SF3B1 mutations are recurrent in chronic lymphocytic leukemia (CLL), particularly enriched in clinically aggressive stereotyped subset #2. To investigate their impact, we conducted RNA-sequencing of 18 SF3B1MUT and 17 SF3B1WT subset #2 cases and identified 80 significant alternative splicing events (ASEs). Notable ASEs concerned exon inclusion in the non-canonical BAF (ncBAF) chromatin remodeling complex subunit, BRD9, and splice variants in eight additional ncBAF complex interactors. Long-read RNA-sequencing confirmed the presence of splice variants, and extended analysis of 139 CLL cases corroborated their association with SF3B1 mutations. Overexpression of SF3B1K700E induced exon inclusion in BRD9, resulting in a novel splice isoform with an alternative C-terminus. Protein interactome analysis of the BRD9 splice isoform revealed augmented ncBAF complex interaction, while exhibiting decreased binding of auxiliary proteins, including SPEN, BRCA2, and CHD9. Additionally, integrative multi-omics analysis identified a ncBAF complex-bound gene quartet on chromosome 1 with higher expression levels and more accessible chromatin in SF3B1MUT CLL. Finally, Cancer Dependency Map analysis and BRD9 inhibition displayed BRD9 dependency and sensitivity in cell lines and primary CLL cells. In conclusion, spliceosome dysregulation caused by SF3B1 mutations leads to multiple ASEs and an altered ncBAF complex interactome, highlighting a novel pathobiological mechanism in SF3B1MUT CLL.
- MeSH
- alternativní sestřih MeSH
- chronická lymfatická leukemie * genetika patologie metabolismus MeSH
- fosfoproteiny * genetika metabolismus MeSH
- lidé MeSH
- mutace * MeSH
- proteiny obsahující bromodoménu MeSH
- restrukturace chromatinu * MeSH
- sestřihové faktory * genetika metabolismus MeSH
- spliceozomy * metabolismus genetika MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.
- MeSH
- antibakteriální látky farmakologie MeSH
- beta-laktamy farmakologie MeSH
- chloramfenikol farmakologie MeSH
- dihydrofolátreduktasa genetika metabolismus MeSH
- Escherichia coli chemie genetika metabolismus MeSH
- guanosintetrafosfát analogy a deriváty metabolismus MeSH
- isoleucin-tRNA-ligasa genetika MeSH
- lékové interakce MeSH
- ligasy antagonisté a inhibitory genetika metabolismus MeSH
- mupirocin farmakologie MeSH
- proteosyntéza účinky léků MeSH
- ribozomy účinky léků metabolismus MeSH
- RNA transferová genetika metabolismus MeSH
- subcelulární frakce chemie účinky léků metabolismus MeSH
- tetracyklin farmakologie MeSH
- thiostrepton farmakologie MeSH
- tolerance léku * MeSH
- trimethoprim farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
