10 research outputs found
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Splicing Factor 3b Subunit 1 (SF3B1) mediates Mitochondrial Iron Overload In Myelodysplastic Syndromes With Ring Sideroblasts By Alternative Splicing Of Mitoferrin-1 (SLC25A37)
Abstract
Splicing factor 3b subunit 1, SF3B1 contributes to the formation of ring sideroblasts (RS) in Myelodysplastic syndromes (MDS). Abnormalities in iron trafficking have been implicated in the pathogenesis of refractory anemia with RS (RARS) and RARS with thrombocytosis (RARS-T). In RARS/-T, light microscopy and traditional iron profiles do not accurately reflect intracellular iron status. SF3B1 mutant (MUT, n=25) and wild type (WT, n=8) RARS patients (pts) have no difference in iron profiles (ferritin (ng/mL): 1244 ng/mL ± 926 vs 1215 ± 1065; total iron binding capacity (ug/dL): 252 ± 80 vs 234 ± 50). We previously reported distinct differences in iron distribution between SF3B1 MUT and WT pts based on transmission electron microscopy (EM). Coarse iron deposits are present in the mitochondria of SF3B1 MUT while smaller iron deposits are found in WT. We hypothesized that SF3B1 mutations affect distinct downstream targets leading to the iron phenotype differences in MUT vs WT. To study these differences, we performed a series of EM, flow cytometric (FC) and RNA-sequencing experiments. Quantification of iron by scoring deposits of iron/grid showed higher amount of mitochondrial iron in MUT vs WT by EM. We used a FC based-approach to quantify the cytoplasmic and mitochondrial iron utilizing the properties of two cell-permeant compounds (calcein-AM and rhodamine-B) being retained in the cytoplasm and the mitochondria, respectively. In keeping with our EM studies, we found that SF3B1 MUT have a higher % of rhodamine-B+ cells compared to WT RARS pts and healthy subjects (77 vs 19 vs 0.87%; n=6) indicating that MUT accumulates more mitochondrial iron compared to WT. Cytoplasmic iron was not different between MUT and WT (57 vs 43%) but higher than healthy subjects (1.5%; p=0.02), suggesting that MUT store more mitochondrial iron compared to WT pts. Sf3b1+/- mice also showed higher iron stores in the mitochondria rather than in the cytosplasm (85 vs 16%; n=4). To understand the factors leading to increased mitochondrial iron, we analyzed the transcriptome of BM cells derived from a homogeneous group of SF3B1 MUT (n=3), WT RARS (n=3), and healthy subjects (n=3). Total RNA (1.5-3ug) was subjected to RNA-sequencing using Illumina HiSeq2000. Twenty-million sequencing reads were interrogated. A comprehensive bioinformatic analysis was conducted on three levels (exon usage, gene expression, pathway analysis). Global gene expression analysis detected significant gene expression changes in 59 genes (FDR < 0.2) between MUT and healthy subjects. Mitochondrial genes linked to iron pathophysiology were well represented in the analysis. A total of 354 genes with exon usage/ gene expression difference in at least 1 exon were investigated. One of the top candidate genes showing alternative splicing is SLC25A37 (chr:8p21.2), a mitochondrial iron importer that mediates Fe2+ incorporation into the mitochondria. SLC25A37 was associated with a 2-4 fold higher expression in MUT vs WT and healthy individuals (mean base=4145 vs 2451 vs 1058). We also investigated other genes important in iron metabolism including SLC25A38, PUS1, and GLRX5 but no differences in both exon usage and gene expression were noted supporting the different nature of acquired and congenital sideroblastic anemia (SA). Some mitochondrial genes important in iron trafficking showed no changes in exon usage but exhibited differences in gene expression suggesting that they are pathways independent of SF3B1 mutations but still contributing to iron metabolism. ALAS2 was down-regulated in MUT vs WT RARS (fold change (FC): 0.39) while both MUT and WT pts exhibited an increased level compared to healthy subjects (FC: 5.0 and 2.0). ABCB7 was down-regulated compared to healthy subjects in both groups (FC:0.45 and 0.42). Using bisulfite sequencing we found that MUT have significantly higher hypermethylation of ALAS2 compared to WT RARS pts (FDR<.01; p<.00036) suggesting that epigenetic modification may explain the reduced ALAS2 expression instead of a splicing defect. SLC25A37 has been linked to an erythropoietic protoporphyria variant but not to congenital or acquired SA. In summary, SF3B1 MUT have increased mitochondrial iron as shown by EM and FC vs WT RARS pts. SF3B1 mutations mediate increased mitochondrial iron and RS formation by the alternative splicing of an iron transporter gene, SLC25A37, a novel pathway of iron-overload in the pathogenesis of MDS with RS.
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Biological Rationale for the Favorable Clinical Outcomes of Patients Carrying SF3B1 Mutations in Myelodysplastic Syndromes with Ring Sideroblasts
Abstract
Abstract 922
SF3B1, a RNA splicing factor is frequently mutated in refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). Aside from its phenotypic importance, SF3B1 has been associated with good outcomes in MDS. Consistent with others, we found favorable survival outcomes in SF3B1 mutants. To explain the clinical phenotypes (good outcomes, anemia, and less progression to acute myeloid leukemia (AML)), we delved into the consequences of SF3B1 dysfunction in MDS. We hypothesized that SF3B1 mutations result in changes in RNA splicing of key genes in erythropoiesis resulting in anemia. Moreover, we postulated that SF3B1 is a founder mutation in RS formation but insufficient as a sole abnormality to induce a deleterious phenotype. The acquisition of genetic/ epigenetic aberrations may trigger the final phenotype. We reported abundant iron deposits in the mitochondria of SF3B1 mutants. We hypothesized that SF3B1 mutations change the composition/ chemical valence of iron leading to free radicals and DNA damage. We performed Energy-dispersive X-ray/ Electron-Energy Loss Spectroscopy on WT and SF3B1 mutants (n=2) finding that Fe2O3 is the most prevalent iron form. Changes in iron might predispose to DNA damage. We used flow cytometry to measure gamma-H2AX, a marker of DNA damage finding higher gamma-H2AX levels in bone marrow of WT (n=4; 26.35±28.36) vs SF3B1 mutants (n=8; 4.19±4.34), healthy subjects (n=4; 6.15±3.74) and other WT low-risk MDS (n=5; 7.6±1.6). Since increased DNA damage induces chromatin remodeling, we found that WT have more condensed chromatin with granular cytoplasm vs SF3B1 mutants (n=2). Further, RNA-sequencing showed alterations in histone deacetylases (HDAC1/HDAC2) and modification (ASXL1) in SF3B1 mutants. This links splicing mutations to epigenetic events and may represent the 2nd hit to induce the disease phenotype. Other targets were interrogated. Since SF3B1 interacts with members of Class II polycomb group (PcG) in Sf3b1+/− mice and PcG genes, like ASXL1/ EZH2 are mutated in MDS, we performed sequencing on PcG Class II genes (RNF2/ PCGF2). No mutation was found in RARS/RARS-T pts (n=34; SF3B1 mutant=26; WT=8). mRNA levels might be changed in these pts. We also elucidated the role of SF3B1 in the phenotype of anemia. RNA-sequencing has been instructive. Ribosomal proteins (RPS14 and RPS17) are implicated in the pathogenesis of 5q- MDS and Diamond-Blackfan anemia. Both RPS14/ RPS17 expression was different in SF3B1 mutant vs WT (n=2). Further, cytokine analysis showed a trend to higher IL-6 levels in WT vs mutants (n=3). IL-6 is associated with anemia and poor response to therapy in some diseases. We assessed the response to erythropoietin (EPO) and found that none of the WT RARS/-T (n=4) responded to EPO while 15/19 (79%) of SF3B1 mutants responded (p=0.008). It's suggested that SF3B1 mutants have better outcomes due to decreased AML transformation. To dissect this point, we looked at chromosomal defects in SF3B1 mutants (n=27) and WT (n=11), finding that 45% of WT carried complex karyotype (del(5q), del(17p) del(7), and i(17p); p=0.0009) while only 18% of mutants carried one additional abnormality (del(20q), inv3, and 16p). We also checked for concomitant mutations in genes of methylation (TET2, DNMT3A, IDH1/2), histone (ASXL1, UTX, EZH2), transcription (RUNX1, JAK2, TP53), signaling (CBL, NRAS, KRAS), and splicing (U2AF1, SRSF2). We analyzed the global mutational status in RARS/RARS-T (n=38; WT, n=11 and SF3B1 mutants, n=27) finding that 5/11 (45%) WT carried in total 6 other methylation (n=2), transcription (n=1), and splicing (n=3) gene mutations. Further, 15/27 (56%) of SF3B1 mutants carried a total of 16 mutations excluding SF3B1 in: methylation (n=9), transcription (n=4), splicing (n=1), histone (n=2). The most prevalent mutated genes (TET2, DNMT3A) predict for good treatment response. In-vitro treatment with decitabine (0.2 uM) showed changes in chromatin condensation, DNA damage, and response in SF3B1 mutants vs WT (n=2) suggesting that the pathogenetic effects of SF3B1 may be elicited through the methylation pathway. Lastly, SF3B1 predicts for good outcomes. SF3B1 may mediate effects on anemia through alterations in ribosomal function, cytokines, and epigenetic changes. Conversely, SF3B1 mutants have better survival and lower risk to AML evolution because of lower tendency to acquire poor prognostic defects.
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No relevant conflicts of interest to declare