Recent advances in defining low- and intermediate-risk myelodysplastic syndromes (MDSs) have emphasized the critical role of molecular characterization using next-generation sequencing (NGS). Molecular profiling significantly enhances diagnostic precision, classification, and risk stratification, thereby informing therapeutic decisions, including the timing of hematopoietic stem-cell transplantation (HSCT). The Molecular International Prognostic Scoring System integrates clinical and molecular data, reclassifying and upstaging a substantial number of patients compared with previous prognostic systems, possibly allowing for more tailored therapeutic interventions. The novel therapeutic agents luspatercept and imetelstat have been particularly impactful. Luspatercept, which is effective in lower-risk (LR)-MDS, especially but not only in SF3B1-mutated cases, promotes late-stage erythroid maturation and transfusion independence (TI). Imetelstat, a telomerase inhibitor, induces TI while demonstrating disease-modifying effects as it significantly reduces mutation allele frequencies in patients who respond. These agents exemplify personalized medicine, emphasizing treatment selection and timing based on individual molecular and clinical features. Current research focuses on optimizing therapeutic strategies and exploring combination treatments to improve clinical outcomes.

Myocardial ischemia/reperfusion injury (MIRI) significantly contributes to the increased mortality associated with cardiovascular diseases; however, there are no effective therapeutic agents. Hydroxysafflor yellow A (HSYA) is a natural small molecule that possesses potent anti-ischemic myocardial injury properties. However, precise molecular targets remain unclear. In this study, we identified splicing factor 3A subunit 1 (SF3A1) as a direct target of HSYA through thermal proteome profiling. SF3A1 participates in spliceosome assembly and it has been reported to mediate the alternative splicing of precursor messenger RNA. Based on this function, we used RNA sequencing to investigate the downstream mechanisms of SF3A1 and found that the most strongly correlated genes and pathways were associated with mitochondrial injury and energy metabolism. Furthermore, the results demonstrated that in MIRI, HSYA could clear the accumulation of reactive oxygen species, thereby restoring mitochondrial polarization and membrane potential. HSYA further drove oxidative phosphorylation in the electron transport chain and promoted ATP synthesis. Additionally, we silenced the SF3A1 gene and confirmed that SF3A1 played a regulatory role in mitochondrial energy metabolism and HSYA-mediated therapeutic effects. In vivo, HSYA also demonstrated significant therapeutic efficacy in acute myocardial mouse infarction models, with the ability to enhance mitochondrial energy metabolism in myocardial tissue. In summary, our research suggests that SF3A1, through the pharmacological targeting of mitochondrial energy metabolism, may emerge as a novel therapeutic target for MIRI. Moreover, specific modulation of SF3A1 by HSYA may provide a new perspective for designing cardiovascular protectants with novel mechanisms, facilitating the development of precise therapies for MIRI.

Alternative splicing (AS) is a fundamental mechanism for enhancing transcriptome diversity and regulating gene expression, crucial for various cellular processes and the development of complex traits. This review examines the role of AS in metabolic disorders, including obesity, weight loss, dyslipidemias, and metabolic syndrome. We explore the molecular mechanisms underlying AS regulation, focusing on the interplay between cis-acting elements and trans-acting factors, and the influence of RNA-binding proteins (RBPs). Advances in high-throughput sequencing and bioinformatics have unveiled the extensive landscape of AS events across different tissues and conditions, highlighting the importance of tissue-specific splicing in metabolic regulation. We discuss the impact of genetic variants on AS, with a particular emphasis on splicing quantitative trait loci (sQTLs) and their association with cardiometabolic traits. The review also covers the regulation of spliceosome components by phosphorylation, the role of m6A modification in AS, and the interaction between transcription and splicing. Additionally, we address the clinical relevance of AS, illustrating how splicing misregulation contributes to metabolic diseases and the potential for therapeutic interventions targeting splicing mechanisms. This comprehensive overview underscores the significance of AS in metabolic health and disease, advocating for further research to harness its therapeutic potential.

U2 small nuclear ribonucleoprotein auxiliary factor 1 (U2AF1) gene is a pivotal splicing factor frequently mutated in various malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). U2AF1 plays a critical role in the recognition and processing of 3' splice sites during pre-mRNA splicing, thereby contributing to the regulation of gene expression and the generation of protein diversity. However, how U2AF1 contributes to the formation and regulation of different categories of chimeric RNAs remains elusive. In this study, we aimed to elucidate the involvement of U2AF1 in chimeric RNA formation and its regulatory impact on different categories of chimeric RNA. Employing knockdown and overexpression strategies in leukemia and esophageal cancer cell lines, we conducted paired-end RNA sequencing following U2AF1 knockdown to assess transcriptomic alterations and their influence on alternative splicing patterns. Subsequently, we utilized the SOAPfuse algorithm to detect and characterize chimeric RNAs from the paired-end RNA sequencing data. Our findings unveiled significant changes in the landscape of chimeric RNA upon U2AF1 knockdown, highlighting its critical role in chimeric RNA formation. This study provides novel insights into how U2AF1 mediates chimeric RNA formation and regulates distinct categories of chimeric RNA within leukemia cell lines. Thereby highlighting its potential as a biomarker for leukemia and other malignancies, promising avenues for future diagnostic and therapeutic developments.

Recurrent somatic mutations in the spliceosome genes SF3B1, SRSF2, and U2AF1 are frequently identified in patients with myeloid neoplasms, such as myelodysplastic syndromes. We characterized the in vivo consequences of expressing two hotspot mutations in U2AF1 that code for the S34F and Q157R substitutions. Our results indicate that the two mutations induce distinct hematopoietic phenotypes in mice, suggesting that the U2AF1 S34F and U2AF1 Q157R mutations should not be conflated as they may impact disease pathogenesis differently in patients. Mice expressing U2af1 S34F have a more severe reduction in their blood and bone marrow cell counts and reduced stem cell repopulating ability, compared to mice expressing U2af1 Q157R. The expression and splicing of target genes are largely unique between the mutations, in both mouse and human samples, potentially driving the phenotypic differences induced by either mutation. The two mutations co-occur with different gene mutations in patients and are not equally represented across myeloid neoplasms, suggesting that multiple mechanisms likely drive U2AF1-mutant disease pathogenesis. Collectively, our results support that U2AF1 S34F and U2AF1 Q157R mutations induce distinct hematopoietic, gene expression, and RNA splicing phenotypes in vivo. Larger population studies will be needed to determine if these phenotypic changes translate into clinico-pathologic differences in patients warranting separate classification.

Plants thrive in dynamic environments by activating sophisticated molecular networks that fine-tune their responses to stress. A key component of these networks is gene regulation at multiple levels, including precursor messenger RNA (pre-mRNA) splicing, which shapes the transcriptome and proteome landscapes. Through the precise action of the spliceosome complex, noncoding introns are removed and coding exons are joined to produce spliced RNA transcripts. While constitutive splicing always generates the same messenger RNA (mRNA), alternative splicing (AS) produces multiple mRNA isoforms from a single pre-mRNA, enriching proteome diversity. Remarkably, 80% of multiexon genes in plants generate multiple isoforms, underscoring the importance of AS in shaping plant development and responses to abiotic and biotic stresses. Recent advances in CRISPR-Cas genome and transcriptome editing technologies offer revolutionary tools to dissect AS regulation at molecular levels, unveiling the functional significance of specific isoforms. In this review, we explore the intricate mechanisms of pre-mRNA splicing and AS in plants, with a focus on stress responses. Additionally, we examine how leveraging AS insights can unlock new opportunities to engineer stress-resilient crops, paving the way for sustainable agriculture in the face of global environmental challenges.

Acute myeloid leukemia, myelodysplasia‑related (AML‑MR), a challenging and aggressive subtype of AML, is characterized by unique genetic abnormalities and molecular features, which contribute to its poor prognosis compared with other AML subtypes. The present review summarizes the current understanding of AML‑MR pathogenesis, highlighting notable advancements in genetic and cytogenetic insights. Critical mutations, such as those in the tumor antigen p53 and additional sex combs like 1 genes, and their role in disease progression and resistance to treatment, are explored. The review further investigates how clonal evolution and cellular microenvironment alterations drive AML‑MR transformation and impact patient outcomes. Despite the poor outlook typically associated with AML‑MR, developments in treatment approaches offer hope. The present review considers the efficacy of novel therapeutic agents, including CPX‑351, hypomethylating agents and targeted molecular therapies. Additionally, innovations in immunotherapy and allogeneic hematopoietic stem cell transplantation are discussed as promising avenues to improve patient survival rates. The challenges of treating AML‑MR, particularly in elderly and pretreated patients, underline the necessity for individualized treatment strategies that consider both the biological complexity of the disease and the overall health profile of the patient. The present review focuses on the mechanisms of AML‑MR transformation, highlighting factors that may offer a crucial theoretical foundation and pave the way for future applications in precision medicine. Future research directions include exploring novel targeted therapies and combination regimens to mitigate the transformation risks and enhance the quality of life of patients with AML‑MR.

To investigate the clinical characteristics and prognosis of mutation spots and concomitant gene mutations in myelodysplastic syndromes (MDS) with SF3B1 mutation (SF3B1mut).

Patients diagnosed with MDS at Shanghai Jiao Tong University School of Medicine Affiliated Sixth People's Hospital from October 2008 to November 2023 were enrolled in this study. SF3B1mut was identified by next-generation sequencing (NGS).

One hundred and seven (8.7%) cases harbored the SF3B1 mutation. The most frequent SF3B1mut, noted in 47.66% of all patients, was the hotspot K700E. K666 and R625 were observed in 24.30% and 9.35%, respectively. Two less frequent mutation subtypes accounted for 5.61% of H662 and 4.67% of E622. Patients with the K666 mutation showed more severe thrombocytopenia (p = 0.032), significantly lower NK cell percentage (p = 0.001), and the Th1/Th2 ratio (p = 0.018) in the bone marrow (BM). The overall survival (OS) in patients with E622 and H662 mutations was significantly longer than that of patients with the R625 mutation (p = 0.045) and the K666 mutation (p = 0.010). Multi-variance analysis showed the SF3B1 mutation involving the K666 hotspot independently predicted overall survival in MDS (HR 2.094, p = 0.050). Notably, most (11/13, 84.6%) of concomitant TP53 mutations were mono-hit, which did not affect the survival of patients in our cohort.

SF3B1mut patients with specific mutation spots and concomitant gene mutations showed distinct clinical features and prognosis. Consequently, a comprehensive study of specific subtypes is of great significance for improving the prognosis of patients with SF3B1 mutations.

RNA splicing, a highly regulated process performed by the spliceosome, is essential for eukaryotic gene expression and cellular function. Numerous cellular stresses, including oncogenic insults, dysregulate RNA splicing, often provoking inflammatory responses and cell death. However, the molecular signals generated by splicing aberrations and the mechanism by which cells sense and respond to these signals remain poorly understood. Here, we demonstrate that spliceosome inhibition induces the widespread formation of left-handed Z-form double-stranded RNA (Z-RNA), predominantly derived from mis-spliced exonic and intronic RNA transcripts in the nucleus. These Z-RNAs are exported to the cytoplasm in a RanGTP-dependent manner. Cytosolic sensing of accumulated Z-RNA by the host sensor Z-DNA-binding protein 1 (ZBP1) initiates cell death, primarily through RIPK3-MLKL-dependent necroptosis. Together, these findings reveal a previously uncharacterized mechanism in which ZBP1-mediated detection of Z-RNA serves as a critical response to global RNA splicing perturbations, ultimately triggering inflammatory cell death.

Immunotherapies for acute myeloid leukemia (AML) and other cancers are limited by a lack of tumor-specific targets. Here we discover that RNA-binding proteins and glycosylated RNAs (glycoRNAs) form precisely organized nanodomains on cancer cell surfaces. We characterize nucleophosmin (NPM1) as an abundant cell surface protein (csNPM1) on a variety of tumor types. With a focus on AML, we observe csNPM1 on blasts and leukemic stem cells but not on normal hematopoietic stem cells. We develop a monoclonal antibody to target csNPM1, which exhibits robust anti-tumor activity in multiple syngeneic and xenograft models of AML, including patient-derived xenografts, without observable toxicity. We find that csNPM1 is expressed in a mutation-agnostic manner on primary AML cells and may therefore offer a general strategy for detecting and treating AML. Surface profiling and in vivo work also demonstrate csNPM1 as a target on solid tumors. Our data suggest that csNPM1 and its neighboring glycoRNA-cell surface RNA-binding protein (csRBP) clusters may serve as an alternative antigen class for therapeutic targeting or cell identification.

Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here, we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic splicing machinery mutations. We utilized feature-barcoded peptide-major histocompatibility complex (MHC) dextramers to isolate neoantigen-reactive T cell receptors (TCRs) from healthy donors, patients with active myeloid malignancy, and following curative allogeneic stem cell transplant. Neoantigen-reactive CD8+ T cells were present in the blood of patients with active cancer and had a distinct phenotype from virus-reactive T cells with evidence of impaired cytotoxic function. T cells engineered with TCRs recognizing SRSF2 mutant-induced neoantigens arising from mis-splicing events in CLK3 and RHOT2 resulted in specific recognition and cytotoxicity of SRSF2-mutant leukemia. These data identify recurrent RNA mis-splicing events as sources of actionable public neoantigens in myeloid leukemias and provide proof of concept for genetically redirecting T cells to recognize these targets.

Distinguishing donor- vs. recipient-derived myelodysplastic neoplasm (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is challenging and has direct therapeutical implications.

Here, we took a translational approach that we used in addition to conventional diagnostic techniques to resolve the origin of MDS in a 38-year-old patient with acquired aplastic anemia and evolving MDS after first allo-HSCT. Specifically, we used single-cell transcriptional profiling to differentiate between donor- and recipient-derived bone marrow cells and established a strategy that additionally allows identification of cells carrying the MDS-associated U2AF1S34Y variant.

The patient exhibited mixed donor chimerism combined with severely reduced erythropoiesis and dysplastic morphology within the granulocytic and megakaryocytic lineage along with the MDS-associated U2AF1S34Y mutation in the bone marrow. Single-cell transcriptional profiling together with targeted enrichment of the U2AF1S34Y-specific locus further revealed that, while the immune compartment was mainly populated by donor-derived cells, myelopoiesis was predominantly driven by the recipient. Additionally, concordant with recipient-derived MDS, we found that U2AF1S34Y-mutated cells were exclusively recipient derived with X but not Y chromosome-specific gene expression.

Our study highlights the clinical potential of integrating high-resolution single-cell techniques to resolve complex cases for personalized treatment decisions.

The study was funded by intramural resources of the Charité - Universitätsmedizin Berlin and the Berlin Institute of Health.

Perturbation of mRNA splicing is commonly observed in human cancers and plays a role in various aspects of cancer hallmarks. Understanding the mechanisms and functions of alternative splicing (AS) not only enables us to explore the complex regulatory network involved in tumour initiation and progression but also reveals potential for RNA-based cancer treatment strategies. This review provides a comprehensive summary of the significance of AS in liver cancer, covering the regulatory mechanisms, cancer-related AS events, abnormal splicing regulators, as well as the interplay between AS and post-transcriptional and post-translational regulations. We present the current bioinformatic approaches and databases to detect and analyse AS in cancer, and discuss the implications and perspectives of AS in the treatment of liver cancer.

Myelodysplastic syndromes and other cancers are often associated with mutations in the U2 snRNP protein SF3B1. Common SF3B1 mutations, including K700E, disrupt SF3B1 interaction with the protein SUGP1 and induce aberrant activation of alternative 3' splice sites (ss), presumably resulting from aberrant U2/branch site (BS) recognition by the mutant spliceosome. Here, we apply a method of U2 IP-seq to profile BS binding across the transcriptome of K562 leukemia cells carrying the SF3B1 K700E mutation. For alternative 3' ss activated by K700E, we identify their associated BSs and show that they are indeed shifted from the WT sites. Unexpectedly, we also identify thousands of additional changes in BS binding in the mutant cells that do not alter splicing. These new BSs are usually very close to the natural sites, occur upstream or downstream, and either exhibit stronger base-pairing potential with U2 snRNA or are adjacent to stronger polypyrimidine tracts than the WT sites. The widespread imprecision in BS recognition induced by K700E with limited changes in 3' ss selection expands the physiological consequences of this oncogenic mutation.

The mechanisms by which somatic mutations of splicing factors, such as U2AF1S34F in lung adenocarcinoma, contribute to cancer pathogenesis are not well understood. Here, we used prime editing to modify the endogenous U2AF1 gene in lung adenocarcinoma cells and assessed the resulting impact on alternative splicing. These analyses identified KRAS as a key target modulated by U2AF1S34F. One specific KRAS mutation, G12S, generates a cryptic U2AF1 binding site that leads to skipping of KRAS exon 2 and generation of a non-functional KRAS transcript. Expression of the U2AF1S34F mutant reverts this exon skipping and restores KRAS function. Analysis of cancer genomes reveals that U2AF1S34F mutations are enriched in KRASG12S-mutant lung adenocarcinomas. A comprehensive analysis of splicing factor/oncogene mutation co-occurrence in cancer genomes also revealed significant co-enrichment of KRASQ61R and U2AF1I24T mutations. Experimentally, KRASQ61R mutation leads to KRAS exon 3 skipping, which in turn can be rescued by the expression of U2AF1I24T. Analysis of genomic and clinical patient data suggests that both U2AF1 mutations occur secondary to KRAS mutation and are associated with decreased overall patient survival. Our findings provide evidence that splicing factor mutations can rescue splicing defects caused by oncogenic mutations. More broadly, they demonstrate a dynamic process of cascading selection where mutational events are positively selected in cancer genomes as a consequence of earlier mutations.

Myeloid malignancies are a spectrum of clonal disorders driven by genetic alterations that cooperatively confer aberrant self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs). Induced pluripotent stem cells (iPSCs) can be differentiated into HSPCs and have been widely explored for modeling hematologic disorders and cell therapies. More recently, iPSC models have been applied to study the origins and pathophysiology of myeloid malignancies, motivated by the appreciation for the differences in human oncogene function and the need for genetically defined models that recapitulate leukemia development. In this review, we will provide a broad overview of the rationale, the challenges, practical aspects, history, and recent advances of iPSC models for modeling myeloid neoplasms. We will focus on the insights into the previously unknown aspects of human oncogene function and cooperativity gained through the use of these models. It is now safe to say that iPSC models are a mainstay of leukemia modeling "toolbox" alongside primary human cells from normal and patient sources.

RNA methylation and the machinery that regulates or "reads" its expression has recently been implicated in the pathogenesis of acute myeloid leukemia (AML) and other hematologic malignancies. Modulation of these epigenetic marks has started to become a reality as several companies around the world seek to leverage this knowledge therapeutically in the clinic. Although the bases of observed activity in AML have been described by numerous groups, the exact context in which these therapies will ultimately be used remains to be properly determined. While context is likely to be of great importance here, a more "global" mechanism of action might allow for more widespread applicability to multiple disease subtypes. In other areas such as the myelodysplastic and other preleukemic syndromes, data remain sparse. Ongoing work is needed to determine whether therapeutic modulation of RNA modifications is a viable and biologically plausible approach in these cases. Regardless of the outcomes, this is an exciting era for "epitranscriptomics" as we navigate a pathway forward. Here, I describe the current knowledge around RNA methylation and hematologic malignancies at the end of 2024 including some of the relevant questions that are yet to be answered.

Minor spliceosome is responsible for recognizing and excising a specific subset of divergent introns during the pre-mRNA splicing process. Mutations in the unique snRNA and protein components of the minor spliceosome are increasingly being associated with a variety of germline and somatic human disorders, collectively termed as minor spliceosomopathies. Understanding the mechanistic basis of these diseases has been challenging due to limited functional information on many minor spliceosome components. However, recently published cryo-electron microscopy (cryo-EM) structures of various minor spliceosome assembly intermediates have marked a significant advancement in elucidating the roles of these components during splicing. These structural breakthroughs have not only enhanced our comprehension of the minor spliceosome's functionality but also shed light on how disease-associated mutations disrupt its functions. Consequently, research focus is now shifting toward investigating how these splicing defects translate into broader pathological processes within gene expression pathways. Here we outline the current structural and functional knowledge of the minor spliceosome, explore the mechanistic consequences of its mutations, and discuss emerging challenges in connecting molecular dysfunctions to clinical phenotypes.

Highly recurrent somatic mutations in the gene encoding the core splicing factor SF3B1 are drivers of multiple cancer types. SF3B1 is a scaffold protein that orchestrates multivalent protein-protein interactions within the spliceosome that are essential for recognizing the branchsite (BS) and selecting the 3' splice site during the earliest stage of pre-mRNA splicing. In this review, we first describe the molecular mechanism by which multiple oncogenic SF3B1 mutations disrupt splicing. This involves perturbation of an early spliceosomal trimeric protein complex necessary for accurate BS recognition in a subset of introns, which leads to activation of upstream branchpoints and selection of cryptic 3' splice sites. We next discuss how specific transcripts affected by aberrant splicing in SF3B1-mutant cells contribute to the initiation and progression of cancer. Finally, we highlight the prognostic value and disease phenotypes of different cancer-associated SF3B1 mutations, which is critical for developing new targeted therapeutics against SF3B1-mutant cancers still lacking in the clinic.

SF3B1 is the most frequently mutated splicing factor in cancer. Mechanistically, such mutations cause missplicing by promoting aberrant 3' splice site usage; however, how this occurs remains controversial. To address this issue, we employed a computational screen of 600 splicing-related proteins to identify those whose reduced expression recapitulated mutant SF3B1 splicing dysregulation. Strikingly, our analysis revealed only two proteins whose loss reproduced this effect. Extending our previous findings, loss of the G-patch protein SUGP1 recapitulated almost all splicing defects induced by SF3B1 hotspot mutations. Unexpectedly, loss of the RNA helicase Aquarius (AQR) reproduced ~40% of these defects. However, we found that AQR knockdown caused significant SUGP1 missplicing and reduced protein levels, suggesting that AQR loss reproduced mutant SF3B1 splicing defects only indirectly. This study advances our understanding of missplicing caused by oncogenic SF3B1 mutations, and highlights the fundamental role of SUGP1 in this process.

U2AF1 is a core component of spliceosome and controls cell-fate specific alternative splicing. U2AF1 mutations have been frequently identified in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) patients, and mutations in U2AF1 are associated with poor prognosis in hematopoietic malignant diseases. Here, by forced expression of mutant U2AF1 (U2AF1 S34F) in hematopoietic and leukemic cell lines, we find that U2AF1 S34F causes increased reactive oxygen species (ROS) production. In hematopoietic cell line, a defect in mitochondrial function and DNA damage response deficiency are found in U2AF1 S34F expressing 32D cells. In leukemic cell line Molm13 cells, U2AF1 mutation leads to resistance to DNA damaging agents. Accumulation of DNA damage is also found in U2AF1 S34F expressing leukemic cells when treated with DNA damage agent. Finally, in our established hematopoietic-specific U2af1 S34F knock-in mice model, U2AF1 mutation leads to the development of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and causes DNA damage accumulation in hematopoietic cells. Our study provides evidence that U2AF1 mutation causes DNA damage response deficiency and DNA damage accumulation in hematopoietic cells, and suggests that mutant U2AF1 induced higher ROS production, resistance to DNA damaging agents and increased genomic instability may contribute to poor prognosis of AML patients with U2AF1 mutations.

Interferon-gamma (IFN-γ) is a critical cytokine that plays a pivotal role in immune system regulation. It is a key mediator of both cellular defense mechanisms and antitumor immunity. As the sole member of the type II interferon family, IFN-γ modulates immune responses by activating macrophages, enhancing natural killer cell function, and regulating gene expression across multiple cellular processes. Alternative splicing is a post-transcriptional gene expression regulatory mechanism that generates multiple mature messenger RNAs from a single gene, dramatically increasing proteome diversity without the need of a proportional genome expansion. This process occurs in 90-95% of human genes, with alternative splicing events allowing for the production of diverse protein isoforms that can have distinct-or even opposing-functional properties. Alternative splicing plays a crucial role in cancer immunology, potentially generating tumor neoepitopes and modulating immune responses. However, how alternative splicing affects IFN-γ's activity is still poorly understood. This review explores how alternative splicing regulates the expression and function of both upstream regulators and downstream effectors of IFN-γ, revealing complex mechanisms of gene expression and immune response modulation. Key transcription factors and signaling molecules of the IFN-γ pathway are alternatively spliced, and alternative splicing can dramatically alter IFN-γ signaling, immune cell function, and response to environmental cues. Specific splice variants can enhance or inhibit IFN-γ-mediated immune responses, potentially influencing cancer immunotherapy, autoimmune conditions, and infectious disease outcomes. The emerging understanding of these splicing events offers promising therapeutic strategies for manipulating immune responses through targeted molecular interventions.

Single-cell technologies offer a unique opportunity to explore cellular heterogeneity in health and disease. However, reliable identification of cell types and states represents a bottleneck. Available databases and analysis tools employ dissimilar markers, leading to inconsistent annotations and poor interpretability. Furthermore, current tools focus mostly on physiological cell types, limiting their applicability to disease. We developed the Cell Marker Accordion, a user-friendly platform providing automatic annotation and unmatched biological interpretation of single-cell populations, based on consistency weighted markers. We validated our approach on multiple single-cell and spatial datasets from different human and murine tissues, improving annotation accuracy in all cases. Moreover, we show that the Cell Marker Accordion can identify disease-critical cells and pathological processes, extracting potential biomarkers in a wide variety of disease contexts. The breadth of these applications elevates the Cell Marker Accordion as a fast, flexible, faithful and standardized tool to annotate and interpret single-cell and spatial populations in studying physiology and disease.

The spliceosome protein, SF3B1, is associated with U2 snRNP during early spliceosome assembly for pre-mRNA splicing. Frequent somatic mutations in SF3B1 observed in cancer necessitates the characterization of its role in identifying the branchpoint adenosine of introns. Remarkably, SF3B1 is the target of three distinct natural product drugs, each identified by their potent anti-tumor properties. Structural studies indicate that SF3B1 conformational flexibility is functionally important, and suggest that drug binding blocks the transition to a closed state of SF3B1 required for the next stage of spliceosome assembly. This model is confounded, however, by the antagonistic property of an inactive herboxidiene analog. In this study, we established an assay for evaluating the thermostability of SF3B1 present in the nuclear extract preparations employed for in vitro splicing studies, to investigate inhibitor interactions with SF3B1 in a functional context. We show that both active and antagonistic analogs of natural product inhibitors affect SF3B1 thermostability, consistent with binding alone being insufficient to impair SF3B1 function. Surprisingly, SF3B1 thermostability differs among nuclear extract preparations, likely reflecting its conformational status. We also investigated a synthetic SF3B1 ligand, WX-02-23, and found that it increases SF3B1 thermostability and interferes with in vitro splicing by a mechanism that strongly resembles the activity of natural product inhibitors. We propose that altered SF3B1 thermostability can serve as an indicator of inhibitor binding to complement functional assays of their general effect on splicing. It may also provide a means to investigate the factors that influence SF3B1 conformation.

Myelodysplastic Neoplasm (MDS) is a cancer associated with aging, often leading to acute myeloid leukemia (AML). One of its hallmarks is hypermethylation, particularly in genes responsible for DNA repair. This study aimed to evaluate the methylation and mutation status of DNA repair genes (single-strand - XPA, XPC, XPG, CSA, CSB and double-strand - ATM, BRCA1, BRCA2, LIG4, RAD51) in MDS across three patient cohorts (Cohort A-56, Cohort B-100, Cohort C-76), using methods like pyrosequencing, real-time PCR, immunohistochemistry, and mutation screening. Results showed that XPA had higher methylation in low-risk MDS compared to high-risk MDS. For double-strand repair genes, ATM displayed higher methylation in patients who transformed to AML (p = 0.016). ATM gene expression was downregulated in MDS compared to controls (p = 0.042). When patients were classified according to the WHO 2022 guidelines, ATM expression progressively decreased from low-risk subtypes (e.g., Hypoplastic MDS) to high-risk MDS and AML. Patients who transformed to AML had a higher 5mC/5hmC ratio compared to those who didn't (p = 0.045). Additionally, poor cytogenetic risk patients had higher tissue methylation scores than those with good risk (p = 0.035). Analysis using the cBioPortal platform identified ATM as the most frequently mutated DNA repair gene, with various mutations, such as frameshift and missense, most of which were classified as oncogenic. The findings suggest that ATM is frequently silenced or downregulated in MDS due to methylation or mutations, contributing to the progression to AML. This highlights ATM's potential role in the disease's advancement and as a target for future therapeutic strategies.

Cancer research remains clinically unmet in many areas due to limited access to patient samples and the lack of reliable model systems that truly reflect human cancer biology. The emergence of patient-derived induced pluripotent stem cells and engineered human pluripotent stem cells (hPSCs) has helped overcome these challenges, offering a versatile alternative platform for advancing cancer research. These hPSCs are already proving to be valuable models for studying specific cancer driver mutations, offering insights into cancer origins, pathogenesis, tumor heterogeneity, clonal evolution, and facilitating drug discovery and testing. This article reviews recent progress in utilizing hPSCs for clinically relevant cancer models and highlights efforts to deepen our understanding of fundamental cancer biology.

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and extensive ITK mRNA alternative splicing. We further introduce the most comprehensive list to date of proteins involved in splicing and leverage cysteine- and protein-directed activity-based protein profiling with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

Alternative splicing (AS) is a mechanism that generates translational diversity within a genome. Equally important is the dynamic adaptability of the splicing machinery, which can give preference to one isoform over others encoded by a single gene. These isoform preferences change in response to the cell's state and function. Particularly significant is the impact of physiological alternative splicing in T lymphocytes, where specific isoforms can enhance or reduce the cells' reactivity to stimuli. This process makes splicing isoforms defining features of cell states, exemplified by CD45 splice isoforms, which characterize the transition from naïve to memory states. Two developments have accelerated the use of AS dynamics for therapeutic interventions: advancements in long-read RNA sequencing and progress in nucleic acid chemical modifications. Improved oligonucleotide stability has enabled their use in directing splicing to specific sites or modifying sequences to enhance or silence particular splicing events. This review highlights immune regulatory splicing patterns with potential significance for enhancing anticancer immunotherapy.

X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in ALAS2, a gene responsible for heme synthesis. Treatments are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients. The absence of a suitable animal model has hindered the development of gene therapy research for this disease. We engineered a conditional Alas2-knockout (KO) mouse model using tamoxifen administration or treatment with lipid nanoparticles carrying Cre-mRNA and conjugated to an anti-CD117 antibody. Alas2-KOBM animals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells, hemoglobin, and hematocrit. In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, characterized by reduced oxidative phosphorylation, mitochondria's function, and activity of key tricarboxylic acid cycle enzymes. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. The IE was associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the liver, spleen, and bone marrow and the formation of ring sideroblasts. To investigate the potential of a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) to direct ALAS2 expression in erythroid cells. Infusion of bone marrow (BM) cells with 0.6 to 1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice improved complete blood cell levels, tissue iron accumulation, and survival rates. These findings suggest our vector could be curative in patients with XLSA.

Somatic mutation and fusion detection in acute myeloid leukemia to determine disease subtype and treatment regime is a common practice, but it's not yet employed in chronic myeloid leukemia (CML). CML is still monitored by routine quantitative determination of the BCR-ABL fusion transcript and treated with tyrosine kinase inhibitors (TKIs). Despite the availability of the three generations of TKIs, resistance and progression in CML pathogenesis suggest a strong role for somatic mutations. The present study aimed to identify the role of somatic mutation profiling in CML patients in disease management. 196 CML patient samples were used in this investigation, comprising 26 CML-BP, 8 CML-AP, and 162 CML-CP samples. Following cytogenetic analysis for confirmation, each sample was sequenced utilizing the Ion Torrent platform by a targeted panel. Of the 196 CML samples, 81 (41.33%) had 125 variations affecting 27 genes, while 115 (58.67%) harboured no mutations. The study revealed that ASXL1 (31.2%), ABL1 (14.4%), and TET2 (8.8%) were the most frequently altered genes. These genes are recognized indicators of CML disease. Few samples found with mutated GATA2, IDH1, NRAS, SETBP1, WT1, PHF6, KIT, etc. and fusions like RUNX1(5)-MECOM (2) and CBFB- MYH11 are indicative of disease progression. The outcome of this study suggests that mutational profiling of CML patients can help in the prognostication of disease. Based on the results of the study, the authors have also provided possible future risk stratification and diagnosis workflow for CML disease.

The online version contains supplementary material available at 10.1007/s12288-024-01808-9.

Lower risk (LR) myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem and progenitor disorders caused by the accumulation of somatic mutations in various genes including epigenetic regulators that may produce convergent DNA methylation patterns driving specific gene expression profiles. The integration of genomic, epigenomic, and transcriptomic profiling has the potential to spotlight distinct LR-MDS categories on the basis of pathophysiological mechanisms. We performed a comprehensive study of somatic mutations and DNA methylation in a large and clinically well-annotated cohort of treatment-naive patients with LR-MDS at diagnosis from the EUMDS registry (ClinicalTrials.gov.NCT00600860). Unsupervised clustering analyses identified six clusters based on genetic profiling that concentrate into four clusters on the basis of genome-wide methylation profiling with significant overlap between the two clustering modes. The four methylation clusters showed distinct clinical and genetic features and distinct methylation landscape. All clusters shared hypermethylated enhancers enriched in binding motifs for ETS and bZIP (C/EBP) transcription factor families, involved in the regulation of myeloid cell differentiation. By contrast, one cluster gathering patients with early leukemic evolution exhibited a specific pattern of hypermethylated promoters and, distinctly from other clusters, the upregulation of AP-1 complex members FOS/FOSL2 together with the absence of hypermethylation of their binding motif at target gene enhancers, which is of relevance for leukemic initiation. Among MDS patients with lower-risk IPSS-M, this cluster displayed a significantly inferior overall survival (p < 0.0001). Our study showed that genetic and DNA methylation features of LR-MDS at early stages may refine risk stratification, therefore offering the frame for a precocious therapeutic intervention.

Endogenous retroelements (EREs), which comprise half of the human genome, play a pivotal role in genome dynamics. Some EREs retained the ability to encode proteins, although most degenerated or served as a source for novel genes and regulatory elements during evolution. Despite ERE repression mechanisms developed to maintain genome stability, widespread pervasive ERE activation is observed in cancer including hematological malignancies. Challenging the perception of noncoding DNA as "junk," EREs are underestimated contributors to cancer driver mechanisms as well as antitumoral immunity by providing innate immune ligands and tumor antigens. This review highlights recent progress in understanding ERE co-option events in cancer and focuses on the controversial debate surrounding their causal role in shaping malignant phenotype. We provide insights into the rapidly evolving landscape of ERE research in hematological malignancies and their clinical implications in these cancers.

The transcription factor NF-κB plays a critical role in the control of innate and adaptive immunity and inflammation. Several recent studies have demonstrated that the mutation of different splicing factor genes, including SF3B1, SRSF2 and U2AF1, in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) result in hyperactive NF-κB signaling through the aberrant splicing of different target genes. The presence of U2AF1 and SF3B1 mutations in the bone marrow cells of MDS and AML patients induces oncogenic isoforms of the target gene IRAK4, leading to hyperactivation of NF-κB signaling and an increase in the fitness of leukemic stem and progenitor cells (LSPCs). The potent IRAK4 inhibitor CA-4948 has shown efficacy in both pre-clinical studies and MDS clinical trials, with splicing factor mutant patients showing the higher response rates. Emerging data has, however, revealed that co-targeting of IRAK4 and its paralog IRAK1 is required to maximally suppress LSPC function in vitro and in vivo by inducing cellular differentiation. These findings provide a link between the presence of the commonly mutated splicing factor genes and activation of innate immune signaling pathways in myeloid malignancies and have important implications for targeted therapy in these disorders.

Immunotherapy has revolutionized cancer treatment, but the limited availability of tumor-specific neoantigens still remains a challenge. The potential of alternative mRNA splicing-derived neoantigens as a source of new immunotherapy targets has gained significant attention. Tumors exhibit unique splicing changes and splicing factor mutations which are prevalent in various cancers and play a crucial role in neoantigen production. We present advances in splicing modulation approaches, including small-molecule drugs, decoy and splice-switching antisense oligonucleotides (SSOs), CRISPR, small interfering RNAs (siRNAs), and nonsense-mediated RNA decay (NMD) inhibition, that can be adapted to enhance antitumor immune responses. Finally, we explore the clinical implications of these approaches, highlighting their potential to transform cancer immunotherapy and broaden its efficacy.