Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations.

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

Attenuated cell cycle and DNA damage response transcriptome signatures and overrepresented cell adhesion processes imply accelerated progression in patients with lower-risk myelodysplastic neoplasms.

Patients with myelodysplastic neoplasms (MDS) are classified according to the risk of acute myeloid leukemia transformation. Some lower-risk MDS patients (LR-MDS) progress rapidly despite expected good prognosis. Using diagnostic samples, we aimed to uncover the mechanisms of this accelerated progression at the transcriptome level. RNAseq was performed on CD34+ ribodepleted RNA samples from 53 LR-MDS patients without accelerated progression (stMDS) and 8 who progressed within 20 months (prMDS); 845 genes were differentially expressed (ІlogFCІ > 1, FDR < 0.01) between these groups. stMDS CD34+ cells exhibited transcriptional signatures of actively cycling, megakaryocyte/erythrocyte lineage-primed progenitors, with upregulation of cell cycle checkpoints and stress pathways, which presumably form a tumor-suppressing barrier. Conversely, cell cycle, DNA damage response (DDR) and energy metabolism-related pathways were downregulated in prMDS samples, whereas cell adhesion processes were upregulated. Also, prMDS samples showed high levels of aberrant splicing and global lncRNA expression that may contribute to the attenuation of DDR pathways. We observed overexpression of multiple oncogenes and diminished differentiation in prMDS; the expression of ZEB1 and NEK3, genes not previously associated with MDS prognosis, might serve as potential biomarkers for LR-MDS progression. Our 19-gene DDR signature showed a significant predictive power for LR-MDS progression. In validation samples (stMDS = 3, prMDS = 4), the key markers and signatures retained their significance. Collectively, accelerated progression of LR-MDS appears to be associated with transcriptome patterns of a quiescent-like cell state, reduced lineage differentiation and suppressed DDR, inherent to CD34+ cells. The attenuation of DDR-related gene-expression signature may refine risk assessment in LR-MDS patients.

Advances in Microflow Cytometry-Based Molecular Detection Methods for Improved Future MDS Cancer Diagnosis.

Myelodysplastic syndromes (MDS) are a rare form of early-stage blood cancer that typically leads to leukemia and other deadly complications. The typical diagnosis for MDS involves a mixture of blood tests, a bone marrow biopsy, and genetic analysis. Flow cytometry has commonly been used to analyze these types of samples, yet there still seems to be room for advancement in several areas, such as the limit of detection, turnaround time, and cost. This paper explores recent advancements in microflow cytometry technology and how it may be used to supplement conventional methods of diagnosing blood cancers, such as MDS and leukemia, through flow cytometry. Microflow cytometry, a more recent adaptation of the well-researched and conventional flow cytometry techniques, integrated with microfluidics, demonstrates significant potential in addressing many of the shortcomings flow cytometry faces when diagnosing a blood-related disease such as MDS. The benefits that this platform brings, such as portability, processing speed, and operating cost, exemplify the importance of exploring microflow cytometry as a point-of-care (POC) diagnostic device for MDS and other forms of blood cancer.

Validation of the Artificial Intelligence Prognostic Scoring System for Myelodysplastic Syndromes in chronic myelomonocytic leukaemia: A novel approach for improved risk stratification.

Chronic myelomonocytic leukaemia (CMML) is a rare haematological disorder characterized by monocytosis and dysplastic changes in myeloid cell lineages. Accurate risk stratification is essential for guiding treatment decisions and assessing prognosis. This study aimed to validate the Artificial Intelligence Prognostic Scoring System for Myelodysplastic Syndromes (AIPSS-MDS) in CMML and to assess its performance compared with traditional scores using data from a Spanish registry (n = 1343) and a Taiwanese hospital (n = 75). In the Spanish cohort, the AIPSS-MDS accurately predicted overall survival (OS) and leukaemia-free survival (LFS), outperforming the Revised-IPSS score. Similarly, in the Taiwanese cohort, the AIPSS-MDS demonstrated accurate predictions for OS and LFS, showing superiority over the IPSS score and performing better than the CPSS and molecular CPSS scores in differentiating patient outcomes. The consistent performance of the AIPSS-MDS across both cohorts highlights its generalizability. Its adoption as a valuable tool for personalized treatment decision-making in CMML enables clinicians to identify high-risk patients who may benefit from different therapeutic interventions. Future studies should explore the integration of genetic information into the AIPSS-MDS to further refine risk stratification in CMML and improve patient outcomes.

Phase 1/2 study of CPX-351 for patients with Int-2 or high risk International Prognostic Scoring System myelodysplastic syndromes and chronic myelomonocytic leukaemia after failure to hypomethylating agents.

Failure after hypomethylating agents (HMAs) is associated with dismal outcomes in higher risk myelodysplastic syndromes (HR-MDS) or chronic myelomonocytic leukaemia (CMML). We aimed to evaluate the safety and preliminary activity of lower doses of CPX-351, a liposomal encapsulation of cytarabine and daunorubicin, in a single-centre, phase 1/2 study for patients with HR-MDS or CMML after HMA failure. Four doses of CPX-351 (10, 25, 50 and 75 units/m2 ) administered on Days 1, 3 and 5 of induction and Days 1 and 3 of consolidation were evaluated. Between June 2019 and June 2023, 25 patients were enrolled (phase 1: n = 15; phase 2: n = 10) including 19 (76%) with HR-MDS and 6 (24%) with CMML. Most common grade 3-4 non-haematological treatment-emergent adverse events were febrile neutropenia (n = 12, 48%) and lung infection (n = 5, 20%). Three patients (age >75) experienced cardiac toxicity at the 75 units/m2 dose. Further enrolment continued at 50 units/m2 . Four- and 8-week mortality were 0% and 8% respectively. The overall response rate was 56% with median relapse-free and overall survivals of 9.2 (95% CI 3.2-15.1 months) and 8.7 months (95% CI 1.8-15.6 months) respectively. These data suggest that lower doses of CPX-351 are safe. Further studies are needed to evaluate its activity.

Inflammatory profile of lower risk myelodysplastic syndromes.

The precise link between inflammation and pathogenesis of myelodysplastic syndrome (MDS) is yet to be fully established. We developed a novel method to measure ASC/NLRP3 protein specks which are specific for the NLRP3 inflammasome only. We combined this with cytokine profiling to characterise various inflammatory markers in a large cohort of patients with lower risk MDS in comparison to healthy controls and patients with defined autoinflammatory disorders (AIDs). The ASC/NLRP3 specks were significantly elevated in MDS patients compared to healthy controls (p < 0.001) and these levels were comparable to those found in patients with AIDs. The distribution of protein specks positive only for ASC was different to ASC/NLRP3 ones suggesting that other ASC-containing inflammasome complexes might be important in the pathogenesis of MDS. Patients with MDS-SLD had the lowest levels of interleukin (IL)-1ß, tumour necrosis factor (TNF), IL-23, IL-33, interferon (IFN) γ and IFN-α2, compared to other diagnostic categories. We also found that inflammatory cytokine TNF was positively associated with MDS progression to a more aggressive form of disease and IL-6 and IL-1ß with time to first red blood cell transfusion. Our study shows that there is value in analysing inflammatory biomarkers in MDS, but their diagnostic and prognostic utility is yet to be fully validated.

Deferasirox, an iron chelator, impacts myeloid differentiation by modulating NF-kB activity via mitochondrial ROS.

The iron chelator deferasirox (DFX) is effective in the treatment of iron overload. In certain patients with myelodysplastic syndrome, DFX can also provide a dramatic therapeutic benefit, improving red blood cell production and decreasing transfusion requirements. Nuclear Factor-kappa B (NF-kB) signalling has been implicated as a potential mechanism behind this phenomenon, with studies focusing on the effect of DFX on haematopoietic progenitors. Here, we examine the phenotypic and transcriptional effects of DFX throughout myeloid cell maturation in both murine and human model systems. The effect of DFX depends on the stage of differentiation, with effects on mitochondrial reactive oxygen species (ROS) production and NF-kB pathway regulation that vary between progenitors and neutrophils. DFX triggers a greater increase in mitochondrial ROS production in neutrophils and this phenomenon is mitigated when cells are cultured in hypoxic conditions. Single-cell transcriptomic profiling revealed that DFX decreases the expression of NF-kB and MYC (c-Myc) targets in progenitors and decreases the expression of PU.1 (SPI1) gene targets in neutrophils. Together, these data suggest a role of DFX in impairing terminal maturation of band neutrophils.

GATA2 deficiency syndrome: A compensatory mechanism gone awry?

In their paper, using zebrafish models, Gioacchino et al. have demonstrated the GATA2 haploinsufficiency, the genetic hallmark of GATA2 deficiency syndrome, promotes erythroid and myeloid cytopenia, and have discovered a self-regulatory mechanism to compensate GATA2 levels and protein function. Commentary on: Gioacchino et al. GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia. Br J Haematol 2024;205:580-593.

Adding the epigenomic signature to the prognostic jigsaw of myelodysplastic neoplasm?

Defining mechanisms of resistance to hypomethylating agents (HMAs) and biomarkers predictive of treatment response remains challenging in myelodysplastic neoplasm (MDS). Currently available prognostic tools that predict overall survival and transformation to acute myeloid leukaemia have not been powered to predict responses to HMAs. Noguera-Castells et al. comprehensively characterized the epigenomic profile in patients with MDS treated with azacitidine and described a methylation signature-based prognostic tool in predicting responses to azacitidine. Commentary on: Noguera-Castells et al. DNA methylation profiling of myelodysplastic syndromes and clinical response to azacitidine: a multicentre retrospective study. Br J Haematol 2024;204:1838-1843.

Monitoring molecular changes in the management of myelodysplastic syndromes.

The ongoing or anticipated therapeutic advances as well as previous experience in other malignancies, including acute myeloid leukaemia, have made molecular monitoring a potential interesting tool for predicting outcomes and demonstrating treatment efficacy in patients with myelodysplastic syndromes (MDS). The important genetic heterogeneity in MDS has made challenging the establishment of recommendations. In this context, high-throughput/next-generation sequencing (NGS) has emerged as an attractive tool, especially in patients with high-risk diseases. However, its implementation in clinical practice still suffers from a lack of standardization in terms of sensitivity, bioinformatics and result interpretation. Data from literature, mostly gleaned from retrospective cohorts, show NGS monitoring when used appropriately could help clinicians to guide therapy, detect early relapse and predict disease evolution. Translating these observations into personalized patient management requires a prospective evaluation in clinical research and remains a major challenge for the next years.

Decreased spermatogonial numbers in boys with severe haematological diseases.

This study examines spermatogonial numbers in testicular samples from 43 prepubertal patients undergoing haematopoietic stem cell transplantation (HSCT). High-dose chemotherapy and/or radiation during HSCT can impact spermatogenesis requiring fertility preservation. Results show that 49% of patients have decreased and 19% severely depleted spermatogonial pool prior to HSCT. Patients with Fanconi anaemia exhibit significantly reduced spermatogonial numbers. Patients with immunodeficiency or aplastic anaemia generally present within the normal range, while results in patients with myelodysplastic syndrome or myeloproliferative neoplasm vary. The study emphasizes the importance of assessing spermatogonial numbers in patients with severe haematological diseases for informed fertility preservation decisions.

Incorporation mutational profile might reduce the importance of blast count in prognostication of low-risk myelodysplastic syndromes.

Addition of molecular data to prognostic models has improved risk stratification of myelodysplastic neoplasms (MDS). However, the role of molecular lesions, particularly in the group of low-risk disease (LR-MDS), is uncertain. We evaluated a set of 227 patients with LR-MDS. Overall survival (OS) and probability of leukaemic progression were the main endpoints. RUNX1 was associated with lower OS and SF3B1 with a reduced risk of death (HR: 1.7, 95% CI, 1.1-2.9; p = 0.05; and HR: 0.23, 95% CI 0.1-0.5; p < 0.001; respectively). TP53 and RUNX1 mutations were predictive covariates for the probability of leukaemic progression (p < 0.001). Blast percentage, neither analysed as categorical (<5% vs. 5%-9%; HR: 1.3, 95% CI, 0.7-2.9; p = 0.2) nor as a continuous variable (HR: 1.07, 95% CI, 0.9-1.1; p = 0.07), had impact on survival or probability of progression (sHR: 1.05, 95% CI, 0.9-1.1; p = 0.2). These results retained statistical significance when analysis was restricted to the definition of LR-MDS according to the WHO 2022 and ICC classifications (<5% blasts). Thus, with the incorporation of molecular data, blast percentage happens to lose clinical significance both for survival and probability of progression in the group of patients with LR-MDS.

Secondary primary malignancies after CD-19 directed CAR-T-cell therapy in lymphomas: A report from the Italian CART-SIE study.

Secondary primary malignancies (SPM) have been reported after anti-BCMA or anti-CD19 chimeric antigen receptor (CAR)-T-cell therapies. While the cytotoxic effect of antecedent therapies, including chemotherapy and radiotherapy, has been well established, few data are available on risk related to CAR-T immunotherapies. The study aimed to analyse the incidence of SPM in 651 patients enrolled in the Italian prospective observational CART-SIE study. SPMs were documented in 4.3% (28/651), and the most frequent SPMs were haematological malignancies. In conclusion, the frequency of SPMs in our cohort of heavily pretreated patients receiving CAR-T was relatively low and consistent with previous studies.

GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia.

The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.

DDX41: exploring the roles of a versatile helicase.

DDX41 is a DEAD-box helicase and is conserved across species. Mutations in DDX41 have been associated with myeloid neoplasms, including myelodysplastic syndrome and acute myeloid leukemia. Though its pathogenesis is not completely known, DDX41 has been shown to have many cellular roles, including in pre-mRNA splicing, innate immune sensing, ribosome biogenesis, translational regulation, and R-loop metabolism. In this review, we will summarize the latest understandings regarding the various roles of DDX41, as well as highlight challenges associated with drug development to target DDX41. Overall, understanding the molecular and cellular mechanisms of DDX41 could help develop novel therapeutic options for DDX41 mutation-related hematologic malignancies.

Expanding the understanding of telomere biology disorder with reports from two families harboring variants in ZCCHC8 and TERC.

Telomere biology disorder (TBD) can present within a wide spectrum of symptoms ranging from severe congenital malformations to isolated organ dysfunction in adulthood. Diagnosing TBD can be challenging given the substantial variation in symptoms and age of onset across generations. In this report, we present two families, one with a pathogenic variant in ZCCHC8 and another with a novel variant in TERC. In the literature, only one family has previously been reported with a ZCCHC8 variant and TBD symptoms. This family had multiple occurrences of pulmonary fibrosis and one case of bone marrow failure. In this paper, we present a second family with the same ZCCHC8 variant (p.Pro186Leu) and symptoms of TBD including pulmonary fibrosis, hematological disease, and elevated liver enzymes. The suspicion of TBD was confirmed with the measurement of short telomeres in the proband. In another family, we report a novel likely pathogenic variant in TERC. Our comprehensive description encompasses hematological manifestations, as well as pulmonary and hepatic fibrosis. Notably, there are no other reports which associate this variant to disease. The families expand our understanding of the clinical implications and genetic causes of TBD.

In Vitro Insights Into the Influence of Marrow Mesodermal/Mesenchymal Progenitor Cells on Acute Myelogenous Leukemia and Myelodysplastic Syndromes.

The study of marrow-resident mesodermal progenitors can provide important insight into their role in influencing normal and aberrant hematopoiesis as occurs in acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS). In addition, the chemokine competency of these cells provides links to the inflammatory milieu of the marrow microenvironment with additional implications for normal and malignant hematopoiesis. While in vivo studies have elucidated the structure and function of the marrow niche in murine genetic models, corollary human studies have not been feasible, and thus the use of culture-adapted mesodermal cells has provided insights into the role these rare endogenous niche cells play in physiologic, malignant, and inflammatory states. This review focuses on culture-adapted human mesenchymal stem/stromal cells (MSCs) as they have been utilized in understanding their influence in AML and MDS as well as on their chemokine-mediated responses to myeloid malignancies, injury, and inflammation. Such studies have intrinsic limitations but have provided mechanistic insights and clues regarding novel druggable targets.

Smaller Cerebellar Lobule VIIb is Associated with Tremor Severity in Parkinson's Disease.

Alterations in the cerebellum's morphology in Parkinson's disease (PD) point to its pathophysiological involvement in this movement disorder. Such abnormalities have previously been attributed to different PD motor subtypes. The aim of the study was to relate volumes of specific cerebellar lobules to motor symptom severity, in particular tremor (TR), bradykinesia/rigidity (BR), and postural instability and gait disorders (PIGD) in PD. We performed a volumetric analysis based on T1-weighted MRI images of 55 participants with PD (22 females, median age 65 years, Hoehn and Yahr stage 2). Multiple regression models were fitted to investigate associations between volumes of cerebellar lobules with clinical symptom severity based on MDS-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) part III score and sub-scores for TR, BR, and PIGD; adjusted for age, sex, disease duration, and intercranial volume as cofactors. Smaller volume of lobule VIIb was associated with higher tremor severity (P = 0.004). No structure-function relationships were detected for other lobules or other motor symptoms. This distinct structural association denotes the involvement of the cerebellum in PD tremor. Characterizing morphological features of the cerebellum leads to a better understanding of its role in the spectrum of motor symptoms in PD and contributes further to identifying potential biological markers.