RESUMEN
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.
RESUMEN
OBJECTIVE: Using a multi-tissue, genome-wide gene expression approach, we recently identified a gene module linked to the extent of human atherosclerosis. This atherosclerosis module was enriched with inherited risk for coronary and carotid artery disease (CAD) and overlapped with genes in the transendothelial migration of leukocyte (TEML) pathway. Among the atherosclerosis module genes, the transcription cofactor Lim domain binding 2 (LDB2) was the most connected in a CAD vascular wall regulatory gene network. Here, we used human genomics and atherosclerosis-prone mice to evaluate the possible role of LDB2 in TEML and atherosclerosis. APPROACH AND RESULTS: mRNA profiles generated from blood macrophages in patients with CAD were used to infer transcription factor regulatory gene networks; Ldlr(-/-)Apob(100/100) mice were used to study the effects of Ldb2 deficiency on TEML activity and atherogenesis. LDB2 was the most connected gene in a transcription factor regulatory network inferred from TEML and atherosclerosis module genes in CAD macrophages. In Ldlr(-/-)Apob(100/100) mice, loss of Ldb2 increased atherosclerotic lesion size ≈2-fold and decreased plaque stability. The exacerbated atherosclerosis was caused by increased TEML activity, as demonstrated in air-pouch and retinal vasculature models in vivo, by ex vivo perfusion of primary leukocytes, and by leukocyte migration in vitro. In THP1 cells, migration was increased by overexpression and decreased by small interfering RNA inhibition of LDB2. A functional LDB2 variant (rs10939673) was associated with the risk and extent of CAD across several cohorts. CONCLUSIONS: As a key driver of the TEML pathway in CAD macrophages, LDB2 is a novel candidate to target CAD by inhibiting the overall activity of TEML.
Asunto(s)
Aterosclerosis/fisiopatología , Enfermedades de las Arterias Carótidas/patología , Quimiotaxis de Leucocito/fisiología , Enfermedad de la Arteria Coronaria/patología , Proteínas con Dominio LIM/fisiología , Factores de Transcripción/fisiología , Migración Transendotelial y Transepitelial/fisiología , Animales , Apolipoproteína B-100/genética , Enfermedades de las Arterias Carótidas/genética , Línea Celular Tumoral , Quimiocina CCL2/farmacología , Enfermedad de la Arteria Coronaria/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Proteínas con Dominio LIM/deficiencia , Proteínas con Dominio LIM/genética , Macrófagos/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/biosíntesis , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Migración Transendotelial y Transepitelial/genéticaRESUMEN
BACKGROUND: The Kruskal-Wallis test is a popular non-parametric statistical test for identifying expression quantitative trait loci (eQTLs) from genome-wide data due to its robustness against variations in the underlying genetic model and expression trait distribution, but testing billions of marker-trait combinations one-by-one can become computationally prohibitive. RESULTS: We developed kruX, an algorithm implemented in Matlab, Python and R that uses matrix multiplications to simultaneously calculate the Kruskal-Wallis test statistic for several millions of marker-trait combinations at once. KruX is more than ten thousand times faster than computing associations one-by-one on a typical human dataset. We used kruX and a dataset of more than 500k SNPs and 20k expression traits measured in 102 human blood samples to compare eQTLs detected by the Kruskal-Wallis test to eQTLs detected by the parametric ANOVA and linear model methods. We found that the Kruskal-Wallis test is more robust against data outliers and heterogeneous genotype group sizes and detects a higher proportion of non-linear associations, but is more conservative for calling additive linear associations. CONCLUSION: kruX enables the use of robust non-parametric methods for massive eQTL mapping without the need for a high-performance computing infrastructure and is freely available from http://krux.googlecode.com.