers per spike in T3 generation. Compared with the control NB1, the average grain numbers per spike significantly increased by 12.6%, 8.3%, 6.5% and 5.8% in the T3 lines JW39-3A, JW1-2B, JW1-1A and JW5-1A, respectively. CONCLUSION: Our study indicated that the expression level of TaCKX2.4 was negatively correlated with the grain number per spike, indicating that the reduced expression of TaCKX2.4 increased grain numbers per spike in wheat.

Response of microRNAs to cold treatment in the young spikes of common wheat.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in biotic and abiotic stresses by regulating their target genes. For common wheat, spring frost damage frequently occurs, especially when low temperature coincides with plants at early floral organ differentiation, which may result in significant yield loss. Up to date, the role of miRNAs in wheat response to frost stress is not well understood. RESULTS: We report here the sequencing of small RNA transcriptomes from the young spikes that were treated with cold stress and the comparative analysis with those of the control. A total of 192 conserved miRNAs from 105 families and nine novel miRNAs were identified. Among them, 34 conserved and five novel miRNAs were differentially expressed between the cold-stressed samples and the controls. The expression patterns of 18 miRNAs were further validated by quantitative real time polymerase chain reaction (qRT-PCR). Moreover, nearly half of the miRNAs were cross inducible by biotic and abiotic stresses when compared with previously published work. Target genes were predicted and validated by degradome sequencing. Gene Ontology (GO) enrichment analysis showed that the target genes of differentially expressed miRNAs were enriched for response to the stimulus, regulation of transcription, and ion transport functions. Since many targets of differentially expressed miRNAs were transcription factors that are associated with floral development such as ARF, SPB (Squamosa Promoter Binding like protein), MADS-box (MCM1, AG, DEFA and SRF), MYB, SPX (SYG1, Pho81 and XPR1), TCP (TEOSINTE BRANCHED, Cycloidea and PCF), and PPR (PentatricoPeptide Repeat) genes, cold-altered miRNA expression may cause abnormal reproductive organ development. CONCLUSION: Analysis of small RNA transcriptomes and their target genes provide new insight into miRNA regulation in developing wheat inflorescences under cold stress. MiRNAs provide another layer of gene regulation in cold stress response that can be genetically manipulated to reduce yield loss in wheat.

Genetic analysis of phytoene synthase 1 (Psy1) gene function and regulation in common wheat.

BACKGROUND: Phytoene synthase 1 (PSY1) is the most important regulatory enzyme in carotenoid biosynthesis, whereas its function is hardly known in common wheat. The aims of the present study were to investigate Psy1 function and genetic regulation using reverse genetics approaches. RESULTS: Transcript levels of Psy1 in RNAi transgenic lines were decreased by 54-76 % and yellow pigment content (YPC) was reduced by 26-35 % compared with controls, confirming the impact of Psy1 on carotenoid accumulation. A series of candidate genes involved in secondary metabolic pathways and core metabolic processes responded to Psy1 down-regulation. The aspartate rich domain (DXXXD) was important for PSY1 function, and conserved nucleotides adjacent to the domain influenced YPC by regulating gene expression, enzyme activity or alternative splicing. Compensatory responses analysis indicated that three Psy1 homoeologs may be coordinately regulated under normal conditions, but separately regulated under stress. The period 14 days post anthesis (DPA) was found to be a key regulation node during grain development. CONCLUSION: The findings define key aspects of flour color regulation in wheat and facilitate the genetic improvement of wheat quality targeting color/nutritional specifications required for specific end products.

Expression of CD40 is a positive prognostic factor of diffuse large B-cell lymphoma treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).

OBJECTIVES: The objective of this study was to investigate the expression level of CD40 and its role in the prognosis of patients with diffuse large B-cell lymphoma (DLBCL) who were treated with rituximab-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). DESIGN AND METHODS: The immunohistochemical expressions of CD40 in 186 well-characterized DLBCL patients were evaluated by tissue microarrays, thereby revealing the relationship of the molecule CD40 with known tumor, patient-related variables, and survival rates. RESULTS: The results showed that CD40 expressions were not statistically different between the germinal center B-cell-like (GCB) type and the non-GCB type. We also analyzed the relationships of CD40 expression with overall survival (OS) and progression-free survival (PFS) in DLBCL patients who were uniformly treated with R-CHOP. A low expression of CD40 compared to high expression is related to poor OS and PFS. CONCLUSION: Our findings indicate that the CD40 level at onset acts as an independent prognostic predictor of DLBCL patients treated with R-CHOP.

MicroRNAs in diffuse large B-cell lymphoma.

The aberrant expression of microRNAs (miRs) has a significant impact on the biological characteristics of lymphocytes, and is important in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). It has been demonstrated, using miR profiling and detecting distinct miR signatures, that certain miRs may accurately distinguish different subtypes and prognostic classifications of DLBCL, as well as distinguish DLBCL from other more indolent lymphomas, including follicular lymphoma. miRs are excellent biomarkers for cancer diagnosis and prognosis. In DLBCL, specific miR expression profiles in the tissues of patients are associated with prognosis and clinical outcome. Over the past decade, there has been substantial investigation concerning the pathogenetic, diagnostic and prognostic roles of miRs in DLBCL. The aim of the present review is to describe the aberrant expression of miRs in DLBCL, and the functions, potential clinical use and possible therapeutic targets of miRs in this disease.