Distinct Iron Architecture in SF3B1-Mutant Myelodysplastic Syndrome Patients Is Linked to an SLC25A37 Splice Variant with a Retained Intron

ORIGINAL ARTICLE Distinct iron architecture in SF3B1-mutant myelodysplastic syndrome patients is linked to an SLC25A37 splice variant with a retained intron

V Visconte1, N Avishai2, R Mahfouz1, A Tabarroki1, J Cowen2, R Sharghi-Moshtaghin2, M Hitomi3, HJ Rogers4, E Hasrouni1, J Phillips1, MA Sekeres1,5, AH Heuer2, Y Saunthararajah1,5, J Barnard6 and RV Tiu1,5

Perturbation in iron homeostasis is a hallmark of some hematologic diseases. Abnormal sideroblasts with accumulation of iron in the mitochondria are named ring sideroblasts (RS). RS is a cardinal feature of refractory anemia with RS (RARS) and RARS with marked thrombocytosis (RARS/-T). Mutations in SF3B1, a member of the RNA splicing machinery are frequent in RARS/-T and defects of this gene were linked to RS formation. Here we showcase the differences in iron architecture of SF3B1-mutant and wild-type (WT) RARS/-T and provide new mechanistic insights by which SF3B1 mutations lead to differences in iron. We found higher iron levels in SF3B1 mutant vs WT RARS/-T by transmission electron microscopy/spectroscopy/ﬂow cytometry. SF3B1 mutations led to increased iron without changing the valence as shown by the presence of Fe2 þ in mutant and WT. Reactive oxygen species and DNA damage were not increased in SF3B1-mutant patients. RNA-sequencing and Reverse transcriptase PCR showed higher expression of a speciﬁc isoform of SLC25A37 in SF3B1-mutant patients, a crucial importer of Fe2 þ into the mitochondria. Our studies suggest that SF3B1 mutations contribute to cellular iron overload in RARS/-T by deregulating SLC25A37.

Leukemia (2015) 29, 188–195; doi:10.1038/leu.2014.170

INTRODUCTION found in RARS and RARS with marked thrombocytosis (RARS/-T) Free iron in the erythroblasts of patients with refractory anemia even though crucial genes of the mitochondrial trafficking have was the first observation that led to the description of side- been found to be differentially regulated including overexpression roblastic anemia (SA).1 Ringed sideroblasts (RS) is a feature of ALAS2 and FTMT (ferritin-encoding gene) and downregulation observed in both congenital and acquired SA cases. RS are of ABCB7 in CD34 þ cells derived from RARS compared with erythroid precursors in the bone marrow (BM) with abnormal iron healthy subjects. The discovery of recurrent somatic mutations in deposition in the mitochondria that appears as a crystalline ring of splicing factor 3b subunit 1 (SF3B1), a component of the RNA- granules around the nucleus. Presence of 15% or more RS, splicing machinery in RARS/-T represents a key breakthrough in 9–11 accompanied by anemia, erythrodysplasia, o5% BM and o1% the understanding of the pathogenesis of RARS/-T. More peripheral blood (PB) blasts, define refractory anemia with RS importantly, our finding that SF3B1 mutations confer a different 12 (RARS). Patients fulfilling criteria for RARS with elevated platelet mitochondrial iron pattern in RARS/-T suggests that SF3B1 may counts are diagnosed as RARS-T. BM iron stores and diffuse be involved in the regulation of downstream proteins of the pattern of iron deposits are often detected and have been mitochondrial network. correlated with the presence of RS.2 During the years, In the present study, we examined the cellular iron phenotypes abnormalities in mitochondrial iron have been implicated in the in SF3B1-mutant and wild-type (WT) RARS/-T patients by perform- pathogenesis of both congenital and acquired SA.3–5 Genetic ing a series of novel electron microscopy techniques combined studies found that heritable mutations in an erythroid-specific with spectroscopy, flow cytometry and RNA-sequencing (RNA- mitochondrial gene, delta aminolevulinate synthase (ALAS2) cause Seq) experiments. Our study suggests that SF3B1-mutant RARS/-T X-linked SA. Mutations in ATP-binding cassette, subfamily B, patients have distinct iron distribution compared with their WT member 7 (ABCB7)—a mitochondrial inner iron transporter—were counterparts and that this is mediated through perturbations in also found in X-linked SA and spinocerebellar ataxia and in cases splicing patterns in key mitochondrial iron proteins, specifically of congenital SA with isodicentric (X)(q13).6 Homozygous SLC25A37. mutations in glutaredoxin 5 (GLRX5), in solute carrier family 25, member 38 (SLC25A38) and in pseudouridine synthase-1 (PUS-1) have been described in congenital SA. Reduced messenger RNA MATERIALS AND METHODS (mRNA) expression levels due to germline polymorphism in Patients and healthy subjects 5,7,8 ferrochelatase (FECH) were found in other cases of SA. No BM and PB cells were obtained from patients with MDS and healthy somatic or germline mutations in the above genes have been controls with written informed consent in accordance with the Declaration

1Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA; 2Department of Materials Science and Engineering, Swagelok Center for Surface Analysis of Materials, Case Western Reserve University, Cleveland, OH, USA; 3Electron Microscopy Facility, Case Western Reserve University, Cleveland, OH, USA; 4Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH, USA; 5Leukemia Program, Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA and 6Department of Quantitative Health Sciences, Cleveland Clinic, OH, USA. Correspondence: Dr RV Tiu, Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Ohio 44195, USA. E-mail: [email protected] Received 15 January 2014; revised 10 April 2014; accepted 19 May 2014; accepted article preview online 23 May 2014; advance online publication, 1 July 2014 Iron and SF3B1 mutations V Visconte et al 189 of Helsinki and approved by the Institutional Review Board of the USA) in 0.5% bovine serum albumin. Percentage of g-H2AX-positive cells Cleveland Clinic. Diagnosis was assigned according to 2008 World Health was analyzed with a CXP software on a FC500 flow cytometer. Organization classification criteria. Reactive oxygen species (ROS) were measured using CellROX Green Flow Cytometry Assay Kit (Life Technologies, Grand Island, NY, USA). PB or 5 Transmission electron microscopy, X-ray energy-dispersive BMMNCs (2–5 Â 10 cells) were washed and maintained in PBS. Cells were spectroscopy and electron energy-loss spectroscopy treated with hydrogen peroxide (H2O2; 1.5%), an inducer of oxidative stress for 10 min at 37 1C and 5% CO2. Untreated cells were used as control. After Fresh BM mononuclear cells (BMMNCs) were isolated using lymphocyte the incubation, cells were washed in PBS once and were stained with separation media (Corning cellgro, Manassas, VA, USA). One million CellROX Green reagent (0.025 mM) for 30 min at room temperature. Cells BMMNCs were first subjected to fixation, washing and dehydration in an were washed in PBS and then analyzed. All the acquisitions were done on ascending-graded alcohol mixture before being embedded in pure epoxy 12 an FC500 flow cytometer (Beckman Coulter, Miami, FL, USA). A total of resin as previously described. Specimens were initially evaluated by light 30 000 events were collected. microscopy of 1 mm plastic sections stained with a combination of toluidine blue and basic fuchsin dyes. Thin sections from selected tissue blocks were cut at 60–80 nm and supported on a 200 mesh copper grids. Sanger sequencing Sections were stained using osmium impregnation (OTOTO technique) in Direct sequencing was performed on SF3B1 (exon13–16; uc002uue.1)9,16 order to increase the stability under the electron beam, and examined and SLC25A37 (exons 1–4; uc003xdo.3) coding regions using an available using a Zeiss Libra 200 transmission electron microscope (Zeiss, set of primers (genome.ucsc.edu). Sequencing analyses were performed Oberkochen, Germany). Electron micrographs were taken using a Gatan using DNA from BM or PB mononuclear cells. Bidirectional sequencing was Orius CCD digital camera (Gatan, Inc., Pleasanton, CA, USA). Sections were performed by standard techniques using an ABI 3730xl DNA analyzer then analyzed to resolve the elemental composition, chemical status and (Applied Biosystems, Foster City, CA, USA). Mutational analysis was oxidation state of iron by scanning TEM (STEM) coupled with X-ray energy- assessed on the basis that alterations are somatic if they were not dispersive spectroscopy (XEDS) and electron energy-loss spectroscopy detected in healthy individuals, in CD3-positive cells (germline source) (EELS). STEM images were acquired with a high-angle annular dark-field derived from patients cells, in published SNP databases (dbSNP, http:// detector. XEDS is a powerful technique that can rapidly determine the www.ncbi.nlm.nih.gov/projects/SNP) and/or they were not reported as elemental composition of a sample. XEDS is the analytical technique most SNPs in previous publications. commonly used for the elemental analysis or chemical characterization of a specimen. The principle of XEDS is based on the fact that when a high- energy electron beam is focused on a specimen of interest, the incident RNA-Sequencing analysis/Transcript Variant detection beam can excite an electron in an inner shell producing the ejection of the latter from the shell and the formation of an electron hole. An electron Total RNA was isolated from BMMNCs of 6 RARS/-T patients (SF3B1 from an outer shell drops down to fill the hole, and the difference in mutant, n ¼ 3; WT, n ¼ 3) and three healthy subjects. RNA (1.5–3 mg) was energy between the higher-energy shell and the lower-energy shell is subjected to RNA-seq using Illumina HiSeq2000 (Otogenetics, Norcross, released in the form of an X-ray, which can be measured by XEDS. Some of GA, USA). Approximately 20 million sequencing reads were generated these emitted X-ray photons will enter the X-ray detector and create a per subject. Hundred base pair paired-end RNA-Seq reads were mapped to the hg19 RefSeq human transcriptome and spliceome by DNAnexus charge pulse that is proportional to the energy of the emitted X-ray 17 photon. The charge pulse is in turn converted to a voltage pulse, which is (http://dnanexus.com) using a Bayesian method where a read was further amplified by a field-effect transistor. The amplified voltage pulse mapped when its posterior probability of mapping exceeded 0.9. is then stored into a multichannel analyzer. As the energy of the X-rays is These filtered posterior probabilities were summed to generate characteristic of the atomic structure of the element from which they were fractional read counts per gene and exon, with probabilities from emitted, the elemental composition of the specimen can be determined splice junction spanning reads counted for each relevant exon. We used using the relative intensity. The Zeiss Libra 200 has a Noran XEDS system rounded gene and exon read counts as inputs for our differential attached to it. This system contains a Li drifted Si detector capable of an expression analyses. For differential gene expression analysis, we used TMM18 normalization energy resolution of 140 eV full-width half-maximum from a Mn Ka line. 19 EELS technique involves the measurement of the energy distribution of and the voom-limma approach from the R package limma version 3.18 electrons that have interacted with a specimen and lost energy due to with R version 3.0.2 in order to perform differential gene expression inelastic scattering. The distribution of transmitted electrons as a function analysis for comparisons of interest. of energy loss is called an energy-loss spectrum. Such spectrum carries For the differential exon usage analysis, we used the R package DEXSeq, invaluable information about the chemical composition of a sample as well version 1.4.0 with R version 2.15.2 to perform differential exon usage as information about bonding, coordination and charge transfer on an analysis for comparison of interest. For the gene set and exon set over/ B under representation analysis, we used the R package goseq version 1.6.0 atomic level due to its high-energy resolution ( 0.5 ev). This ability to 20 achieve such a high-energy resolution, when compared with XEDS, (http://www.bioconductor.org/packages/release/bioc/html/goseq.html) to perform over and under representation analysis of gene sets taking provides the ability to not only identify the elements present but also to 12 determine their chemical state. This technique is complementary to EDS length bias into account. analysis, as it provides the qualitative analysis of light elements. A detailed For differential transcript analysis, we first realigned the raw RNA-Seq description of both techniques can be found elsewhere.13,14 reads to the human transcriptome from Ensembl Release 73 using the STAR aligner21 version 2.3.1. Transcriptome-aligned reads were then passed to BitSeq version 0.7022 to generate 100 draws of fractional Flow cytometry transcript counts per subject. For each set of draws of the transcript counts, 18 19 Fresh PB or BMMNCs (3–5 Â 105 cells) were treated with calcein-AM we used TMM normalization with the voom-limma approach in the (0.05 mM) (Cat. #17783; Sigma-Aldrich, St Louis, MO, USA) and commercially R package limma version 3.16.3 to get differential transcript estimates and available rhodamine B (1 mM) (Cat. #79754; Sigma-Aldrich) for 20 min at variances, then combined the estimates and variances across draws using 23 room temperature. Cells were washed and reconstituted in PBS before flow the multiple imputation combining rules to get our final fold change (FC) cytometry acquisition. RhoNox-1 was prepared in-house by chemical estimates and P-values for each comparison of interest. This approach modification of commercial rhodamine B as previously described in order accounts for uncertainty about the transcript counts, which in general are to selectively detect Fe2 þ .15 For the experiment conducted with RhoNox-1, not directly observed. 3 Â 105 BMMNCs were incubated in Iscove’s modified Dulbecco’s serum- free medium with FeO4S (100 mM) for 30 min at 37 1C, 5% CO2. After incubation, cells were washed and incubated with RhoNox-1 (2 mM) for Reverse transcriptase PCR 30 min at room temperature. Cells were washed before acquisition. FlowJo BM cells were isolated from healthy subjects (n ¼ 2), SF3B1 mutant (n ¼ 2) (Tree Star, Ashland, OR, USA) was used for the analysis of the RhoNox-1 and WT (n ¼ 2) RARS/-T patients and used as a source of RNA. RNA was experiment. Double strands DNA breaks were measured by staining PB or isolated using NucleoSpin RNA II (Macherey-Nagel, Bethlehem, PA, USA) BMMNCs with an antibody to H2AX phosphorylated on serine residue 139 and converted to cDNA using iScript RT Supermix (Bio-Rad Laboratories, (g-H2AX). Cells (2–5 Â 105) were fixed with 2% paraformaldehyde, Hercules, CA, USA). mRNA levels of the SLC25A37 normal and splice variant permeabilized with 90% cold methanol and stained with Alexa 488- with a retained intron were measured using specific primer sets.24 conjugated g-H2AX antibody (Cell Signaling Technology, Danvers, MA, Amplified PCR products were visualized using a 1.2% agarose gel.

& 2015 Macmillan Publishers Limited Leukemia (2015) 188 – 195 Iron and SF3B1 mutations V Visconte et al 190 RESULTS staining procedures, and oxygen and carbon, which represent SF3B1-mutant RARS/-T patients showed more abundant iron organic materials. Iron was detected in the area of interest (area deposits in the mitochondria #1) (Figures 2b and e) and was absent in area #2 (Figure 2c) We analyzed the iron profile of SF3B1 mutant (n ¼ 25) and WT confirming that the presence of iron was specific. Except for (n ¼ 8) RARS patients, finding no difference in iron parameters like oxygen, carbon and iron, all others elements were used in the ferritin (1244 ng/ml±926 vs 1215 ng/ml±1065) and total iron- staining process to enhance the image contrast and maintain the binding capacity (252 mg/dl±80 vs 234 mg/dl±50). Traditional stability of the sample under high current electron beam. Iron was iron profile measurements do not accurately reflect intracellular also shown in cells of WT RARS patients (Figures 2d and e) but at iron status. Focusing our attention on the ultrastructure of BM cells lower intensity and amounts when compared with mutant patient derived from RARS patients, we previously reported the presence (Figure 2b). The elemental composition of the iron deposits in of more iron deposits in SF3B1 mutant compared with WT RARS SF3B1 mutant and WT RARS patients were similar (Figures 2b and patients using TEM.12 In order to test whether SF3B1 mutations e). Microanalysis of iron deposits using EELS showed that the lead to iron overload in cells of RARS/-T patients, we isolated fresh spectrum peak is close to 708 eV, the peak shape and position 2 þ BMMNCs from SF3B1 WT (n ¼ 2) and mutant (n ¼ 3) RARS/-T confirm the presence of divalent iron (Fe ) in the deposits patients and analyzed stained sections for elemental composition, (Figure 2f). We also compared the peak and shape of the iron iron chemical status and oxidation state of iron by using a found in the deposits of erythroblasts from RARS/-T with the ones microanalysis technique, which combines STEM, XEDS and EELS. of the iron in different elements and prove that both peak and 2 þ 25 As illustrated in the representative case in Figures 1a and b, the shape identified Fe (Supplementary Figures S1a and b). patient carrying SF3B1 mutation (K700E) showed more iron deposits in the mitochondria (white spots indicated with arrows) compared with the WT RARS/-T patient. A dark-field STEM image SF3B1 mutations lead to divalent iron overload in the depicts the presence and configuration of iron deposits in the mitochondria of RARS/-T patients mitochondria as evidenced by visualizing the outer membrane of We next compared the mitochondrial and cytoplasmic iron the mitochondria in an erythroblast of an SF3B1-mutant patient content between SF3B1 mutant and WT RARS/-T patients using (Figure 1c). Iron appears as a perinuclear ring of blue granules by a flow cytometric approach. BMMNCs were loaded with two iron- traditional Prussian blue staining. Prussian blue staining identified binding fluorochromes, like calcein-AM and rhodamine B. Dot 68% of RS in the BM (Figure 1d). plots illustrate the percentage of rhodamine B and calcein-AM- labeled cells from a representative SF3B1 WT and mutant patient. For calcein-AM-positive cells, two live gates (O4 and P) were set Divalent iron (Fe2 þ ) is the most prevalent iron form of the iron based on the presence of two populations with different deposits in RARS/-T patients fluorescence (Figure 3a, upper panels). BMMNCs from SF3B1 In order to clarify the chemical status of iron in its mitochondrial mutant (n ¼ 3) accumulate more cytoplasmic chelatable iron as deposits of BMMNCs in SF3B1 mutant and WT RARS/-T patients, shown by a slight increase in the percentage of calcein-AM- we compared their STEM images (Figures 2a and d). XEDS was positive cells compared with WT RARS patients (n ¼ 3), although used to confirm the presence of iron in the deposits by comparing this increase was not statistically significant (P gate: 57.4%±21.4 the area of iron deposits (area #1; Figure 2a) to the other area in vs 38.4%±11.1; P ¼ 0.24). Using rhodamine B, we analyzed the the cell (area #2; Figure 2a). As shown in Figures 2b and e, XEDS mitochondrial iron content and found that SF3B1 mutant (n ¼ 4) identified a spectrum of elements present in the scanning area. has significantly higher amount of mitochondrial iron as shown by Both areas contained nickel, cadmium and lead resulting from the an increase in the percentage of rhodamine B-positive cells

Figure 1. SF3B1 mutations lead to a different iron pattern in cells from RARS/-T patients. TEM analysis on BM sections (1 mm) shows abundant iron deposits in an SF3B1 mutant (a) compared with WT (b) (white arrow). (c) STEM image shows that iron deposits are localized in the mitochondria of cells from an SF3B1-mutant RARS patient. (d) Light microscopy image of a Prussian blue stained BM aspirate of a patient with RARS shows that iron-laden mitochondria appears in the shape of perinuclear blue granules in the erythroid precursors. RS were scored as 68%. Electron micrographs were taken with a Gatan Orius CCD digital camera.

Leukemia (2015) 188 – 195 & 2015 Macmillan Publishers Limited Iron and SF3B1 mutations V Visconte et al 191

Figure 2. SF3B1 mutations do not lead to a change in the chemical valence of the iron in RARS/-T patients. A dark-field STEM image shows the presence of deposits in SF3B1 mutant (a) and WT (d). (b, e) Chemical analysis of iron deposits by X-ray energy dispersive spectrometry confirmed the presence of iron (Fe) peak in area 1 (orange rectangle) (b, c) compared with area 2 (blue rectangle). Area 2 was used as negative control and no iron deposits were found (c). (f) EELS spectrum of Fe deposits is close to 708 eV and confirms that the iron deposits are ferrous ions (Fe2 þ )inSF3B1 mutant and WT patients. Electron micrographs were taken with a Gatan Orius CCD digital camera.

Figure 3. SF3B1 mutations lead to an increase in mitochondrial iron content in RARS/-T patients. BMMNCs from SF3B1 mutant and WT RARS/-T patients were labeled with cytoplasmic calcein-AM, and mitochondrial rhodamine B dyes. Cells were then washed, analyzed by flow cytometry and dot plots generated. SF3B1-mutant patients show a slight but insignificant increased level of cytoplasmic iron (calcein-AM-positive cells) (a; upper right panel) and a significant marked increased mitochondrial iron (a; lower right panel) compared with WT patients. (b) Bar graph represents the mean±s.d. of the percentage of positive cells for rhodamine B and calcein-AM in SF3B1 mutant (n ¼ 3) (gray bars) vs WT (n ¼ 3) (white bars) RARS/-T patients. Nonparametric two-tailed test was used to assess significance. *Po0.05.

(82.3%±10.2 vs 23.6%±14.9; P ¼ 0.004) compared with WT RARS ROS and DNA damage were not increased in SF3B1 mutant patients (n ¼ 2) (Figure 3b). As an internal control, calcein-AM and compared with SF3B1 WT RARS/-T patients rhodamine B fluorescence were measured in BMMNCs from We and others have reported that SF3B1 mutant show better healthy subjects (n ¼ 3) and were found to be at lower levels clinical outcomes compared with WT RARS patients.10,12,16 With compared with patients cells (calcein-AM: 1.50%±0.53; rhoda- the intent of explaining the biological rationale of better mine B: 0.87±0.29) (data not shown). We then chemically outcomes in SF3B1 mutants, we focused our attention in modified in-house a commercially available rhodamine B as elucidating the consequences of iron overload. Accumulation of already reported.15 Modified rhodamine B (RhoNox-1) is a labile iron can cause cellular and DNA damage. Using flow selective fluorescent probe for divalent iron (Fe2 þ ). We first cytometry, we measured phosphorylated H2AX (g-H2AX) as a treated BMMNCs from an SF3B1 mutant and WT RARS patient with marker of double-strand DNA breaks. Increased percentages in the FeO4S (100 mM), as exogenous source of divalent iron for 30 min number of g-H2AX-positive cells proportionally correspond to and thereafter utilized RhoNox-1 (2 mM) in order to detect increased DNA damage. We found that PB or BM cells from SF3B1 endogenous labile divalent iron. As shown in Figures 4a and b, mutant (n ¼ 8; BM/PB ¼ 4/4) have significant less DNA damage an increase in the basal level of mitochondrial labile Fe2 þ was compared with WT (n ¼ 3; BM/PB ¼ 2/1) RARS patients, and found in the SF3B1 mutant vs WT patient in RhoNox-1-labeled healthy subjects (n ¼ 4; BM/PB ¼ 4/0) as shown by the lower untreated cells (geometric mean fluorescence index: 32.1 vs 24.9; percentage of g-H2AX-positive cells (4.19±4.34 vs 12.2±4.33 vs 1.28-fold) and in RhoNox-1-labeled FeO4S-treated cells (geometric 6.15±3.74; P ¼ 0.01) (Figure 5a). Iron overload leads to the mean fluorescence index: 55.7 vs 33.9; 1.64-fold) confirming our increased production of ROS, especially hydroxyl radicals. Using a previous results and also suggesting that SF3B1 mutants might be CellROX green reagent, we did not find a difference in ROS levels more sensitive to exogenous supply of iron. between SF3B1 mutant (n ¼ 4) and WT (n ¼ 3) RARS/-T patients in

& 2015 Macmillan Publishers Limited Leukemia (2015) 188 – 195 Iron and SF3B1 mutations V Visconte et al 192

Figure 4. SF3B1 mutations lead to an increase in ferrous iron deposition in the mitochondria of RARS/-T patients. (a) BMMNCs from an SF3B1 mutant and a WT RARS patient were incubated in serum-free cell culture media with FeO4S (100 mM) for 30 min at 37 1C5%CO2. Cells were washed after the incubation and labeled with the chemical-modiﬁed rhodamine B (RhoNox-1, 2 mM) for 20 min at room temperature. FeO4S- untreated cells were used as control. Histograms show the shifts in the geometric mean ﬂuorescence index (MFI) in SF3B1 mutant (right) compared with WT (left). (b) Bar graph shows an increase in the geometric MFI value representing the increase in ferrous iron in untreated and FeO4S-treated of SF3B1 mutant (gray bars) and WT (white bars) RARS patients.

SF3B1 mutations lead to alternative usage of a specific isoform of SLC25A37 (Mitoferrin-1) It has been reported that SF3B1 mutations affect genes of the mitochondrial network.12,26 Since SF3B1 is a splicing factor, it may elicit its effect by affecting the regulation of downstream targets via alternative splicing. We thus performed RNA-Seq analysis of BM cells from three SF3B1 mutant (K700E, n ¼ 2; H662Q, n ¼ 1), three WT RARS patients and three healthy subjects. A comprehensive bioinformatic analysis was conducted on three levels: gene, transcript and exon. Global differential gene expression analysis detected changes in 45 genes (false discovery rateo0.1) between SF3B1 mutant and healthy subjects. No genes showed differential expression (false discovery rateo0.1) between SF3B1 mutant and WT RARS patients (Supplementary Table S1). Known key players in the pathogenesis of SA were interrogated: ALAS2 was upregulated in SF3B1 mutant vs WT RARS patients (FC ¼ 1.46) and in SF3B1 mutant vs healthy subjects (FC ¼ 1.72). ABCB7 was downregulated compared with healthy subjects in both groups (FC ¼ 0.42, P ¼ 0.037 for SF3B1 mutants and FC ¼ 0.49, P ¼ 0.044 for WT RARS) in accordance with previously published data in the literature.3 Global differential exon usage analysis detected 271 genes (Supplementary Table S2) with at least one exon showing changes in differential usage (false discovery rateo0.10) between SF3B1 mutants and WT RARS patients. This analysis showed that one of the top candidate genes was SLC25A37 (P ¼ 4.7e À 4, false discovery rate ¼ 0.089). An increased mRNA expression level of SLC25A37 was found in SF3B1 mutant (red line) compared with WT (blue line) RARS/-T patients (FC ¼ 1.98) (Figure 6a) as well as compared with healthy subjects (FC ¼ 3.31), although neither Figure 5. SF3B1 mutations do not lead to an increased double comparison was significant. Differential transcript analysis showed strands DNA damage and oxidative stress. (a) BM or PB mono- that SLC25A37 transcript ENST00000518881 (Ensembl transcript nuclear cells from SF3B1 mutant and WT RARS patients and healthy subjects were fixed in 2% paraformaldehyde, following by permea- id), which contains a retained intron, was more highly expressed in bilization with 90% cold methanol. Anti-H2AX-Alexa 488-labeled SF3B1 mutants vs WT RARS patients (FC ¼ 5.6, P ¼ 0.034) and vs antibody was used to measure phosphorylated H2AX as marker of healthy subjects (FC ¼ 22.7, P ¼ 2.6e À 4) (Figure 6b). This tran- chromatin-based DNA damage. Bar graph shows the increase in script was expressed on average at about a tenth of the level of H2AX as mean±s.d. of the percentage of positive cells for SLC25A37’s major protein-coding transcript (ENST00000519973), phosphorylated H2AX in SF3B1 mutant (n ¼ 8), WT (n ¼ 3) RARS/-T which is relatively abundant and in contrast did not show patients. Healthy subjects (n ¼ 4) were used as control. (b) Bar graph evidence of differential transcript expression. A representative represents the mean±s.d. of the percentage of positive cells for scheme of the major protein-coding transcript and the transcript CellROX Green reagent shows no difference in ROS production in with a retained intron is shown in Figure 6c. The differences in untreated and H O -treated BM/PBMNCs derived from SF3B1 mutant 2 2 expression levels of the SLC25A37 isoform in SF3B1 mutant, SF3B1 (n ¼ 3) and WT (n ¼ 3) RARS/-T patients. Nonparametric two-tailed test was used to assess significance. *Po0.05. WT and healthy subjects were further confirmed by reverse transcriptase PCR (Supplementary Figure S2). SLC25A37 is a gene located on chromosome 8p21.2 with a full-length cDNA com- posed by 4 exons and 12 known transcripts, two of which are normal conditions (93.8±7.9 vs 83.2±15.9; P ¼ 0.35). Similarly, we protein coding. Sanger sequencing of SLC25A37 coding region did did not find any difference under conditions of oxidative stress by not detect any somatic mutations in a total of 33 patients (SF3B1 stimulating the cells with H2O2 (90.4±14.3 vs 70.4±40.0; P ¼ 0.46) mutant/WT: 21/12). SLC25A37 or better known as Mitoferrin-1 is a (Figure 5b). protein localized in the inner membrane of the mitochondria,

Leukemia (2015) 188 – 195 & 2015 Macmillan Publishers Limited Iron and SF3B1 mutations V Visconte et al 193

Figure 6. RNA-Seq analysis detects a different expression pattern in SLC25A37 gene in SF3B1 mutant compared with WT RARS/-T patients. Total RNA was isolated from BMMNCs of SF3B1 mutant (n ¼ 3) and WT (n ¼ 3) RARS/-T patients. RNA from healthy subjects was used as control (n ¼ 3). RNA sequencing was performed by Otogenetics. (a) A screenshot of fitted expression counts showed an increased mRNA expression level (1.98-FC) of SLC25A37 in SF3B1 mutants (red line) compared with WT (blue line) RARS/-T patients. Area in magenta color also indicates the differentially used exon. (b) Screenshot illustrates the different isoforms of SLC25A37. SLC25A37 (#_004; ENSEMBL_518881) represents an isoform characterized by a retained intron with increased differential usage in SF3B1-mutant patients. (c) Scheme of the normal and truncated protein and domains of SLC25A37 were illustrated based on structural domains and sequence annotations collected from protein structure and modifications in publicly available databases (www.uniprot.org; http://www.phosphosite.org). which acts as importer of Fe2 þ into the mitochondria in erythroid We reported that SF3B1 haploinsufficiency leads to the RS precursors. SLC25A37 protein has been linked to FECH and phenotype in MDS and other non-MDS BM failure conditions, Abcb10 in the regulation of the erythroid heme biosynthesis.27 and we studied the potential link between SF3B1 mutations and A known transcription factor GATA-1, which represents a marker of the mitochondrial pathways and how it contributes to the disease hematopoietic maturation, seems to regulate the import of Fe2 þ biology and phenotype.12,26,28–30 Other studies suggested the by SLC25A37. We found a trend toward increased mRNA relationship between SF3B1 mutations and mitochondrial proteins expression levels of ABCB10 (FC ¼ 2.27), FECH (FC ¼ 1.43) and but did not delve into the specific mechanisms or pathways that GATA-1 (FC ¼ 1.55) in SF3B1 mutant vs WT RARS/-T patients, contribute to iron accumulation or disease phenotype in SF3B1- although none of them reached statistical significance. There were mutant MDS.10,31 Using TEM, we found that within RARS/-T, no significant differences in the levels of any of the RNA-variant sideroblasts of SF3B1-mutant patients have distinct ultrastructural transcripts of ABCB7 and ALAS2. iron distribution characterized by abundant iron deposits We also investigated other genes important in iron metabolism compared with those of WT patients.12 In the present in congenital SA including SLC25A38, PUS-1 and GLRX5 but no manuscript, we confirmed that TEM is able to detect a differences in exon usage, gene expression and RNA-variant difference in the iron content between mutant and WT RARS/-T transcripts were noted, supporting the differences in the patients not appreciated by conventional Prussian blue staining. pathogenesis of iron accumulation between acquired and The finding led us to think that the differences in iron is connected congenital SA. with the differences in better survival outcomes of SF3B1 mutant vs WT RARS/-T patients.10,12,16 Thus, we hypothesized that SF3B1 mutations may have effects on downstream targets implicated in DISCUSSION the Fenton reaction whereby ferrous iron (Fe2 þ ) is converted in Ring sideroblasts (RS) are erythroid precursors containing iron ferric iron (Fe3 þ ) via superoxide ion. It has been reported that deposits, most commonly detected by Prussian blue staining and SF3B1 mutations might affect the mitochondrial network inducing visualized by light microscopy as a perinuclear ring of blue iron accumulation at different levels.32 We used XEDS/EELS granules. TEM clarified that these deposits are specifically localized techniques, which have the power to qualitatively and in the mitochondria of erythroid precursors. RS are present in 25% quantitatively discriminate the presence of specific elements, of patients with MDS and rarely seen in other conditions. This primarily used in the evaluation of surface materials. This is the feature is a diagnostic criterion of RARS/-T. In 2011, the discovery first time that both techniques were used to elucidate the iron of mutations in a pre-mRNA splicing factor called splicing factor 3b, characteristics in a hematologic neoplasm like MDS, specifically in subunit 1 (SF3B1) in 60–82% of RARS/-T patients added an RARS/-T. This is an important distinction from the two prior studies additional molecular criterion to identify RARS/-T subtypes. that used both techniques separately and consequently affects

& 2015 Macmillan Publishers Limited Leukemia (2015) 188 – 195 Iron and SF3B1 mutations V Visconte et al 194 the results and conclusions of those studies. In the two previously intron retention have been identified in several human disorders published papers,33,34 the authors concluded that ferric iron and may be used as a diagnostic marker of diseases.36 It is of further (Fe3 þ ) was the most prevalent form of iron. The difference interest that intron retention is the least frequent alternative between Fe2 þ and Fe3 þ is very narrow and is highly dependent splicing outcome.37 Wang et al.24 speculated that the presence of on the spectral intensity and the peak shape of the iron. The an abnormal transcript of SLC25A37 containing an insert of intron 2 differences in spectral intensity between Fe2 þ and Fe3 þ can be with a stop codon contributes to the phenotype observed in some appreciated by looking at the major and minor peaks (L3 and L2) variants of erythropoietic protoporphyria that do not carry the of iron. For the L3 peak, the expected spectral intensity of Fe2 þ typical FECH mutation, ALAS2 mutation and FECH polymorphism. In edge peak has a maximum occurring at 707.5 eV while the this study Wang et al. found that erythropoietic protoporphyria expected Fe3 þ edge major peak occurs at 710 eV. For the L2 peak, patients with increased abnormal SLC25A37 expression have the expected spectral intensity of Fe2 þ edge peak has a maximum reduced FECH activity. Our study investigated the role of of B720 eV while the expected Fe3 þ edge peak is beyond 720 SLC25A37 in a disease with a different natural history from usually at the range of 722–723 eV.25 In addition to the observed erythropoietic protoporphyria in which no differences in FECH chemical shifts, the distinction between Fe2 þ and Fe3 þ is also were found. The invariable presence of SLC25A37 intron retention dependent on the L2 and L3 edge shapes. Based on these in our SF3B1-mutated cases suggests that it was under positive chemical properties, we again confirmed that ferrous iron (Fe2 þ ) selection because of its functionality. The presence of this abundant is the most prevalent form of iron present in the cellular iron isoform of SLC25A37 suggests that the iron delivered into the deposits of either SF3B1 mutant or WT RARS/-T (Figure 2). Using mitochondria is altered in SF3B1-mutant RARS/-T patients. Our data flow cytometry, we verified that the increased amount of iron in also indicate that SF3B1 mutations may induce deregulation of SF3B1-mutant patients was more pronounced in the mitochondria crucial iron importers that work at the initial steps in the delivery compared with the cytoplasm. In addition, we used a modified of iron into the mitochondria rather than in the successive steps of fluorescent dye (RhoNox-1), which is able to specifically recognize iron incorporation. The specific splice variant with a retained intron only Fe2 þ , and confirmed that the deposits in SF3B1 mutant and of SLC25A37 may translate to a truncated protein that can be a WT patients contain divalent iron. In normal physiologic acid base novel mediator of iron overload in SF3B1 mutant RARS/-T patients and oxygen tension states, Fe2 þ is soluble and oxidized to Fe3 þ , and a potential therapeutic target in MDS with SF3B1 mutations. which when hydrolyzed, leads to the formation of insoluble Fe3 þ Indeed, the predictive putative protein translated from hydroxides, which have redox activity and capacity to induce ENST00000518881 would have 218 amino acids and would retain damage. In our study, the oxidative stress measured by ROS levels the first 70 amino-acid residues of the WT protein. Conventional was not different between SF3B1 mutant and WT RARS/-T patients protein properties tools (UniProt2 and ProteinPredict) show that suggesting that oxidative stress is similar irrespective of the cause this mutant protein would have an altered three dimensional of mitochondrial iron overload. Furthermore, the lower levels of structure with features of higher level of structural disorder. double-stranded DNA damage observed in SF3B1 mutant However, the protein would retain the ability to anchor the inner compared with SF3B1 WT RARS/-T patients and healthy subjects mitochondrial membrane. Moreover, it will probably not affect the may be due to upregulated antioxidant defenses and DNA repair function of Abcb10, as it would retain the Abcb10-binding motif of mechanisms in the BM progenitors of patients harboring SF3B1 the WT SLC25A37 protein.38 Finally, a plausible speculation about mutations. the function for this neomorphic protein would be a role in Fe2 þ We thus utilized RNA-Seq as the best methodology to appreciate storage in the mitochondria similar to that of frataxin.39 the effects of SF3B1 on other pathways. When we interrogated genes with mitochondrial function or location, their mRNA expression levels were significantly different between SF3B1 CONFLICT OF INTEREST mutant, WT RARS/-T patients and healthy subjects. As many known The authors declare no conflict of interest. iron transport systems are located within the mitochondrial machinery, we first focused our analysis on genes with relevant iron transport functions. Interestingly, the mRNA level of SLC25A37 (Mitoferrin-1), a protein implicated in the mitochondrial iron ACKNOWLEDGEMENTS delivery in erythroid cells, was found to be significantly over- We would like to thank Dr Andrea N. Ladd of the Department of Cellular and expressed in SF3B1 mutant compared with both WT RARS/-T Molecular Medicine, Cleveland Clinic Lerner Institute, Cleveland OH and Dr Velizar patients and healthy subjects. It has been shown that iron Shivarov of the Laboratory of Hematopathology and Immunology, National accumulation, heme and iron sulfur cluster synthesis are reduced Hematology Hospital, Sofia, Bulgaria for their helpful recommendations and careful by silencing SLC25A37 in mouse 3T3 fibroblasts.35 Our study revisions of the manuscript. This work was supported in full or partially by Cleveland Clinic Seed Support, and Scott Hamilton CARES grant (RVT) provides some new answers to the queries surrounding the formation of RS in RARS/-T as previously proposed when SF3B1 mutations were first linked to the formation of RS.12 It was speculated that SF3B1 mutations lead to iron accumulation by AUTHOR CONTRIBUTIONS affecting several key iron trafficking pathways including ABCB7, VV conceived the study, performed experiments, analyzed the data and wrote protoporphyrin IX, FECH, iron sulfur cluster proteins and the manuscript; NA performed and analyzed electron microscopy experiments; mitochondrial electron respiratory chain.32 In our study, none of RM performed experiments and provided important insights to the manuscript; these pathways were significantly different between SF3B1 mutant AT collected data and performed experiments; JC analyzed and interpreted the and WT RARS/-T patients suggesting that other distinct mechanisms electron microscopy results; JP synthesized the RhoNox-1 compound; RS-M may be responsible for these changes. Through the application of a analyzed the electron microscopy results; MH designed protocols of slides comprehensive bioinformatic analysis in RARS/-T subtypes, we preparation for electron microscopy; HJR reviewed BM pathology and edited identified a statistically significant increase in a specific RNA splice the manuscript; EH performed experiments; MAS provided patients; AHH variant of SLC25A37 with a retained intron in SF3B1 mutant provided fundamental feedbacks and long track experience on material science compared with WT RARS/-T patients and healthy individuals. This to the manuscript; YS provided patients and edited the manuscript; JB analyzed finding is important because, through a novel bioinformatic the RNA-Seq data and provided important suggestions; RVT conceived the approach of RNA-sequencing analysis, we were the first to study, designed the experimental approach, reviewed the clinical data, identify and link an alternatively spliced mitochondrial gene in interpreted the data, provided the patients and wrote the manuscript; all MDS with RS. Alternative splicing, presence of splice products and authors read and approved the manuscript.

Leukemia (2015) 188 – 195 & 2015 Macmillan Publishers Limited Iron and SF3B1 mutations V Visconte et al 195 REFERENCES 20 Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for 1 Bjorkman SE. Chronic refractory anemia with sideroblastic bone marrow; a study RNA-seq: accounting for selection bias. Genome Biol 2010; 11: R14. of four cases. Blood 1956; 11: 250–259. 21 Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S et al. STAR: ultrafast 2 Dorion RP, Alomari M, Wood GC. Predicting the presence or absence universal RNA-seq aligner. Bioinformatics 2013; 29: 15–21. of ringed sideroblasts in patients suspected of having a myelodysplastic 22 Glaus P, Honkela A, Rattray M. Identifying differentially expressed transcripts from syndrome and increased iron stores: a simple observation. Leukemia 2001; 15: RNA-seq data with biological variation. Bioinformatics 2012; 28: 1721–1728. 1793–1795. 23 Barnard J, Rubin DB. Miscellanea. Small-sample degrees of freedom with multiple 3 Pellagatti A, Cazzola M, Giagounidis AA, Malcovati L, Porta MG, Killick S et al. imputation. Biometrika 1999; 86: 948–955. Gene expression profiles of CD34 þ cells in myelodysplastic syndromes: 24 Wang Y, Langer NB, Shaw GC, Yang G, Li L, Kaplan J et al. Abnormal mitoferrin-1 involvement of interferon-stimulated genes and correlation to FAB subtype and expression in patients with erythropoietic protoporphyria. Exp Hematol 2011; 39: karyotype. Blood 2006; 108: 337–345. 784–794. 4 Boultwood J, Pellagatti A, Nikpour M, Pushkaran B, Fidler C, Cattan H et al. The role 25 Garvie LAJ, Buseck PR. Ratios of ferrous to ferric iron from nanometre-sized areas of the iron transporter ABCB7 in refractory anemia with ring sideroblasts. PLoS in minerals. Nature 1998; 396: 667–670. One 2008; 3: e1970. 26 Visconte V, Mahfouz RZ, Tabarroki A, Hasrouni E, Rogers HJ, Saunthararajah Y et al. 5 Cazzola M, Invernizzi R. Ring sideroblasts and sideroblastic anemias. Haematologica BCL-2 family of genes is a key regulator in the pathogenesis of SF3B1 mutant and 2011; 96: 789–792. wild type MDS with ring sideroblasts and represents a novel drug target in this 6 Sato K, Torimoto Y, Hosoki T, Ikuta K, Takahashi H, Yamamoto M et al. Loss of disease. (ASH Annual Meeting). Blood 2013; 122:263. ABCB7 gene: pathogenesis of mitochondrial iron accumulation in erythroblasts in 27 Chen W, Dailey HA, Paw BH. Ferrochelatase forms an oligomeric complex refractory anemia with ringed sideroblast with isodicentric (X)(q13). Int J Hematol with mitoferrin-1 and Abcb10 for erythroid heme biosynthesis. Blood 2010; 116: 2011; 93: 311–318. 628–630. 7 Camaschella C, Campanella A, De Falco L, Boschetto L, Merlini R, Silvestri L et al. 28 Visconte V, Mahfouz RZ, Barnard J, Tabarroki A, Zhang L, Hasrouni E et al. Splicing The human counterpart of zebrafish shiraz shows sideroblastic-like microcytic factor 3b subunit 1 (SF3B1) mediates mitochondrial iron overload in myelodys- anemia and iron overload. Blood 2007; 110: 1353–1358. plastic syndromes with ring sideroblasts by alternative splicing of mitoferrin-1 8 Caudill JS, Imran H, Porcher JC, Steensma DP. Congenital sideroblastic anemia (SLC25A37). (ASH Annual Meeting). Blood 2013; 122: 1555. associated with germline polymorphisms reducing expression of FECH. 29 Visconte V, Makishima H, Maciejewski JP, Tiu RV. Emerging roles of the spliceo- Haematologica 2008; 93: 1582–1584. somal machinery in myelodysplastic syndromes and other hematological 9 Visconte V, Makishima H, Jankowska A, Szpurka H, Traina F, Jerez A et al. SF3B1, a disorders. Leukemia 2012; 26: 2447–2454. splicing factor is frequently mutated in refractory anemia with ring sideroblasts. 30 Visconte V, Tabarroki A, Rogers HJ, Hasrouni E, Traina F, Makishima H et al. SF3B1 Leukemia 2012; 26: 542–545. mutations are infrequently found in non-myelodysplastic bone marrow failure 10 Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D et al. syndromes and mast cell diseases but, if present, are associated with the ring Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N Engl J Med sideroblast phenotype. Haematologica 2013; 98: e105–e107. 2011; 365: 1384–1395. 31 Nikpour M, Scharenberg C, Liu A, Conte S, Karimi M, Mortera-Blanco T et al. The 11 Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R et al. Frequent transporter ABCB7 is a mediator of the phenotype of acquired refractory anemia pathway mutations of splicing machinery in myelodysplasia. Nature 2011; 478: with ring sideroblasts. Leukemia 2013; 27: 889–896. 64–69. 32 Gattermann N. SF3B1 and the riddle of the ring sideroblast. Blood 2012; 120: 12 Visconte V, Rogers HJ, Singh J, Barnard J, Bupathi M, Traina F et al. SF3B1 3167–3168. haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic 33 Rademakers LH, Koningsberger JC, Sorber CW, Baart de la Faille H, Van Hattum J, syndromes. Blood 2012; 120: 3173–3186. Marx JJ. Accumulation of iron in erythroblasts of patients with erythropoietic 13 Williams D, Carter CB. Transmission Electron Microscopy: A Textbook For Material protoporphyria. Eur J Clin Invest 1993; 23: 130–138. Science. Chapter 39 (High-Energy Loss Spectra and Images). Plenum Press: New 34 Grasso JA, Myers TJ, Hines JD, Sullivan AL. Energy-dispersive X-ray analysis of the York, USA, 1996, pp 715–716. mitochondria of sideroblastic anaemia. Br J Haematol 1980; 46: 57–72. 14 Tan H, Verbeeck J, Abakumov A, Van Tendeloo G. Oxidation state and chemical 35 Paradkar PN, Zumbrennen KB, Paw BH, Ward DM, Kaplan J. Regulation of mito- shift investigation in transition metal oxides by EELS. Ultramicroscopy 2012; 116: chondrial iron import through differential turnover of mitoferrin 1 and mitoferrin 2. 24–33. Mol Cell Biol 2009; 29: 1007–1016. 15 Hirayama T, Okuda K, Nagasawa H. A highly selective turn-on fluorescent probe 36 Michael IP, Kurlender L, Memari N, Yousef GM, Du D, Grass L et al. Intron retention: for iron(II) to visualize labile iron in living cells. Chem Sci 2013; 4: 1250–1256. a common splicing event within the human kallikrein gene family. Clin Chem 16 Traina F, Visconte V, Elson P, Tabarroki A, Jankowska AM, Hasrouni E et al. 2005; 51: 506–515. Impact of molecular mutations on treatment response to DNMT inhibitors in 37 Galante PA, Sakabe NJ, Kirschbaum-Slager N, de Souza SJ. Detection and eva- myelodysplasia and related neoplasms. Leukemia 2013; 28:78–87. luation of intron retention events in the human transcriptome. RNA 2004; 10: 17 Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying 757–765. mammalian transcriptomes by RNA-Seq. Nat Methods 2008; 5: 621–628. 38 Chen W, Paradkar PN, Li L, Pierce EL, Langer NB, Takahashi-Makise N et al. 18 Oshlack A, Robinson MD, Young MD. From RNA-seq reads to differential Abcb10 physically interacts with mitoferrin-1 (Slc25a37) to enhance its stability expression results. Genome Biol 2010; 11:220. and function in the erythroid mitochondria. Proc Natl Acad Sci USA 2009; 106: 19 Smyth G. Limma: linear models for microarray data. In Gentleman R, Carey V, 16263–16268. Dudoit S, Irizarry R, Huber W (eds.) Bioinformatics and Computational Biology 39 Lane DJ, Richardson DR. Frataxin, a molecule of mystery: trading stability for Solutions using R and Bioconductor. Springer: New York, USA, 2005, pp 397–420. function in its iron-binding site. Biochem J 2010; 426: e1–e3.

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