Nuclear inclusions of pathogenic ataxin-1 induce oxidative stress and perturb the protein synthesis machinery

Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management

Modulation of IL-6/STAT3 signaling axis in CD41FOXP32 T cells represents a potential antitumor mechanism of azacitidine

CD+1 T cells orchestrate immune responses and are actively engaged in shaping tumor immunity. Signal transducer and activator of transcription (STAT) signaling controls the epigenetic tuning of CD+1 T-cell differentiation and polarization, and perturbed STAT signaling networks in CD+1 T cells subvert antitumor immunity in malignancies. Azacitidine (AZA), the mainstay therapy for high-risk myelodysplastic syndromes (HR-MDS), affects CD+1 T-cell polarization and function, but whether this contributes to AZA efficacy is currently unknown. By using functional proteomic, transcriptomic, andmutational analyses in 73 HR-MDS patients undergoing AZA therapy, we demonstrate that responding patients exhibited a coordinated CD+1 T-cell immune response and downregulated the inflammatory cytokine signaling pathways in CD+1 T cells after AZA, in contrast to nonresponders who upregulated the same pathways. We further observed an AZA-mediated downregulation of intereukin-6 (IL-6)- induced STAT3 phosphorylation in CD+1FOXP32 conventional T cells (Tcons) that correlated independently with better response and survival, whereas it was also not associated with the mutation number and profile of the patients. The AZA-induced downregulation of IL-6/STAT3 axis in Tcons restored the STAT signaling architecture in CD+1 T-cell subsets, whereas STAT signaling networks remained disorganized in patients who upregulated IL-6/STAT3 activity in Tcons. Given the pivotal role of CD+1 T cells in adaptive immunity, our findings suggest that the downregulation of the IL-6/STAT3 pathway in Tcons potentially constitutes a previously unrecognized immune-mediatedmechanism of action of AZA and sets the scene for developing rational strategies of AZA combinations with IL-6/STAT3 axis inhibitors. © 2021 by The American Society of Hematology

Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness. What's new? In chronic lymphocytic leukemia (CLL), cases with unmutated immunoglobulin receptors (U-CLL) are generally associated with inferior outcome, albeit still displaying considerable heterogeneity. Might such differences in CLL progression be explained by epigenetics? In this study, the authors found that an unusually aggressive subset of CLLs called subset #8 has a distinctive DNA-methylation profile. They also found that p63 is a novel pro-survival factor for CLL cells. These molecular studies may lead to new prognostic biomarkers, and possibly new therapeutic targets, for CLL