University of Groningen
Genetic defects in myeloid malignancies and preleukemic conditions Berger, Gerbrig
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record
Publication date: 2019
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA): Berger, G. (2019). Genetic defects in myeloid malignancies and preleukemic conditions. Rijksuniversiteit Groningen.
Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Download date: 26-09-2021 (Submitted) 3. AB Mulder, S Ogawa, JHA Martens, JH Jansen and E Vellenga and JH Jansen JHA Martens, S Ogawa, AB Mulder, risk and result from an aberrant an aberrant from risk and result G Berger, M Gerritsen, TN Koorenhof-Scheele, G Yi, LI Kroeze, G Yi, LI Kroeze, TN Koorenhof-Scheele, M Gerritsen, G Berger, heme-metabolism gene program Ringsideroblasts in acute myeloid myeloid in acute Ringsideroblasts M Stevens-Kroef, K Yoshida, E van den Berg, H Schepers, G Huls, G Huls, H Schepers, den Berg, E van K Yoshida, M Stevens-Kroef, leukemia are associated with adverse adverse with associated are leukemia
Chapter 3
Abstract Ringsideroblasts (RS) emerge from aberrant erythroid differentiation, resulting in excessive mitochondrial iron accumulation. This is a characteristic feature of myelodysplastic syndromes (MDS) with mutations in spliceosome gene SF3B1, but RS is also observed in patients with acute myeloid leukemia (AML). We therefore characterized the presence of RS in a cohort of AML patients. The RS-AML subgroup was enriched with patients in the ELN adverse-risk category (55%). In line with this finding, 35% of all RS-AML cases had complex cytogenetic aberrancies, and TP53 was most recurrently mutated in this cohort (42%), followed by DNMT3A (29%), RUNX1 (21%) and ASXL1 (19%). In contrast to RS-MDS, the incidence of SF3B1 mutations was low (8%). Whole-exome sequencing and SNP array analysis on a subset of patients showed that the RS phenotype did not result from a single-gene defect. Shared genetic defects between erythroblasts and total mononuclear cell fraction within the same 3 patient indicate common ancestry for the erythroid lineage and the myeloid blast cells in RS-AML patients. RNA sequencing of CD34+ AML cells revealed differential gene expression between RS-AML and a separate AML cohort, including genes involved in megakaryocytic/erythroid differentiation and mRNA splicing. Furthermore, several heme-metabolism-related genes were found to be upregulated in RS-AML, as was observed in SF3B1mut MDS. These results demonstrate that erythroblasts share ancestry with malignant myeloid blast cells in RS-AML. Although the genetic background of RS- AML differs from that of RS-MDS, downstream effector pathways may be comparable, providing a possible explanation for presence of RS in AML.
ingsideroblasts (RS) are erythroid in low-risk MDS, which is characterized precursor cells that accumulate by a stable clinical course and a low risk R excessive mitochondrial iron of leukemic transformation.(8,13) As a and can be observed in bone marrow core component of the U2 small nuclear smears associated with multiple medical ribonucleoprotein particle (snRNP), conditions.(1) Acquired presence of RS is a SF3B1 is essential for pre-RNA splicing. characteristic feature in myelodysplastic (14) The molecular mechanism by which syndrome (MDS) subtypes, including SF3B1 mutations result in RS formation MDS with single lineage dysplasia is not yet fully understood. A proposed (MDS-RS-SLD), multilineage dysplasia mechanism is that specific patterns of (MDS-RS-MLD) and in combination missplicing result in altered expression with the presence of thrombocytosis of genes that are essential for correct (MDS/MPN-RS-T).(2) Non-malignant programming of erythropoiesis.(15,16) causes of RS include several drugs, The relationship between toxins, alcohol, copper deficiency and genetic defects in SF3B1 and the RS- congenital sideroblastic anemia.(3) This phenotype is not one-to-one; in 10-20% latter group comprises conditions of the MDS-RS patients no mutation caused by inborn defects in genes in the SF3B1 gene is detected.(8-12) that operate in several mitochondrial Moreover, RS can also be present in a pathways, including ALAS2, ABCB7, subset of AML patients(17), while SF3B1 SLC25A38 and HSPA9.(4-7) mutations are infrequent findings in In MDS, the RS phenotype is this disease.(10,17,18) Besides SF3B1, strongly correlated with mutations in the only other correlation between splicing factor 3B subunit 1 (SF3B1), a gene defect and the RS phenotype with an incidence higher than 80%.(8-12) was described for PRPF8, for which SF3B1 mutations are usually observed mutations are reported in ~3% of the
66 Ringsideroblasts in acute myeloid leukemia
cases.(19) Other spliceosomal genes that lymphoprep (PAA, Cölbe, Germany) are more frequently mutated in myeloid according to standard procedures. malignancies, including SRSF2, U2AF1, To separate cell fractions, following ZRSR2, have not been implicated in the thawing of viably frozen MNCs, cells RS-phenotype.(9) were washed and stained with a panel In the present study we deter- of antibodies and sorted for purification mined the prevalence of ringsideroblasts of different cell fractions (antibodies in various ontogenic AML subtypes and against CD3, CD34, CD71 and the association of the RS phenotype in CD235a surface markers (conjugates AML with adverse risk characteristics. CD3-FITC (cat. 345763), CD34-APC To identify the landscape of genomic (cat. 555824) or CD34-PE Cy7 (cat. defects that underlies the RS phenotype 348811) or CD34-FITC (cat. 345801, in AML, we performed whole exome BioLegend, Uithoorn, the Netherlands), sequencing, targeted sequencing and CD71-BV786 (cat. 563768) or CD71- SNP-array analysis. Finally, to identify APC (cat. 551374) or CD71-PE (cat. 3 differential expression of genes asso- 555537) and CD235a-BV421 (cat. ciated with the RS phenotype in AML, 562938) or CD235a-APC (cat. 551336), we performed RNA sequencing on antibodies were obtained from BD CD34+-selected AML cells. Bioscience (Breda, The Netherlands) unless otherwise indicated)). Analysis and Material & methods sorting of various cell fractions was performed on MoFlo XDP or Astrios Patients and data collection For this (Dako Cytomation, Carpinteria, CA, USA). study, we collected data from 126 AML Single viable cells were selected based and high-risk MDS (≥10% bone marrow on forward and side scatter profiles in blasts) who were diagnosed between combination with negativity for DAPI January 2000 and April 2018 at the or PI (both Sigma-Aldrich, Saint Louis, University Medical Center Groningen. MO, USA). Blast fraction was defined as The inclusion criterion was the reported CD34 positive, T cell fraction as CD3 presence of ringsideroblasts in the positive and erythroblast fraction as diagnostic bone marrow smear. Patients CD71/CD235a positive. Sorting purity with previously reported MDS-RS was defined as ≥95% and confirmed by were excluded. Diagnosis and risk reanalysis. classification was revised based upon DNA isolation and World Health Organization classification amplification. Genomic DNA from (2016)(2) and European Leukemia Net various cell fractions was extracted with (ELN) recommendations (2017)(20). Bone the NucleoSpin Tissue kit (Macherey- marrow (BM) and/or peripheral blood Nagel, Düren, Germany) according to (PB) from patients were biobanked after the manufacturer’s instructions. In informed consent for investigational use. case of insufficient yield, a maximum The study was conducted in accordance of 70ng DNA was amplified using the with the Declaration of Helsinki and Qiagen REPLI-g kit (Qiagen, Venlo, the institutional guidelines and regulations. Netherlands) in one reaction, according Morphologic and cytogenetic analyses to the manufacturer’s protocol. were accomplished following standard Targeted deep sequencing procedures. using a myeloid gene panel. Targeted Sorting of cell fractions. sequencing of DNA derived from BM The mononuclear cell (MNC) fraction or PB samples obtained at diagnosis was from BM and/or PB was obtained by carried out using the myeloid TruSight density gradient centrifugation using sequencing panel (Illumina, San Diego,
67 Chapter 3
CA, USA). Library preparation was per- (Agilent Technologies, Santa Clara, CA, formed according to the manufacturer’s USA), massively parallel sequencing protocol (Illumina). Aligning and filter- was performed on enriched exome ing of sequence data was performed fragments using the HiSeq 2500 using NextGENe version 2.3.4.2 (Soft- platform (Illumina, San Diego, CA, USA). Genetics, Pennsylvania, US). Cartagenia Alignment of sequences and calling of Bench Lab NGS (Agilent, Santa Clara, mutations was executed our previously CA, USA) was used for analysis of the described in-house pipelines(9,21), with resulting vcf file. Sequencing artifacts minor modifications. The resultant data were excluded using a threshold of 5%. file was analyzed for the presence of A minimal variant read depth of 20 germline variants in genes that have been reads was set as criterion. Variants that previously implicated in sideroblastic frequently occur in the general healthy anemia (including ALAS2, SLC25A38, population (>2% 1000 Genome Phase 1, FECH, MFRN1, ISCU, ISCA1/2, 3 ESP6500 and dbSNP, and >5% Genome NIFS, HSP70, HSPA9, HSCB, GLRX5, of the Netherlands) were excluded from ABCB7, IRBP1, GLRX5, PUS1, further analysis. YARS2).(23) Subsequently, DNA isolated Microarray-based genomic from sorted autologous T cells was used profiling. Microarray-based genomic as a constitutive reference to exclude profiling on MNC and erythroblast germline variants. Filtering strategy fractions was carried out with the and variant calling was performed as CytoScan HD array platform (Affymetrix, previously described.(24) Inc., Santa Clara, CA, USA) in agreement Confirmation of mutations in with the manufacturer’s reference. erythroblasts. For a subset of patients, Data analysis was performed using the presence of somatic variants in Chromosome Analysis Suite software TP53 and SRSF2 identified by WES package (Affymetrix) and annotations were validated and quantified in the of reference genome build GRCh37 erythroblast fraction using amplicon- (hg19). Comprehensive analysis based deep sequencing on an Ion and interpretation of the obtained Torrent Personal Genome Machine microarray genomic profiling data was (Thermo Fisher Scientific, Waltham, performed using a previously described MA, USA). The sequencing procedure filtering pipeline and according to using an automated robotic workflow criteria that have been described was performed as previously described previously.(21) Aberrations meeting (Sandmann, PlosOne, 2017). The obtained these criteria were included for genomic sequencing data was mapped to the profiling and described in accordance reference genome build GRCh37 (hg19). with the standardized ISCN 2016 Variant calling was performed using the nomenclature system.(22) Visualization SeqNext module of the Sequence pilot of the resultant genomic profiles was software, version 4.2.2 (JSI Medical performed using NEXUS software Systems, Ettenheim, Germany). (Nexus Copy Number 8.0, BioDiscovery, RNA Extraction and Illumina El Segundo, CA, USA). high-throughput sequencing. RNA Whole exome sequencing. was isolated by separation of the Whole exome sequencing (WES) aqueous phase by TRIzol Reagent to an average depth of 143x was (Thermo Fisher) according to the performed on DNA isolated from manufactures protocol. The aqueous diagnostic BM-MNC (n=13) or PB- phase was subsequently mixed 1:1 with MNC (n=3) samples. Following exome 70% ethanol and isolation was continued capturing using Human All Exon V5 using the RNeasy mini kit (Qiagen)
68 Ringsideroblasts in acute myeloid leukemia
Table 1 – Clinical characteristics of AML with RS phenotype
All RS cases 1-4% RS 5-14% RS ≥15% RS
Total 126 53 42% 28 22% 45 36% Age (years) Median 67 67 63 70 Range 32-87 40-81 32-78 43-87 Sex Male 84 67% 32 60% 20 71% 32 71% Female 42 33% 21 40% 8 29% 13 29% Type of disease de novo AML 70 56% 35 66% 12 43% 23 51% 3 sAML 20 16% 6 11% 8 29% 6 13% t-MN 17 14% 7 13% 3 11% 7 16% Other 19 15% 5 9% 5 18% 9 20% WHO diagnosis AML with MDS- 47 37% 14 26% 16 57% 17 38% related changes AML NOS 24 19% 13 25% 3 11% 8 18% t-MN 17 14% 7 13% 3 11% 7 16% MDS-EB2 17 14% 5 9% 4 14% 8 18% AML with recurrent 16 13 % 13 25 % 1 4% 2 4% abnormalities Other 5 4% 1 2% 1 4% 3 7% ELN risk score Favorable 12 10% 11 21% 1 4% 0 0% Intermediate 6 5% 4 8% 2 7% 0 0% Intermediate* 34 27% 17 32% 6 21% 11 24 % Adverse 71 56% 21 40% 18 64% 32 71% Unknown 3 2% 0 0% 1 4% 2 4% BM blasts, % Median 32% 34% 24% 32% Range 10%-91% 10-88% 11-88% 10-91% Erythroblasts, % Median 21% 23% 21% 17% Range 1-64% 4-64% 2-50% 1-59%
Data denoted as number (percentage), unless otherwise stated. * No evaluation for ASXL1, RUNX1 and TP53. Abbreviations: AML – acute myeloid leukemia, t-MN – therapy-related myeloid neoplasm, NOS – not otherwise specified, sAML- secondary AML, MDS-EB2 – myelodysplastic syndrome with excess blasts type 2, ELN– European Leukemia Net, WHO – World Health Organization, BM – bone marrow, RS - ringsideroblasts
69 Chapter 3
including performing on-column a heatmap. To assess the enriched DNaseI treatment. RNA libraries annotation for deregulated genes, were prepared using the KAPA RNA we performed functional enrichment HyperPrep Kit with RiboErase (HMR) analysis with the Metascape tool, and according to the manufactures protocol those terms showing p-values below (KR1351 – v1.16, Roche Sequencing 0.01 were considered significantly over- Solutions). In short; 25ng -1ug input represented. RNA was depleted from ribosomal Statistical Analysis. Bivariate RNA by oligo hybridization, RNaseH correlations were made using a Pearson treatment and DNase digestion. rRNA- correlation (continuous variables) or depleted RNA was fragmented to ~200 Spearman correlation (categorical bp fragments and first strand synthesis variables). A p-value <0.05 was used to was performed using random primers. define statistical significance. Statistical Second strand synthesis was performed calculations were performed using 3 using dUTP for strand specificity. After Prism version 6.0. adapter ligation, library amplification was performed and the number of Results cycles was dependent on the amount of starting material. Fragment sizes and Clinical Characteristics quality was checked on a bioanalyser In our previous study on clonal evolution using a high sensitivity DNA Chip in therapy-related myeloid neoplasms (Agilent). Samples were sequenced on (t-MNs)(24), ringsideroblasts (RS) were the Illumina HiSeq 2000. Finally, each observed frequently in bone marrow eligible library was subjected to 2×43 smears. To study the RS phenotype in bp paired-end sequencing (PE43) on an more detail in a more comprehensive Illumina NextSeq 500 system. group of myeloid neoplasms (MNs), in RNA-Seq analysis. The hg19 the present study we collected clinical reference genome was first indexed data was collected for on a cohort of by STAR aligner with UCSC gene patients (N = 126) consisting of group annotation. The resulting RNA- of 126 AML and high-risk MDS seq reads were mapped to the hg19 (≥10% BM blasts) patients hereafter genome using STAR with two- also indicated as ‘AML patients’. pass mode, and the gene-level read Patients reported here with RS (≥1%) counts were enumerated at the same in the diagnostic bone marrow smear, time. The DESeq2 tool was used to excluded those with a documented examine differentially expressed genes prior clinical history of MDS-RS. The by conducting pair-wise comparison median blast percentage in this cohort between different groups. Only genes was 32% (range 10-91%), two-third of with a Benjamini-Hochberg-adjusted the patients were male and the median p-value <0.1 and fold change >1.5 were age at diagnosis was 67 years (range considered significantly deregulated. 32-87). The majority of patients were Principal component analysis (PCA) diagnosed with de novo AML (55.6%) and t-Distributed Stochastic Neighbor and AML with myelodysplasia-related Embedding (t-SNE) were used to probe changes was the most common WHO the transcriptomic relationships between (2016) subtype (37.3%, Table 1). these groups. To group differential Although highest RS percentages were genes with similar expression patterns, observed in cases with lower blast counts, the k-means clustering approach the blast count did not significantly was performed based on z-score correlate with RS percentage (Pearson normalization and then displayed as r = 0.16, p = 0.07; Figure 1A). Patients
70 Ringsideroblasts in acute myeloid leukemia
igure
were placed into subgroupes based on their RS percentage; 1-4% RS, 5-14% r RS and ≥15% RS (Table 1). Groups were comparable regarding blast percentage, erythroblast percentage and age. The group of AML patients with an RS phenotype was enriched with patients Ringsideroblasts in the ELN adverse risk category (55%) (Figure 1B). The proportion of adverse risk patients increased with increasing RS percentage (Table 1, Figure 1B). one marro blasts The subgroup with ≥15% RS, which represents the minimum required percentage for WHO MDS-RS diagnosis in absence of SF3B1 mutations(2), had 3 not patients in the ELN favorable risk category (Figure 1B). ll cases R Mutational and chromosomal defects observed in association with RS phenotype Complex cytogenetic aberrancies R R (defined as 3 or more abnormalities) as a orable d erse detected by conventional karyotyping ntermediate nkno n were observed in 35% of all RS-cases ntermediate (total n=126, Figure 2A), with increasing Figure 1 – Clinical data. incidences associated with higher RS A) Correlation between blast percentage and ringsideroblast percentages (respectively 21%, 39% and percentage, both determined in diagnostic bone marrow 49% for the individual three groups smear. Boxplot represents median and range of blast (data not shown). A subset of 48 patients percentage in the RS cohort. B) Pie charts representing risk (de novo AML, n=27; sAML, n=9; t-MN, classification (ELN 2017) for total RS cohort (‘all cases’) n=6; MDS-EB2, n=6) was analyzed for and three igure subgroups based on RS percentage (Table 1) mutations in a panel of genes that are *Mutational status for ASXL1, RUNX1 and TP53 unknown. recurrently mutated in MNs using NGS Abbreviations: RS – ringsideroblasts, ELN – European methods (either panel-based or WES). Leukemia Net. In this subset, 22 patients had ≥15% RS, 17 patients had 1-4% RS and 9 patients had 5-14%. In addition, screening for with de novo AML, one with t-MN and mutations in CEBPa, FLT3 and NPM1 one with sAML. No mutations in these was conducted by conventional RT-PCR. frequently affected genes were identified Ringsideroblasts Ringsideroblasts In accordance with cytogenetic findings, in two patients. The incidence of certain mutations in the TP53 gene (42%), mutations tended to segregate with RS which frequently coincide with genetic percentages: NPM1 mutations were CBL KIT TP53 TET2 FLT3 IDH1IDH2 ETV6 JAK2 WT1 instability, were found most recurrently mainly observed in cases withASXL1 lowNPM1 SRSF2RS BCOR NRASSF3B1 STAG2U2AF1EAH2CEBPa GATA2 IKZF1 ZRSR2 com le del del del RUNX1 SETBP1 BCORL PTPN11 mutated gene in this subset (Figure 2B). percentages (30% vs. 6%DNMT3A in >15% RS NOTCH1 Other frequently mutated genes include group). In contrast TP53 and GATA2 RUNX1 (21%), NPM1 (17%) and the mutations were detected especially in epigenetic modifiers DNMT3A (29%), patients with higher RS percentages ASXL1 (19%) and TET2 (16%). SF3B1 (73% vs. 12% in the 1-4% RS group) mutations were detected in 4 cases; two (data not shown).
71 igure
r
Ringsideroblasts
one marro blasts
ll cases R
R R
a orable d erse ntermediate nkno n ntermediate
Chapter 3
igure
Ringsideroblasts Ringsideroblasts
CBL KIT TP53 TET2 FLT3 IDH1IDH2 ETV6 JAK2 WT1 ASXL1NPM1 SRSF2 BCOR NRASSF3B1 STAG2U2AF1EAH2CEBPa GATA2 IKZF1 ZRSR2 com le del del del RUNX1 SETBP1 BCORL PTPN11 DNMT3A NOTCH1 Figure 2. Chromosomal and molecular defects observed in association with RS phenotype A) Frequency of commonly observed cytogenetic defects in myeloid neoplasms (MNs) in RS-cohort, subdivided by 3 ringsideroblast (RS) percentage at diagnosis, n=126 B) Frequency of mutations in genes commonly mutated in MNs as detected by NGS, subdivided by RS percentage. ASXL1, CALR, CBL, DNMT3A, EZH2, IDH1, IDH2, JAK2, KIT, NRAS, RUX1, SF3B1, SRSF2, TET2, TP53 and WT1 n=48, BCOR, BCORL, ETV6, GATA2, GNAS, IKZF1, NOTCH1, PTPN11, SETBP1, STAG2, U2AF1 and ZRSR2 n=45. For CEBPa (n=83), FLT3 (n=103) and NPM1 (n=93) results of conventional RT-PCR are displayed.
Genome-wide screening for genetic 1). However no such mutations were defects detected. Based on WES, a median of To identify possible genetic defects 19 (range 9-34) somatically acquired underlying the RS phenotype that are mutations were found per patient, of not covered by panel-based sequencing, which a median 2.5 (range 0-10) were 15 patients of the RS cohort were observed in genes frequently mutated in selected for genome-wide analysis myeloid malignancies (Figures 3A, 3B and based on high RS percentage (≥13%) Supplementary table 2). The total number and material availability. This group of mutations did not correlate with was supplemented with one patient age (Pearson r = 0.07, p = 0.78, Figure (RS022) that had 15% RS at AML 3C). The vast majority of observed relapse following autologous stem mutations concerned nonsynonymous cell transplantation, while no RS were point mutations (81%, Figure 3D). TP53 observed at initial AML diagnosis. All was most frequently affected in this 16 patients belonged to the adverse risk cohort (in 12/16 patients), followed group according to ELN criteria. Using by DNMT3A (4/16) and SRSF2 WES, samples were first analyzed for (3/16) (Figure 3A). In this cohort, one the occurrence of germline mutations mutation was detected in SF3B1 and in genes associated with congenital no mutations were observed in PRPF8. sideroblastic anemia (supplementary table For patient RS022, who had 15% RS and
Figure 3. Genetic defects detected by whole exome sequencing and SNP-array analysis. A) Overview; for each patients, all mutations in genes known to be recurrently mutated in myeloid malignancies detected by WES are depicted as well as cytogenetic abnormalities detected by SNP array analysis B) Number of acquired mutations per patients as determined by WES. In black, the number of mutations in genes that have been previously implicated in pathogenesis of myeloid malignancies; in grey the number of mutations in genes that have not been previously implicated in myeloid malignancies. C) Correlation between age and the number of mutations . The Pearson correlation coefficient was determined. D) Distribution of the different types of alterations detected in the total set of patients with RS-phenotype. E) SNP array results overview, figure created using Nexus software.
72 Ringsideroblasts in acute myeloid leukemia
R R R R R R R R R R R R R R R R TP53 on myeloid malignancy associated genes DNMT3A yeloid malignancy associated genes ASXL1 TET2 GATA2 SF3B1 SRSF2 SF1 EP300 umber o mutations IDH1 IDH2 WT1 SETBP1 RUNX1 R R R R R R R R R R R R R R R R NRAS NF1 CBL ETV6 CEBPA 3 BCOR PRPF8 r
umber o mutations nonsynonymous sto gain rames i t deletion s licing rames i t insertion non rames i t deletion utation romotry sis de novo eletion t rec ain PRPF8 in ol ed
73 Chapter 3
several cytogenetic abnormalities in the of erythroblast mutations and the relapse sample while both were absent at percentage of RS (Pearson r = -0.4343, initial presentation, no mutations were p = 0.20) (Figure 4B). SNP array analysis detected in known leukemia-associated on both fractions revealed that most of genes (Figure 3A). However, the observed the cytogenetic defects were shared mutation in BUB1, a key player in the by the MNC fraction and erythroblast mitotic spindle checkpoint, may explain populations. However differences were the chromosomal aberrancies observed also detected in 4/6 patients (Figure in this relapse sample (Supplementary 4C). Observed differences consisted of table 2). In addition to WES, SNP either novel aberrancies, exemplified array analysis was used to identify by the gain of chromosome 11 in the chromosomal defects (Supplementary EBs of patient RS06, or discrepancies figure 1). Scattering of one or more in observed frequency of cytogenetic chromosomes, a process called defects, as observed for the 7q deletion 3 chromothripsis, was detected in 9/16 in patient RS09 (0.6 in EBs vs. 0.25 in cases. Deletions of chromosomes 5q, total MNCs) (Figure 4C). The frequency (parts of) chromosome 7, chromosome of cytogenetically aberrant clones 12p, chromosome 17p and (partial) gain within the erythroblast fractions did not of chromosome 8 were other frequently correlate to RS percentages in these four observed chromosomal aberrancies patients (data not shown). No hotspot (Figures 3A and 3E). In 6/16 cases the mutations in exons 13-16 of SF3B1 17p deletion involved the locus for were detected in sorted EB fractions TP53, and for 4/16 cases this deletion (data not shown). involved the locus of PRPF8 (marked As erythroblasts of RS-MN by asterisks in Figure 3A). Together, patients were observed as part of the these data indicate that no single gene malignant clone, we hypothesized that defect underlies the RS phenotype in RS could be eliminated upon treatment. this cohort: RS in AML is associated We therefore analyzed follow-up BM with a variety of adverse risk genetic smears including RS percentage, which defects. was available for 17 patients of the RS cohort. Eight patients received intensive Erythroblasts share genetic defects chemotherapy (median interval between with leukemic clones both evaluations 2 months (range 1-5)), An intriguing aspect of RS in MNs six were treated using hypomethylating is that erythroid differentiation takes agents (median interval 4.3 months place to a certain extent, while myeloid (2.5-12) and three patients received a differentiation is completely blocked combination of both (median interval resulting in blast accumulation. To 16 months (5-20) (Supplementary table determine whether RS are part of the 3)). Of this group, 65% responded to malignant leukemic clone, VAFs of TP53 therapy (defined as complete or partial and SRSF2 mutations detected by WES response) and 35% did not respond in the total MNC fraction were detected (defined as no response or relapse). in sorted erythroblast populations of Patients who responded showed a 8 patients. VAFs strongly correlated marked decrease in RS percentage at between both cell fractions (Pearson follow-up evaluation, whereas the RS r = 0.94, p = 0.0002) (Figure 4A). No percentage in non-responders was fairly correlation was observed between VAFs stable or increased (Figure 4D).
74 Ringsideroblasts in acute myeloid leukemia
R c R R c R c Ringsideroblasts R c R R c R c R c delins R R c del ariant allele re uency R c ariant allele re uency R c
ryt roblasts Ringsideroblasts ryt roblasts R R r otal s 3 R R R R R R raction R R R R R R R R R R r R R
Ringsideroblasts relati e c ange R otal s aseline ime oint raction
Fig 4. Genetic defects in erythroblast of patients with RS-phenotype A) VAFs of TP53 and SRSF2 mutations detected in MNC fractions (determined by WES) and erythroblast fraction (determined by targeted sequencing). B) Correlation between VAFs in erythroblast fractions (VAF indicated on left y-axis) and observed RS percentages (percentage indicated on right y-axis), different colors represent individual mutations (as indicated in legend of figure 4A). C) Differences observed in SNP array results between MNC- and EB fractions, figure displays screenshots taken from Nexus software. D) Relative changes in RS percentage of patients who received treatment, the RS percentage at follow- up examination (timepoint 2) are displayed relative to the RS percentages determined at diagnosis (baseline). Blue lines indicate patients who responded to therapy and red lines indicate patients who did not respond (see supplementary table 3).
Upregulated genes in RS-AML are background regarding cytogenetic and associated with megakaryocytic/ genetic defects (part of the Blueprint erythroid differentiation and mRNA study (Yi et al, in press)), SF3B1-mutated splicing MDS CD34+ samples (GSE63569,(16)) To investigate transcriptional and three AML samples containing differences that underlie the RS- SF3B1 mutations (TCGA and Blueprint phenotype in AML, RNA sequencing dataset and 1 of our own samples). was performed on CD34+ selected PCA and t_SNE analysis based on AML of six patients with ≥13% RS gene expression revealed clustering (Supplementary table 4). The results of ‘general’ AML samples on one side were compared to normal bone marrow of the plot and NBM samples and (NBM) CD34+ (GSE63569,(16)), samples MDS samples on the other side (Figure from 36 AML patients with a mixed 5A). RS-AML samples were located
75 Chapter 3
c romatin organi ation R R regulates genes in ol ed in megakaryocyte di erentiation and latelet unction megakaryocyte di erentiation regulation o cellular catabolic rocess li id biosynt etic rocess tem lated transcri tion initiation iral rocess cellular res onse to ormone stimulus R mR licing co actor biosynt etic rocess tetra yrrole biosynt etic rocess omeostasis o number o cells cell mor ogenesis in ol ed in di erentiation regulation o cellular locali ation negati e regulation o catabolic rocess − neural tube de elo ment regulation o li id metabolic rocess stem cell o ulation maintenance telomere organi ation − negati e regulation o rotein modi ication rocess − − − log t− o n cell acti ation in ol ed in immune res onse cytokine roduction cell acti ation in lammatory res onse R ytokine ignaling in mmune system R da ti e mmune ystem regulation o innate immune res onse ositi e regulation o ydrolase acti ity sa like rece tor signaling at ay R oll ike Rece tors ascades endocytosis 3 leukocyte c emota is R esicle mediated trans ort ka a kinase ka a signaling sa steoclast di erentiation − macro age acti ation dendritic cell migration res onse to tumor necrosis actor cell ro ection assembly − regulation o rotein kinase acti ity − − log eneral R
Fig 5. RS-AML transcription program A) PCA and t-SNE plots using RNA-seq results of the indicated cell types. For the following cell types previously published RNA-seq was included: NBM (GSE63569, (16)), General_AML patients (Blueprint study, (Yi et al, submitted)), SF3B1_MDS (GSE63569, (16) ) and SF3B1_AML (combination of TCGA dataset, Blueprint study and own data), B) Biological process enrichment for genes up- and downregulated in RS-AML as compared to a general AML cohort.
in between these populations, as well in megakaryocyte differentiation as the SF3B1 mutated AML samples. (Supplementary table 5). Several Compared to NBM CD34+, 1196 genes upregulated genes that are included in were identified to be upregulated in this gene ontology (GO) term, including AML with RS-phenotype. Functionally, GATA1, GATA2, KLF1, AHSP and this gene set was highly enriched for TRIM58, are also involved in the epigenetic and protein modifications. regulation of erythroid differentiation Processes affected by downregulated (Supplementary table 5). Also, EPOR, the genes (n=1309) include cell cycle- gene encoding for the erythropoietin related pathways (supplementary figure receptor was upregulated in RS-AML. 2). To investigate gene expression In addition, functional annotation patterns that are correlated to RS- revealed that mRNA splicing is affected. AML but not to AML in general, we This GO term includes SF3A1, SF3A2, compared gene expression of RS- SF1, SNRPC, which are all genes AML to data derived from a cohort of involved in splice site selection, and AML patients. Functional annotation SRSF8, SAP18 and MAGOH which of the 1464 upregulated genes in are important for the regulation of RS-phenotype AML demonstrated alternative mRNA splicing (data not chromatin organization and stem cell shown). The 2223 downregulated genes maintenance as affected processes are mainly involved in immune-related (Figure 5B). Furthermore, this analysis processes (Figure 5B). revealed enrichment for genes involved
76 Ringsideroblasts in acute myeloid leukemia
Similarities with SF3B1 mutated MN regulated in all three groups (Figure samples 6D) (Supplementary table 6). Examples As the presence of RS is strongly of upregulated genes included ALAS2, associated with SF3B1 mutations HBB and NFE2, SC4A1, which in MDS, we determined whether are all players in heme metabolism RS-phenotype AML samples share (Supplementary table 5). Functional similarities with SF3B1mut MNs. Using annotation revealed erythrocyte-related K-means clustering, we visualized processes as highest for this gene set differentially expressed genes between (Figure 6D). Commonly down regulated RS-phenotype AML (n=6), NBM genes strongly enrich for interleukin-10 CD34+ cells (n=5) and both SF3B1mut pathway signaling (Figure 6D). Gene AML (n=3) and SF3B1mut MDS (n=8). expression of PRPF8 and SF3B1 was This analysis revealed 6 major clusters not lower in RS-phenotype AML as of differentially expressed genes compared to NBM (supplementary figure (Figures 6A and 6B). Clusters 1 and 6, 3). Together, this demonstrates that RS- 3 enriched for genes associated with the phenotype AML samples shares a gene GO terms cell cycle-related processes expression signature with SF3B1mut and protein modifications, respectively, samples, but that these AMLs also have which distinguished the AML samples unique characteristics that distinguish from MDS and NBM samples (Figure them from SF3B1mut MNs. 6B and supplementary figure 3). Cluster 4, which is enriched for the GO terms Discussion cellular communication and trafficking, seems to be specific for NBM samples In this study, we characterized the (supplementary figure 3). Cluster 5 presence of RS in a cohort of patients represents 655 genes that are specifically diagnosed with acute myeloid leukemia upregulated in SF3B1mut-MDS samples. or high-risk MDS. We observed that Functionally, these genes are involved RS-AML is enriched for ELN adverse in erythroid development including iron risk disease, including associated genetic metabolism (Figure 6C). In three RS- and chromosomal defects. Clinically, phenotype AML samples, RS05, RS11 higher RS percentage at diagnosis is and RS19, cluster 5 showed a pattern accompanied with higher incidence of comparable to the MDS samples (Figure poor risk disease characteristics. Unlike 6A). In contrast, the three SF3B1mut- MDS, no single gene defect was found AML samples and samples RS01, RS06 that underlies the RS phenotype in AML. and RS08 showed a lower expression of Gene expression analysis indicated that cluster 5 genes (Figure 6A). This division RS-AML, as compared to a general AML within the RS-AML group cannot be cohort, is characterized by upregulation linked to differences regarding RS, blasts of genes enriched for megakaryocytic/ or erythroblast percentage or based on erythroid differentiation and mRNA genetic mutations (Supplementary table splicing. 4). Although RS are generally To identify gene expression regarded as a specific feature of certain differences that are shared by RS- MDS subtypes, the reported incidence phenotype AML samples and SF3B1mut, of 25% indicates that RS is a rather we compared gene expression of all common finding in AML.(17) In contrast gene sets to NBM and determined to reports on MDS-RS subtypes, the overlapping gene sets. A total mutations in spliceosome gene SF3B1 of 47 genes were consistently up were rarely observed in our RS-AML regulated and 125 genes were down cohort. The paucity of this mutation in
77 Chapter 3
cluster cluster cluster
ression luster
ed e n og
mali − o r cluster cluster cluster luster − n ression
ed e
luster og n
mali
luster o r n R R R mut mut mut mut mut mut luster luster n co actor metabolic rocess n eme metabolic rocess gas trans ort R ryt rocytes take u o ygen and release carbon dio ide cellular rotein com le disassembly eryt rocyte di erentiation luster cellular amino acid metabolic rocess drug trans ort n multicellular organismal omeostasis res onse to metal ion res onse to iron ion 3 R rans ort o small molecules transition metal ion omeostasis iral li e cycle res onse to to ic substance cell mor ogenesis in ol ed in di erentiation
R itoc ondrial iron sul ur cluster biogenesis
R R R R R R R com le negati e regulation o su ramolecular iber organi ation SF3B1 mut SF3B1 mut latelet acti ation R log
n R ryt rocytes take u o ygen and release carbon dio ide de ense res onse to irus el er ty e immune res onse myeloid cell di erentiation R o n R R da ti e mmune ystem R isual ototransduction mut o n mut R o tosis regulation o metal ion trans ort log o n n R nterleukin signaling negati e regulation o a o totic signaling at ay res onse to to ic substance cellular res onse to li id negati e regulation o binding transcri tion actor acti ity regulation o cell acti ation c ronic in lammatory res onse ositi e regulation o rogrammed cell deat sa ka a signaling at ay ibrinolysis r yt mic rocess amino acid trans ort R emokine rece tors bind c emokines blood essel mor ogenesis ositi e regulation o at cell di erentiation brain de elo ment regulation o rotein serine t reonine kinase acti ity kidney de elo ment mut o n mut regulation o ibroblast migration log Fig 6 Comparison between RS-AML and SF3B1mut AML and MDS A) Heatmap of gene expression by supervised k-means clustering in RS-AML, SF3B1mut AML and SF3B1mut MDS versus control NBM cells, B) Expression of the 6 clusters identified in A) for individual groups, C) Biological process enrichment for cluster 5 as identified in A), for other clusters see supplementary figure 3), D) Gene overlap of common up- and downregulated genes between RS-AML, SF3B1mut AML and SF3B1mut MDS versus control. Biological process enrichment is shown for commonly up- and downregulated genes.
RS-AML might be explained by the low expression of PRPF8. tendency towards AML transformation Although we did not identify from MDS subtypes with SF3B1 a single gene defect that underlies the mutations(8,13). Also, we did not identify RS phenotype in AML, panel-based and genetic mutations in PRPF8, the only whole exome sequencing revealed a high other gene that has been associated with incidence of adverse-risk mutations in the a RS phenotype.(19) In our cohort 25% RS-AML cohort, including DNMT3A, (4/16) patients had a deletion of the RUNX1 and ASXL1. Mutations in chromosomal locus of PRPF8, but we TP53 were observed most recurrently, did not observe significantly lower gene particularly in patients with >15% RS
78 Ringsideroblasts in acute myeloid leukemia
at AML diagnosis. These results are in as being upregulated in RS-AML, agreement with a previous study using while both become downregulated a restricted gene panel to analyze the during myeloid differentiation(31). These RS-phenotype in AML patients.(17) The results suggest that the erythroid high incidence of poor-risk cytogenetic differentiation program is more active aberrancies, including chromothripsis, in RS-AML, either by a more active probably reflects the high frequency transcriptional program in HSCs or by of TP53 mutations.(25) Interestingly, higher contribution of MEPs to the RS have also been reported in relation CD34+ pool, resulting in the potential to MNs in Li-Fraumeni patients with a of (aberrant) erythroid differentiation congenital TP53 mutation.(26) Defects to a certain stage. in TP53 also occur frequently in acute In MDS, SF3B1 mutations pre- pure erythroid leukemia (AEL). Besides sumably result in RS formation by in- having a high frequency of TP53 terfering with mRNA splicing, resulting mutations, AML with RS-phenotype in differential gene expression.(8,15,16) Al- 3 resembles AEL regarding higher age though RS-AML is genetically distinct at diagnosis (median age 68 years), a from RS-MDS, downstream mecha- male predominance and presence of nisms that result in aberrant erythroid complex chromosomal aberrancies. differentiation may be similar. Func- (27) However, unlike AEL, erythroid tional annotation analysis demonstrated differentiation in AML-RS does not stop mRNA splicing was an affected process at the proeythroblastic stage. Instead, in RS-AML compared to a general AML accumulation of immature myeloid cohort. For the future it is of relevance blast cells is present in AML-RS, while to determine whether splicing defects this is rare in AEL.(27) similar to RS-MDS occur in RS-AML. The presence of RS in our This would be of particular interest AML cohort is not restricted to a as spliceosome inhibitors represent a specific ontogenic subtype(28), indicating promising strategy to specifically target that RS in AML arises from a common MNs with spliceosome mutations.(32) If mechanism that goes beyond disease RS-AML expose splicing defects com- ontogeny. Also, it has been reported parable to RS-MDS, spliceosome inhib- that the presence of RS on its own is not itors could be an interesting treatment predictive for overall survival in AML option for these often adverse-risk AML patients,(17) suggesting that RS-AML is that generally poorly benefit from cur- not a distinct disease entity. However, rent treatment modalities.(18,33) we observed several differences based on gene expression that seem to be In conclusion, we have demonstrated specific for RS-AML. As compared that ringsideroblasts are a frequent to a general AML cohort, RS-AML is finding in AML, particularly in relation characterized by higher expression of to adverse risk genetic defects. We genes involved in megakaryocytic and revealed that erythroblasts share erythroid differentiation. Both lineages ancestry with malignant myeloid blast are derived from the megakaryocyte- cells in RS-AML. Although the genetic erythrocyte progenitor (MEP) cell. background of RS-AML differs from (29) The transcription factors GATA1 that of RS-MDS, downstream effector and GATA2 are involved in erythroid pathways may be comparable, providing lineage restriction, partly via a possible explanation for presence of stimulation of erythropoietin receptor RS in AML. expression(30) which was also reported
79 Chapter 3
References
1. Ohba R, Furuyama K, Yoshida K, et Frequent pathway mutations of splicing myeloid leukemia. Haematologica. 2017; al. Clinical and genetic characteristics machinery in myelodysplasia. Nature. of congenital sideroblastic anemia: 2011; 18. Papaemmanuil E, Gerstung M, comparison with myelodysplastic syndrome Bullinger L, et al. Genomic Classification with ring sideroblast (MDS-RS). Annals of 10. Malcovati L, Papaemmanuil E, Bowen and Prognosis in Acute Myeloid Leukemia. hematology. 2013; DT, et al. Clinical significance of SF3B1 NEJM. 2016; mutations in myelodysplastic syndromes 2. Arber DA, Orazi A, Hasserjian R, et al. and myelodysplastic/myeloproliferative 19. Kurtovic-Kozaric A, Przychodzen The 2016 revision to the World Health neoplasms. Blood. 2011; B, Singh J, et al. PRPF8 defects cause Organization classification of myeloid neo- missplicing in myeloid malignancies. plasms and acute leukemia. Blood. 2016; 11. Patnaik MM, Lasho TL, Hodnefield Leukemia. 2015; JM, et al. SF3B1 mutations are prevalent 3 3. Sheftel AD, Richardson DR, Prchal J, in myelodysplastic syndromes with ring 20. Dohner H, Estey E, Grimwade D, et et al. Mitochondrial iron metabolism and sideroblasts but do not hold independent al. Diagnosis and management of AML in sideroblastic anemia. Acta haematologica. prognostic value. Blood. 2012; adults: 2017 ELN recommendations from 2009; an international expert panel. Blood. 2017; 12. Damm F, Kosmider O, Gelsi-Boyer 4. Cotter PD, Baumann M, Bishop DF. V, et al. Mutations affecting mRNA 21. da Silva-Coelho P, Kroeze LI, Yoshida Enzymatic defect in "X-linked" sideroblastic splicing define distinct clinical phenotypes K, et al. Clonal evolution in myelodysplastic anemia: molecular evidence for erythroid and correlate with patient outcome in syndromes. Nat Commun. 2017; delta-aminolevulinate synthase deficiency. myelodysplastic syndromes. Blood. 2012; Proceedings of the National Academy of 22. Simons A, Shaffer LG, Hastings RJ. Sciences of the United States of America. 13. Greenberg PL, Tuechler H, Schanz J, et Cytogenetic Nomenclature: Changes in 1992; al. Revised international prognostic scoring the ISCN 2013 Compared to the 2009 system for myelodysplastic syndromes. Edition. Cytogenetic and genome research. 5. Allikmets R, Raskind WH, Hutchinson A, Blood. 2012; 2013; et al. Mutation of a putative mitochondrial iron transporter gene (ABC7) in X-linked 14. Chen M, Manley JL. Mechanisms of 23. Fleming MD. Congenital sideroblastic sideroblastic anemia and ataxia (XLSA/A). alternative splicing regulation: insights from anemias: iron and heme lost in Human molecular genetics. 1999; molecular and genomics approaches. Na- mitochondrial translation. Hematology ture reviews Molecular cell biology. 2009; American Society of Hematology Education 6. Guernsey DL, Jiang H, Campagna DR, et Program. 2011; al. Mutations in mitochondrial carrier fami- 15. Alsafadi S, Houy A, Battistella A, et al. ly gene SLC25A38 cause nonsyndromic au- Cancer-associated SF3B1 mutations affect 24. Berger G, Kroeze LI, Koorenhof-Scheele tosomal recessive congenital sideroblastic alternative splicing by promoting alternative TN, et al. Early detection and evolution anemia. Nature genetics. 2009; branchpoint usage. Nat Commun. 2016; of preleukemic clones in therapy-related myeloid neoplasms following autologous 7. Schmitz-Abe K, Ciesielski SJ, Schmidt PJ, 16. Dolatshad H, Pellagatti A, Liberante SCT. Blood. 2018; et al. Congenital sideroblastic anemia due FG, et al. Cryptic splicing events in the iron to mutations in the mitochondrial HSP70 transporter ABCB7 and other key target 25. Fontana MC, Marconi G, Feenstra homologue HSPA9. Blood. 2015; genes in SF3B1-mutant myelodysplastic JDM, et al. Chromothripsis in acute myeloid syndromes. Leukemia. 2016; leukemia: Biological features and impact on 8. Papaemmanuil E, Cazzola M, Boultwood survival. Leukemia. 2017. J, et al. Somatic SF3B1 mutation in 17. Martin-Cabrera P, Jeromin S, Perglerova myelodysplasia with ring sideroblasts. K, et al. Acute myeloid leukemias with ring 26. Talwalkar SS, Yin CC, Naeem RC, et NEJM. 2011; sideroblasts show a unique molecular al. Myelodysplastic syndromes arising in signature straddling secondary acute patients with germline TP53 mutation and 9. Yoshida K, Sanada M, Shiraishi Y, et al. myeloid leukemia and de novo acute Li-Fraumeni syndrome. Arch Pathol Lab
80 Ringsideroblasts in acute myeloid leukemia
Med. 2010; 29. Akashi K, Traver D, Miyamoto T, et al. development, and leukemia. Blood. 1997; A clonogenic common myeloid progenitor 27. Reinig EF, Greipp PT, Chiu A, et al. De that gives rise to all myeloid lineages. 32. Seiler M, Yoshimi A, Darman R, et novo pure erythroid leukemia: refining the Nature. 2000; al. H3B-8800, an orally available small- clinicopathologic and cytogenetic character- molecule splicing modulator, induces istics of a rare entity. Modern pathology : 30. Zon LI, Youssoufian H, Mather C, et al. lethality in spliceosome-mutant cancers. an official journal of the United States and Activation of the erythropoietin receptor Nature medicine. 2018; Canadian Academy of Pathology, Inc. 2018; promoter by transcription factor GATA-1. Proceedings of the National Academy of 33. Rucker FG, Schlenk RF, Bullinger L, 28. Lindsley RC, Mar BG, Mazzola E, et Sciences of the United States of America. et al. TP53 alterations in acute myeloid al. Acute myeloid leukemia ontogeny is 1991; leukemia with complex karyotype correlate defined by distinct somatic mutations. with specific copy number alterations, Blood. 2015; 31. Tenen DG, Hromas R, Licht JD, et al. monosomal karyotype, and dismal Transcription factors, normal myeloid outcome. Blood. 2012; 3
Supplementary tables
Supplementary table 1: Genes implicated in congenital sideroblastic anemia
ABCB7 ISCA1 ALAS2 ISCA2 FECH ISCU GLRX5 MFRN1 HSCB NIFS HSP70 PUS1 HSPA9 SLC25A38 IRBP1 YARS2 IRBP2
81 Chapter 3 p.G102C p.D224E p.V1192D p.R335H p.H2706Q p.L769Q p.K454X p.D104N p.S331T p.V56E p.F871_ K872delins p.R105X p.R1659C p.R353H p.G62E p.V660A p.P33S p.M1L p.R287H p.P244L p.R178C p.R359C p.V450M p.G14D p.K690Q p.A93T p.47_55del p.675_676del p.F369fs p.G642fs p.L957fs p.L198fs p.G312fs p.A356fs c.G304T c.C672A c.T3575A c.G1004A c.T8118A c.T2306A c.A1360T c.G310A c.3955-2A>T c.G992C c.T167A c.1655-1G>A c.2611_2612insAG c.C313T c.C4975T c.G1058A c.G185A c.T1979C c.C97T c.A1T c.G860A c.C731T c.C532T c.C1075T c.G1348A c.G41A c.A2068C c.G277A c.141_164del c.2025_2027del c.1104_1105 insCCCG c.1927dupG c.2871dupA c.594delG c.934delG c.1066delG 6p21.33 1p12 5q12.3 10q26 17q25.3 10q26.13 20p11.23 6p12.3 11q23.3 19q13.33 4q24 1p36.12 Xp11.4 12p13.2 22q13.33 20p12.3 19q13.33 17p12 17p13.1 17p13.1 16q24.3 15q25.1 12q12 11q23.2 10q23.1 9q34.11 8q12.1 3p25.3 17q25.1 5p13.3 21q22.12 20q11.21 4q24 19q13.11 3 11q23.3 14q23.3 ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain ns SNV ns SNV ns SNV stopgain stopgain ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV nonfs del nonfs del fs in s fs ins fs ins fs del fs del fs del exonic exonic exonic exonic exonic exonic exonic exonic splicing exonic exonic splicing exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic HIST1H4I FAM46C ERBB2IP EIF3A DNAH17 CTBP2 CSRP2BP C6orf141 TET2 BCL9L C19orf73 HSPG2 BCOR ETV6 SBF1 PROKR2 CLEC11A CDRT1 TP53 ASGR2 TUBB3 ABHD17C ABCD2 HTR3B GRID1 URM1 TGS1 CAV3 SRSF2 ZFR RUNX1 ASXL1 TET2 CEBPA CBL GPHN 9 8 7 6 5 4 3 1 2 9 8 7 6 5 4 3 1 2 10 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 RS011 RS010 BM-MNC BM-MNC Amino acid change p.G1491R p.S840P p.G56S p.E390K p.R291C p.H92L p.R1095Q p.D1083E p.P1485A p.G120D p.V181M p.R279Q p.V358A p.A298P p.A366V p.R148G p.L52M p.V928M p.R77H p.R266L p.P391fs p.F711fs p.D587fs p.L454F p.P44Q p.T1014M p.T1014M p.P95L p.G870S p.R77C p.R318X p.N182fs p.E208A Coding sequence change c.G4471A c.T2518C c.G166A c.G1168A c.C871T c.A275T c.G3284A c.C3249A c.C4453G c.G359A c.G541A c.G836A c.T1073C c.G892C c.C1097T c.A442G c.C154A c.G2782A c.G230A c.G797T c.1173delC c.2132delT c.1760_1764del c.C1360T c.C131A :c.A150T c.C3041T c.C284T c.G2608A c.C229T c.C952T c.544delA c.A623C Chr. 2p23.3 2p16.3 2q13 2q14.1 6q14.3 6q22.32 7q11.22 7q31.1 9q34.3 10q22.2 11q12.1 11q12.2 13q12.13 14q23.1 16q12.2 17p13.1 17q11.2 20q13.32 Xp11.23 Xq25 2p23.3 5q35.3 19q13.31 2p13.3 3q29 9p13.3 17q21.2 17q25.1 18q12.3 22q11.21 Xp21.2 22q13.2 1p36.31 V Type of Type variant ns SNV ns SN ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV frameshift deletion frameshift deletion frameshift deletion ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain frameshift deletion ns SNV Location of gene exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic Gene CAD FBXO11 ANAPC1 DPP10 HTR1E HINT3 AUTS2 LAMB4 COL5A1 MYOZ1 DTX4 DDB1 PABPC3 C14orf39 IRX6 TP53 ANKRD13B ZNF831 PQBP1 DCAF12L1 DNMT3A ZFP62 ZNF45 ANTXR1 CPN2 GLIPR2 TOP2A SRSF2 SETBP1 LRRC74B MAGEB1 EP300 PLEKHG5 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 10 11 12 13 14 15 16 17 18 19 20 10 11 No
RS001 RS002 RS003 Patient BM-MNC BM-MNC BM-MNC Supplementary table 2: WES results Supplementary table 2:
82 Ringsideroblasts in acute myeloid leukemia p.V140M p.Q1084X p.R153H p.N676D p.P34S p.S375T p.G2402D p.D1815N p.P290S p.C211W p.G53R p.V8M p.W329R p.S1662N p.S325G p.R101H p.I3181T p.E430K p.W52X p.Y73X p.P1617A p.N1616fs p.Y274C p.K431N p.A362T p.G1770R p.V585I p.N90S p.Q153R p.V533M p.W476X p.M1254V p.M1481L p.H62N p.A29P p.G388D p.N64I p.T92M p.K289M c.G418A c.C3250T c.G458A c.A2026G c.C100T c.T1123A c.G7205A c.G5443A c.C868T c.C633G c.G157A c.G22A c.T985C c.G4985A c.A973G c.G302A c.T9542C c.G1288A c.G155A c.T219G c.C4849G c.4848_4849insT c.A821G c.G1293T c.G1084A c.G5308C c.G1753A c.A269G c.A458G c.G1597A c.G1427A c.A3760G c.A4441T c.C184A c.G85C c.G1163A c.A191T c.1328-1G>C c.C275T c.A866T 17p13.1 16p11.2 15q26.1 15q24.2 15q13.3 11q13.2 10q26.2 7p22.1 3q27.1 3q21.2 3p26.1 1q44 1q21.3 1q21.1 3p12.3 1p36.13 1p36.13 19q13.31 17p13.1 17p13.1 4q24 4q24 15q24.2 11q13.2 9q22.2 3q25.2-q26.2 11q13.2 9q21.32 15q24.2 19q13.2 2p21 1q21.2 21q22.3 11p15.4 17p13.3 1p32-p31 21q22.1 19p13.3 3q26.32 18q11.2 3 ns SNV stopgain ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain stopgain ns SNV fs ins ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV splicing ns SNV ns SNV exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic splicing exonic exonic TP53 SRCAP MESP2 GOLGA6D GREM1 KDM2A MKI67 TNRC18 THPO ITGB5 GRM7 OR2T27 SHE PDE4DIP ZNF717 CROCC UBR4 ZNF285 TP53 TP53 TET2 TET2 SNX33 SLC3A2 SLC28A3 SI SF1 RMI1 RASGRF1 PRX PKDCC PDE4DIP PCNT OR52E8 OR1E1 MACF1 KRTAP13-3 KHSRP KCNMB2 HRH4 9 8 7 6 5 4 3 1 2 16 15 14 13 12 11 10 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 RS018 BM-MNC p.D146N p.R291C p.R1400H p.G510C p.V350L p.A16V p.F173S p.V91G p.N91S p.S10T p.A545V p.V144L p.S653L p.A371V p.R2316Q p.C44F p.P4L p.G121S p.Y498C p.E79K p.R951X p.Q72R p.M9T p.H47P p.S1035I p.C106Y p.G260D p.P2742L p.R368C p.R211X p.P269fs p.A300fs p.N135T p.S19F p.R1263W p.S647P p.V353M c.G436A c.C871T c.G4199A c.G1528T c.G1048T c.C47T c.T518C c.T272G c.A272G c.G29C c.C1634T c.G430T c.C1958T c.C1112T c.G6947A c.G131T c.C11T c.G361A c.A1493G c.G235A c.C2851T c.3497-1G>A c.A215G c.T26C c.A140C c.G3104T c.G317A c.G779A c.C8225T c.C1102T c.C631T c.806delC c.898dupG c.A404C c.C56T c.C3787T c.T1939C c.G1057A 1q23.3 1p36.13 1q21.3 2p23.3 2q37.1 3q27.1 4q12 5q35.3 6q22.1 6q24.1 7p22.3 8p11.21 9p11.2 15q25.3 16p13.3 17p13.1 19p13.11 19p13.11 Xp22.31 Xq26.1 2q31.1 17q11.2 6q14.1 7q11.23 8q21.3 16p11.2 17p13.1 17p11.2 17q11.2 22q11.23 6q13 Xp11.23 3q21.3 2p23.3 2p21 7p13 10q25.1 11p15.4 ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain
ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain frameshift deletion frameshift insertion ns SNV ns SNV ns SNV ns SNV ns SNV exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic splicing exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic ITLN1 IGSF21 TCHH DNMT3A SP140 THPO PAICS SLC34A1 GOPC ADGRG6 FAM20C VDAC3 CNTNAP3B KLHL25 CACNA1H TP53 CHERP TSSK6 ANOS1 RAB33A LRP2 NF1 UBE3D CLDN4 CNBD1 TAOK2 TP53 FLII NF1 SMARCB1 RIMS1 TFE3 GATA2 PRR30 EPAS1 HECW1 CFAP43 TRIM34,TRIM- 6-TRIM34 1 3 4 6 7 8 9 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 10
RS004 RS005 BM-MNC BM-MNC
83 Chapter 3 p.T331S p.R19H p.I768L p.C1173S p.S271X p.A365fs p.R417C p.E810fs p.K105E p.R590W p.H277Q p.N297S p.R244X p.K666N p.T5095S p.S137A p.Q61H p.P402A p.V40F p.R150W p.R288C p.V1280M p.E278X p.V178M p.I23T p.G1094S p.K302R p.G191D p.Q425K p.I674T p.F142fs p.I143fs p.R614G p.R1388Q p.W2X p.H36R c.A991T c.G56A c.A2302C c.G3518C c.C812A c.1093_1094- insTCGG c.C1249T c.2429delA c.A313G c.C1768T c.C831G c.A890G c.C730T c.G1998T c.C15284G c.T409G c.296+3G>C c.A183C c.C1204G c.G118T c.C448T c.C862T c.G3838A c.G832T c.858-5C>T c.G532A c.T68C c.G3280A c.A905G c.G572A c.829+5A>T c.C1273A c.T2021C c.425_426insC c.427_430del c.A1840G c.G4163A c.G6A c.A107G 18q23 17q21.31 14q23.1 12p13.1 12p13.32 11p13 11p13 10q26.3 7q36.3 7q21.3 6p21.33 3q21.3 3p14.3 2q33.1 2q31.2 1q31.1 1p13.2 20q13.33 19p13.3 17p13.1 17p13.1 15q21.2 14q32.33 14q32.33 14q24.2 12q21.31 11q14.3 8q24.22 6q21 5q31.3 4q24 3q26.31 3q23 3p22.3 3p22.3 2p23.3 Yq11.222 1q23.3 3 17p13.1 ns SNV ns SNV ns SNV ns SNV stopgain fs ins ns SNV fs del ns SNV ns SNV ns SNV ns SNV stopgain ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV fs ins fs del ns SNV ns SNV stopgain ns SNV exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic splicing exonic exonic exonic exonic exonic exonic exonic splicing exonic exonic exonic exonic exonic splicing exonic exonic exonic exonic exonic exonic exonic exonic GALR1 SOST ARID4A GRIN2B CCND2 WT1 WT1 JAKMIP3 WDR60 PPP1R9A VWA7 GATA2 ABHD6 SF3B1 TTN PRG4 KCNQ2 NRAS AMH TP53 TP53 LEO1 TDRD9 XRCC3 SMOC1 METTL25 TRIM49 ADCY8 PDSS2 KIAA0141 SLC9B1 SPATA16 U2SURP CMTM6 CMTM6 DNMT3A KDM5D FCRLB TP53 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 17 16 15 14 13 12 11 10 20 19 18 17 16 15 14 13 12 11 10 18 17 RS020 RS019 PB-MNC PB-MNC p.T341I p.L697F p.T352M p.A2V p.G217S p.S55X p.Q370fs p.N817S p.N695S p.E264K p.G375C p.E315K p.R10Q p.P95H p.Y91C p.L367S p.D90fs p.I420T p.N3158S p.T1613M p.R132C p.T524K p.H439Y p.R198C p.I366M p.N243S p.N155S p.T188M p.H61R p.V963I p.R72H p.S762G p.R354X p.Q514X p.R1337X c.C1022T c.C2089T c.C1055T c.C5T c.G649A c.C164G c.1109_1122del c.A2450G c.A2084G c.G790A c.G1123T c.G943A c.G29A c.C284A c.A272G c.T1100C c.114+1G>A c.269dupA c.T1259C c.A9473G c.C4838T c.C394T c.C1571A c.C1315T c.C592T c.C1098G c.A728G c.A464G c.C563T c.A182G c.G2887A c.G215A c.A2284G c.C1060T c.C1540T c.C4009T c.562+1G>T c.3268-1G>C 14q21.1 15q25.2 16q24.3 17q21.31 19p13.2 17p13.1 21q22.12 1p36.33 5q11.2 5q13.2 5q33.1 12q21.33 15q26.1 17q25.1 19p13.11 Xp11.22 5q21.1 2p23.3 1p36.31 1q42.3 2q21.2 2q34 3p22.3 3q27.1 4q28.3 9p24.2 11p15.5 14q32.31 16p11.2 17p13.1 18q21.2 Xp21.1 Xq27.2 8p21.1 16p13.3 18q21.2 12p13.31 Xq28 ns SNV ns SNV ns SNV ns SNV ns SNV stopgain frameshift deletion ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV
frameshift insertion ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain stopgain stopgain
exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic splicing exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic splicing splicing LRFN5 EFTUD1 VPS9D1 C17orf53 FCER2 TP53 RUNX1 MIB2 MAP3K1 MRPS27 SYNPO CCER1 IDH2 SRSF2 FCHO1 SMC1A PPIP5K2 DNMT3A NOL9 LYST NCKAP5 IDH1 ARPP21 HTR3E PCDH10 GLIS3 PSMD13 DYNC1H1 ZNF629 TP53 DCC FAM47C MAGEC1 ELP3 CAPN15 DCC ING4 AFF2 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 10 11 10 11 12 13 14 15 16 17 18 19 20 21
RS006 RS007 BM-MNC BM-MNC
84 Ringsideroblasts in acute myeloid leukemia p.R4730Q p.H54Y p.P91A p.M194T p.R250C p.P900S p.Q937X p.V188A p.V97M p.L505V p.S11220R p.R116W p.L321R p.A261V p.A588fs p.P1359S p.G349V p.S714R p.A755V p.K203N p.E374K p.E363Q p.K346N p.P65S p.G362E p.A13V p.G3606V p.L747fs p.P120L p.R354H p.V399M p.E370K p.G427V p.N286H c.G14189A c.C160T c.C271G c.T581C c.C748T c.C2698T c.C2809T c.T563C c.G289A c.C1513G c.T33660G c.C346T c.T962G c.C782T c.1763delC c.C4075T c.G1046T c.T2142G c.C2264T c.G609T c.G1120A c.G1087C c.G1038C c.C193T c.G1085A c.C38T c.G10817T c.2241delG c.C359T c.G1061A c.G1195A c.G1108A c.G1280T c.A856C 19q13.2 16p13.3 15q11.2 9q31.1 4p12 2q13 2q11.2 21q21.1 1q24.2 1q42.13 19p13.2 17p13.1 16q22.1 16p13.3 16p13.3 15q24.2 12q13.13 11q24.3 10q26.3 9q34.3 8p21.3 8p21.3 8p21.3 7p21.2 6p21.32 4p15.31 2q32.1 2p22.2 Xq13.1 1q25.1 2p23.1 Xp22.11 19q13.31 19q13.12 3 ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV stopgain ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV fs del ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV fs del ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic RYR1 CASKIN1 CYFIP1 OR13C3 GUF1 BUB1 TBC1D8 CHODL GPR161 ADCK3 MUC16 TP53 ATP6V0D1 SRL BAIAP3 CSPG4 SCN8A NFRKB KNDC1 LCN1 LPL LPL LPL AGMO KIFC1 NCAPG FSIP2 CEBPZ CITED1 SERPINC1 CAPN14 ZNF645 IRGC ZNF781 9 8 7 6 5 4 3 1 2 9 8 7 6 5 4 3 1 2 21 20 19 18 17 16 15 14 13 12 11 10 21 20 19 18 RS022 RS021 BM-MNC BM-MNC p.G642fs p.R271S p.A1003T p.L70F p.A156P p.V29F p.R151C p.R156C p.V11M p.D160Y p.V127I p.C537Y p.R2049C p.K2010N p.G549D p.A65T p.R26Q p.V266I p.R961H p.V353 p.I63T p.A58V p.E105K p.S471L p.P361A p.V220F c.1927dupG c.A813T c.G3007A c.C208T c.G466C c.G85T c.C451T c.C466T c.G31A c.G478T c.G379A c.G1610A c.C6145T c.G6030T c.G1646A c.G193A c.G77A c.G796A c.G2882A c.G1057A c.T188C c.C173T c.G313A c.C1412T c.C1081G c.G658T 20q11.21 1p34.1 1p21.1 2p11.2 3p21.31 6p22.1 6p12.2 8q24.3 17p13.1 18p11.21 1p36.22 2q31.1 3p21.1 6p12.1 8p12 10q23.33 11p14.1 11q12.1 11q12.2 11q23.3 17p13.1 17q12 17q25.3 21q22.11 21q22.3 Xq12 frameshift insertion ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV ns SNV exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic exonic ASXL1 DPH2 COL11A1 CD8A ELP6 OR2J3 EFHC1 OPLAH TP53 CIDEA SPSB1 SP3 STAB1 DST UNC5D SLC35G1 KCNA4 OR5M9 DAGLA CADM1 TP53 GPR179 FSCN2 IFNAR1 DIP2A HEPH 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 10 11 12 13 14 15 16
RS008 RS009 PB-MNC BM-MNC ns, non synonymous; fs del, frameshift deletion; fs ins, frameshift insertion frameshift fs ins, deletion; frameshift fs del, non synonymous; ns,
85 Chapter 3 Treatment Cluster 5 Down Up Down Down Up 3 Up Daunorubicin + cytarabine Azacitidine followed by daunorubicin + cytarabine by Azacitidine followed Idarubicin + cytarabine Idarubicin + cytarabine Idarubicin + cytarabine Azacitidine Daunorubicin + cytarabine, followed bij azacitidine 1year followed Daunorubicin + cytarabine, Azacitidine Azacitidine Azacitidine Daunorubicine + cytarabine, followed by mercaptopurine, azacitidine, azacitidine, mercaptopurine, by followed Daunorubicine + cytarabine, decitabine Daunorubicine + cytarabine Azacitidine Daunorubicine + cytarabine Idarubicin + cytarabine Idarubicine + cytarabine Azacitidine R PR PR CR CR CR CR CR CR CR CR CR NR NR NR NR NR* Outcome Recurrent mutations Recurrent TP53 TP53, GATA2 TP53, IDH2, RUNX1, SRSF2 RUNX1, IDH2, TP53, ASXL1 TP53, TP53, TP53, TET2, TET2, TET2, SF1 TET2, TP53, TP53, TP53, TP53, TP53, DNMT3A TP53, 5 4 3 8 1 4 1 20 16 12 2.5 1.5 4.5 3.5 2.5 3.5 1.5 Interval (months) AML de novo AML de novo AML de novo AML de novo AML de novo AML de novo ELN 2017 35% 28% 32% 21% 64% 47% 36% 65% 43% 25% 58% 30% 21% 30% 10% 19% 39% Erythro- blasts (%) blasts nd 9% 1% 15 36 44 20 22 13 38% 25% 28% 29% 36% 27% 27% 23% 43% 39% 14% 19% 29% 29% Sidero- blasts (%) blasts % Ringsideroblasts 1% 2% 2% 3% 5% 2% 8% 3% 3% 3% 1% 1% (%) 24% 21% 18% 14% 14% Blasts Timepoint 2: Follow-up Timepoint 2: 1% 0% 0% 1% 0% 2% 0% 6% 7% 0% 13% 25% 56% 39% 33% 55% 25% 16 21 17 27 10 24 RS (%) % Erythro-blasts 33% 50% 24% 25% 16% 16% 29% 29% 30% 12% 17% 35% 15% 40% 54% 33% 59% Erythro- blasts (%) blasts 33 37 69 35 20 15 % Blasts 1% 7% 20% 48% 27% 17% 49% 43% 44% 49% 45% 35% 15% 27% 11% 24% 13% Sidero- blasts (%) blasts (%) 31% 22% 21% 40% 33% 20% 36% 37% 12% 15% 69% 28% 47% 12% 24% 15% 10% 59 78 68 74 70 72 Blasts Timepoint 1: Diagnosis Timepoint 1: Age at diagnosis Age 8% 7% 9% 12% 15% 17% 22% 25% 25% 34% 44% 50% 50% 74% 61% 77% 85% RS (%) F F M M M M Sex Diagnosis de novo AML de novo t-MN sAML sAML de novo AML de novo de novo AML de novo de novo AML de novo de novo AML de novo MDS-EB2 MDS-EB2 de novo AML de novo t-MN sAML t-MN t-MN MDS-EB2 MDS-EB2 UPN RS071 RS068 RS108 RS093 RS001 RS028 RS042 RS122 RS061 RS045 RS006 RS021 RS074 RS012 RS018 RS037 RS002 UPN RS001 RS005 RS006 RS008 RS011 RS019 Supplementary table 3: Treatment strategies and responses strategies Treatment Supplementary table 3: PR – CR – complete remission, NR – no response, RS – ringsideroblasts, UPN – unique patient number, Abbreviations: 7 upon cytogenetic evaluation. based on persistentNR* no response of monosomy presence nd – not determined R – relapse, partial remission, RS-AML patients included for RNA-seq analysis Supplementary table 4:
86 Ringsideroblasts in acute myeloid leukemia
Supplementary table 5: Upregulated genes within GO:0030219 megakaryocyte differentiation Input ID Description Biological Process (GO) GO:1901835 positive regulation of deadenylation-independent decapping of nuclear-transcribed mRNA;GO:1901834 regulation of ZFP36 ZFP36 ring finger protein deadenylation-independent decapping of nuclear-transcribed mRNA;GO:0060213 positive regulation of nuclear-transcribed mRNA poly(A) tail shortening GO:0060265 positive regulation of respiratory burst involved in inflammatory response;GO:0060266 negative regulation of respiratory RPS19 ribosomal protein S19 burst involved in inflammatory response;GO:0060264 regulation of respiratory burst involved in inflammatory response ZFP36 ring finger protein GO:0045657 positive regulation of monocyte differentiation;GO:0060710 chorio-allantoic fusion;GO:0097403 cellular response to ZFP36L1 like 1 raffinose PCBP2 poly(rC) binding protein 2 GO:0075522 IRES-dependent viral translational initiation;GO:0019081 viral translation;GO:0039694 viral RNA genome replication glycoprotein Ib platelet GP1BA GO:0007597 blood coagulation, intrinsic pathway;GO:0042730 fibrinolysis;GO:0045652 regulation of megakaryocyte differentiation subunit alpha ITGA2B integrin subunit alpha 2b GO:0045652 regulation of megakaryocyte differentiation;GO:0070527 platelet aggregation;GO:0002576 platelet degranulation zinc finger protein, FOG GO:0060377 negative regulation of mast cell differentiation;GO:0002295 T-helper cell lineage commitment;GO:0003195 tricuspid valve ZFPM1 family member 1 formation GO:0035854 eosinophil fate commitment;GO:0010725 regulation of primitive erythrocyte differentiation;GO:0030221 basophil GATA1 GATA binding protein 1 differentiation TAL bHLH transcription TAL1 factor 1, erythroid GO:0030221 basophil differentiation;GO:0060375 regulation of mast cell differentiation;GO:0060217 hemangioblast cell differentiation differentiation factor GO:0035854 eosinophil fate commitment;GO:1903589 positive regulation of blood vessel endothelial cell proliferation involved in GATA2 GATA binding protein 2 sprouting angiogenesis;GO:0010725 regulation of primitive erythrocyte differentiation transforming growth GO:0048298 positive regulation of isotype switching to IgA isotypes;GO:0090190 positive regulation of branching involved in ureteric TGFB1 factor beta 1 bud morphogenesis;GO:0048296 regulation of isotype switching to IgA isotypes histone cluster 1 H3 family GO:0038111 interleukin-7-mediated signaling pathway;GO:0006335 DNA replication-dependent nucleosome assembly;GO:0098761 HIST1H3I member i cellular response to interleukin-7 3 MYC proto-oncogene, GO:0090096 positive regulation of metanephric cap mesenchymal cell proliferation;GO:0090095 regulation of metanephric cap MYC bHLH transcription factor mesenchymal cell proliferation;GO:0090094 metanephric cap mesenchymal cell proliferation involved in metanephros development GO:0033089 positive regulation of T cell differentiation in thymus;GO:0045586 regulation of gamma-delta T cell EGR3 early growth response 3 differentiation;GO:0042492 gamma-delta T cell differentiation GO:1902036 regulation of hematopoietic stem cell differentiation;GO:0045652 regulation of megakaryocyte differentiation;GO:1901532 SETD1A SET domain containing 1A regulation of hematopoietic progenitor cell differentiation SOS Ras/Rac guanine GO:2000973 regulation of pro-B cell differentiation;GO:1904693 midbrain morphogenesis;GO:1905456 regulation of lymphoid SOS1 nucleotide exchange progenitor cell differentiation factor 1 runt related transcription GO:0043378 positive regulation of CD8-positive, alpha-beta T cell differentiation;GO:0043376 regulation of CD8-positive, alpha-beta T RUNX3 factor 3 cell differentiation;GO:0043371 negative regulation of CD4-positive, alpha-beta T cell differentiation macrophage migration GO:0061078 positive regulation of prostaglandin secretion involved in immune response;GO:0090238 positive regulation of arachidonic MIF inhibitory factor acid secretion;GO:0090323 prostaglandin secretion involved in immune response histone cluster 1 H3 family GO:0038111 interleukin-7-mediated signaling pathway;GO:0006335 DNA replication-dependent nucleosome assembly;GO:0098761 HIST1H3A member a cellular response to interleukin-7 GO:0002326 B cell lineage commitment;GO:0033152 immunoglobulin V(D)J recombination;GO:1902036 regulation of hematopoietic TCF3 transcription factor 3 stem cell differentiation GO:0003257 positive regulation of transcription from RNA polymerase II promoter involved in myocardial precursor cell SRF serum response factor differentiation;GO:0046016 positive regulation of transcription by glucose;GO:0045059 positive thymic T cell selection Jun proto-oncogene, GO:0045657 positive regulation of monocyte differentiation;GO:0043922 negative regulation by host of viral transcription;GO:0043923 JUN AP-1 transcription factor positive regulation by host of viral transcription subunit GO:1902164 positive regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 ZNF385A zinc finger protein 385A class mediator;GO:0006977 DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest;GO:0072431 signal transduction involved in mitotic G1 DNA damage checkpoint histone cluster 1 H3 family GO:0038111 interleukin-7-mediated signaling pathway;GO:0006335 DNA replication-dependent nucleosome assembly;GO:0098761 HIST1H3C member c cellular response to interleukin-7 GO:1902187 negative regulation of viral release from host cell;GO:0006977 DNA damage response, signal transduction by p53 class PML promyelocytic leukemia mediator resulting in cell cycle arrest;GO:0050713 negative regulation of interleukin-1 beta secretion ASH2 like, histone lysine GO:0045652 regulation of megakaryocyte differentiation;GO:1904837 beta-catenin-TCF complex assembly;GO:0051568 histone H3-K4 ASH2L methyltransferase complex methylation subunit GO:0021702 cerebellar Purkinje cell differentiation;GO:0043973 histone H3-K4 acetylation;GO:0021694 cerebellar Purkinje cell layer LDB1 LIM domain binding 1 formation GO:0003270 Notch signaling pathway involved in regulation of secondary heart field cardioblast proliferation;GO:0072144 glomerular NOTCH1 notch 1 mesangial cell development;GO:0003252 negative regulation of cell proliferation involved in heart valve morphogenesis histone cluster 1 H4 family GO:0045653 negative regulation of megakaryocyte differentiation;GO:0034080 CENP-A containing nucleosome assembly;GO:0006335 HIST1H4K member k DNA replication-dependent nucleosome assembly histone cluster 1 H4 family GO:0045653 negative regulation of megakaryocyte differentiation;GO:0034080 CENP-A containing nucleosome assembly;GO:0006335 HIST1H4J member j DNA replication-dependent nucleosome assembly histone cluster 1 H4 family GO:0045653 negative regulation of megakaryocyte differentiation;GO:0034080 CENP-A containing nucleosome assembly;GO:0006335 HIST1H4L member l DNA replication-dependent nucleosome assembly histone cluster 1 H4 family GO:0045653 negative regulation of megakaryocyte differentiation;GO:0034080 CENP-A containing nucleosome assembly;GO:0006335 HIST1H4B member b DNA replication-dependent nucleosome assembly nuclear receptor GO:0042921 glucocorticoid receptor signaling pathway;GO:0031958 corticosteroid receptor signaling pathway;GO:0030520 intracellular NCOA6 coactivator 6 estrogen receptor signaling pathway GO:2000570 positive regulation of T-helper 2 cell activation;GO:2000569 regulation of T-helper 2 cell activation;GO:0035712 T-helper 2 PRKCQ protein kinase C theta cell activation GO:0060088 auditory receptor cell stereocilium organization;GO:0045541 negative regulation of cholesterol biosynthetic SOD1 superoxide dismutase 1 process;GO:0032287 peripheral nervous system myelin maintenance histone cluster 1 H3 family GO:0038111 interleukin-7-mediated signaling pathway;GO:0006335 DNA replication-dependent nucleosome assembly;GO:0098761 HIST1H3H member h cellular response to interleukin-7 protein arginine GO:0051572 negative regulation of histone H3-K4 methylation;GO:0034970 histone H3-R2 methylation;GO:0043985 histone H4-R3 PRMT6 methyltransferase 6 methylation GO:0060213 positive regulation of nuclear-transcribed mRNA poly(A) tail shortening;GO:0060211 regulation of nuclear-transcribed trinucleotide repeat TNRC6C mRNA poly(A) tail shortening;GO:1900153 positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent containing 6C decay WAS protein family GO:0051497 negative regulation of stress fiber assembly;GO:0038096 Fc-gamma receptor signaling pathway involved in WASF2 member 2 phagocytosis;GO:0032232 negative regulation of actin filament bundle assembly scribbled planar cell GO:0039502 suppression by virus of host type I interferon-mediated signaling pathway;GO:0039503 suppression by virus of host innate SCRIB polarity protein immune response;GO:0039563 suppression by virus of host STAT1 activity ABL proto-oncogene 1, GO:0002333 transitional one stage B cell differentiation;GO:0051281 positive regulation of release of sequestered calcium ion into ABL1 non-receptor tyrosine cytosol;GO:1905555 positive regulation blood vessel branching kinase GO:1901030 positive regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway;GO:1901028 ATP synthase inhibitory ATPIF1 regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway;GO:1904925 positive regulation of factor subunit 1 autophagy of mitochondrion in response to mitochondrial depolarization
87 Chapter 3
GO:0035407 histone H3-T11 phosphorylation;GO:0007257 activation of JUN kinase activity;GO:0030889 negative regulation of B cell PKN1 protein kinase N1 proliferation BCR, RhoGEF and GTPase GO:0043314 negative regulation of neutrophil degranulation;GO:1902564 negative regulation of neutrophil activation;GO:0043313 BCR activating protein regulation of neutrophil degranulation mediator complex GO:0060750 epithelial cell proliferation involved in mammary gland duct elongation;GO:0070562 regulation of vitamin D receptor MED1 subunit 1 signaling pathway;GO:2000347 positive regulation of hepatocyte proliferation histone cluster 1 H4 family GO:0045653 negative regulation of megakaryocyte differentiation;GO:0034080 CENP-A containing nucleosome assembly;GO:0006335 HIST1H4C member c DNA replication-dependent nucleosome assembly megakaryocyte and C6orf25 platelet inhibitory GO:0035855 megakaryocyte development;GO:0030220 platelet formation;GO:0036344 platelet morphogenesis receptor G6b GO:0035378 carbon dioxide transmembrane transport;GO:0060586 multicellular organismal iron ion homeostasis;GO:0015670 carbon RHAG Rh associated glycoprotein dioxide transport ubiquitin associated and GO:0050860 negative regulation of T cell receptor signaling pathway;GO:0050858 negative regulation of antigen receptor-mediated UBASH3A SH3 domain containing A signaling pathway;GO:0050856 regulation of T cell receptor signaling pathway MPL proto-oncogene, MPL GO:1905221 positive regulation of platelet formation;GO:1905219 regulation of platelet formation;GO:1990959 eosinophil homeostasis thrombopoietin receptor GO:0038111 interleukin-7-mediated signaling pathway;GO:0002360 T cell lineage commitment;GO:2001240 negative regulation of IL7 interleukin 7 extrinsic apoptotic signaling pathway in absence of ligand GO:1904141 positive regulation of microglial cell migration;GO:1902228 positive regulation of macrophage colony-stimulating factor CSF1 colony stimulating factor 1 signaling pathway;GO:1904139 regulation of microglial cell migration GO:0060135 maternal process involved in female pregnancy;GO:0030218 erythrocyte differentiation;GO:0034101 erythrocyte KLF1 Kruppel like factor 1 homeostasis GO:0001869 negative regulation of complement activation, lectin pathway;GO:0001868 regulation of complement activation, lectin SERPING1 serpin family G member 1 pathway;GO:0045916 negative regulation of complement activation CBFA2/RUNX1 GO:0045820 negative regulation of glycolytic process;GO:2001170 negative regulation of ATP biosynthetic process;GO:0006110 CBFA2T3 translocation partner 3 regulation of glycolytic process 3 GO:0072016 glomerular parietal epithelial cell development;GO:1901248 positive regulation of lung ciliated cell FOXJ1 forkhead box J1 differentiation;GO:0002897 positive regulation of central B cell tolerance induction alpha hemoglobin AHSP GO:0020027 hemoglobin metabolic process;GO:0030218 erythrocyte differentiation;GO:0034101 erythrocyte homeostasis stabilizing protein pro-apoptotic WT1 GO:1901300 positive regulation of hydrogen peroxide-mediated programmed cell death;GO:0050860 negative regulation of T cell PAWR regulator receptor signaling pathway;GO:0042986 positive regulation of amyloid precursor protein biosynthetic process GO:2001205 negative regulation of osteoclast development;GO:0035583 sequestering of TGFbeta in extracellular matrix;GO:0035582 FBN1 fibrillin 1 sequestering of BMP in extracellular matrix GO:0070963 positive regulation of neutrophil mediated killing of gram-negative bacterium;GO:1900135 positive regulation of renin F2RL1 F2R like trypsin receptor 1 secretion into blood stream;GO:0070962 positive regulation of neutrophil mediated killing of bacterium NOTCH regulated ankyrin GO:1902367 negative regulation of Notch signaling pathway involved in somitogenesis;GO:1902366 regulation of Notch signaling NRARP repeat protein pathway involved in somitogenesis;GO:1902359 Notch signaling pathway involved in somitogenesis nuclear receptor subfamily GO:0034144 negative regulation of toll-like receptor 4 signaling pathway;GO:0070859 positive regulation of bile acid biosynthetic NR1D1 1 group D member 1 process;GO:0034143 regulation of toll-like receptor 4 signaling pathway GO:1905719 protein localization to perinuclear region of cytoplasm;GO:0050687 negative regulation of defense response to RNF26 ring finger protein 26 virus;GO:0070979 protein K11-linked ubiquitination ZFP36 ring finger protein GO:1900153 positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay;GO:0061158 3’-UTR- ZFP36L2 like 2 mediated mRNA destabilization;GO:1900151 regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay GO:1905403 negative regulation of activated CD8-positive, alpha-beta T cell apoptotic process;GO:2000562 negative regulation of CD4- ARG2 arginase 2 positive, alpha-beta T cell proliferation;GO:2000666 negative regulation of interleukin-13 secretion GO:0060509 type I pneumocyte differentiation;GO:0071409 cellular response to cycloheximide;GO:0097533 cellular stress response to KLF2 Kruppel like factor 2 acid chemical protein tyrosine GO:0034164 negative regulation of toll-like receptor 9 signaling pathway;GO:0034163 regulation of toll-like receptor 9 signaling PTPRS phosphatase, receptor pathway;GO:0048671 negative regulation of collateral sprouting type S tripartite motif containing GO:0061931 positive regulation of erythrocyte enucleation;GO:0061930 regulation of erythrocyte enucleation;GO:0043131 erythrocyte TRIM58 58 enucleation TNF receptor superfamily GO:2000663 negative regulation of interleukin-5 secretion;GO:2000666 negative regulation of interleukin-13 secretion;GO:2001180 TNFRSF21 member 21 negative regulation of interleukin-10 secretion zinc finger MIZ-type GO:0007296 vitellogenesis;GO:0045582 positive regulation of T cell differentiation;GO:0045621 positive regulation of lymphocyte ZMIZ1 containing 1 differentiation toll like receptor adaptor GO:0034128 negative regulation of MyD88-independent toll-like receptor signaling pathway;GO:0034127 regulation of MyD88- TICAM1 molecule 1 independent toll-like receptor signaling pathway;GO:0035666 TRIF-dependent toll-like receptor signaling pathway CD59 molecule (CD59 GO:0001971 negative regulation of activation of membrane attack complex;GO:0001969 regulation of activation of membrane attack CD59 blood group) complex;GO:0045916 negative regulation of complement activation dual specificity tyrosine GO:0035617 stress granule disassembly;GO:0043518 negative regulation of DNA damage response, signal transduction by p53 class DYRK3 phosphorylation regulated mediator;GO:0043516 regulation of DNA damage response, signal transduction by p53 class mediator kinase 3 GO:1904283 negative regulation of antigen processing and presentation of endogenous peptide antigen via MHC class I;GO:0002626 HLA-H homeostatic iron regulator negative regulation of T cell antigen processing and presentation;GO:1904282 regulation of antigen processing and presentation of endogenous peptide antigen via MHC class I JunB proto-oncogene, JUNB AP-1 transcription factor GO:0001829 trophectodermal cell differentiation;GO:0060716 labyrinthine layer blood vessel development;GO:0046697 decidualization subunit GO:0045671 negative regulation of osteoclast differentiation;GO:0002762 negative regulation of myeloid leukocyte TOB2 transducer of ERBB2, 2 differentiation;GO:0045670 regulation of osteoclast differentiation CLPTM1, transmembrane GO:0033081 regulation of T cell differentiation in thymus;GO:0033077 T cell differentiation in thymus;GO:0045580 regulation of T cell CLPTM1 protein differentiation transforming growth GO:0042704 uterine wall breakdown;GO:0060364 frontal suture morphogenesis;GO:0010936 negative regulation of macrophage TGFB3 factor beta 3 cytokine production eukaryotic translation GO:0045993 negative regulation of translational initiation by iron;GO:0010999 regulation of eIF2 alpha phosphorylation by EIF2AK1 initiation factor 2 alpha heme;GO:0046986 negative regulation of hemoglobin biosynthetic process kinase 1 GO:0090267 positive regulation of mitotic cell cycle spindle assembly checkpoint;GO:0090266 regulation of mitotic cell cycle spindle PCID2 PCI domain containing 2 assembly checkpoint;GO:1903504 regulation of mitotic spindle checkpoint GO:0002296 T-helper 1 cell lineage commitment;GO:0002295 T-helper cell lineage commitment;GO:0045063 T-helper 1 cell SPN sialophorin differentiation phosphatidylinositol-5- GO:2000786 positive regulation of autophagosome assembly;GO:0035855 megakaryocyte development;GO:2000785 regulation of PIP4K2A phosphate 4-kinase type autophagosome assembly 2 alpha GO:0045736 negative regulation of cyclin-dependent protein serine/threonine kinase activity;GO:1903204 negative regulation of FBXO7 F-box protein 7 oxidative stress-induced neuron death;GO:1903599 positive regulation of autophagy of mitochondrion family with sequence GO:1905035 negative regulation of antifungal innate immune response;GO:1905034 regulation of antifungal innate immune FAM3A similarity 3 member A response;GO:0061760 antifungal innate immune response poly(A) binding protein PABPC4 GO:0043488 regulation of mRNA stability;GO:0061515 myeloid cell development;GO:0061013 regulation of mRNA catabolic process cytoplasmic 4
88 Ringsideroblasts in acute myeloid leukemia
Supplementary table 6: Common upregulated genes Input ID Description Biological Process (GO) PRKCQ protein kinase C theta GO:2000570 positive regulation of T-helper 2 cell activation;GO:2000569 regulation of T-helper 2 cell activation;GO:0035712 T-helper 2 cell activation PARP9 poly(ADP-ribose) polymerase GO:0034356 NAD biosynthesis via nicotinamide riboside salvage pathway;GO:0060335 positive regulation of interferon-gamma- family member 9 mediated signaling pathway;GO:2001034 positive regulation of double-strand break repair via nonhomologous end joining ALAS2 5’-aminolevulinate synthase 2 GO:0006782 protoporphyrinogen IX biosynthetic process;GO:0032364 oxygen homeostasis;GO:0033483 gas homeostasis NFE2 nuclear factor, erythroid 2 GO:0045652 regulation of megakaryocyte differentiation;GO:0006337 nucleosome disassembly;GO:0031498 chromatin disassembly NCF4 neutrophil cytosolic factor 4 GO:0048010 vascular endothelial growth factor receptor signaling pathway;GO:0045730 respiratory burst;GO:0006801 superoxide metabolic process ADCY7 adenylate cyclase 7 GO:0006171 cAMP biosynthetic process;GO:0071377 cellular response to glucagon stimulus;GO:0003091 renal water homeostasis HBG2 hemoglobin subunit gamma 2 GO:0015671 oxygen transport;GO:0042744 hydrogen peroxide catabolic process;GO:0015669 gas transport SLC4A1 solute carrier family 4 GO:0051453 regulation of intracellular pH;GO:0030641 regulation of cellular pH;GO:0015701 bicarbonate transport member 1 HBA2 hemoglobin subunit alpha 2 GO:0015671 oxygen transport;GO:0015701 bicarbonate transport;GO:0042744 hydrogen peroxide catabolic process HBB hemoglobin subunit beta GO:0030185 nitric oxide transport;GO:0045429 positive regulation of nitric oxide biosynthetic process;GO:0070527 platelet aggregation SLC29A3 solute carrier family 29 GO:1901642 nucleoside transmembrane transport;GO:0015858 nucleoside transport;GO:1901264 carbohydrate derivative member 3 transport 3 PFKFB4 6-phosphofructo-2-kinase/ GO:0045821 positive regulation of glycolytic process;GO:2001171 positive regulation of ATP biosynthetic process;GO:0006110 fructose-2,6-biphosphatase 4 regulation of glycolytic process SLC43A1 solute carrier family 43 GO:1902475 L-alpha-amino acid transmembrane transport;GO:0015804 neutral amino acid transport;GO:0015807 L-amino acid member 1 transport B ATF basic leucine zipper ATF-like GO:0072540 T-helper 17 cell lineage commitment;GO:0002295 T-helper cell lineage commitment;GO:0045064 T-helper 2 cell transcription factor differentiation PML promyelocytic leukemia GO:1902187 negative regulation of viral release from host cell;GO:0006977 DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest;GO:0050713 negative regulation of interleukin-1 beta secretion IFITM1 interferon induced GO:0046597 negative regulation of viral entry into host cell;GO:0046596 regulation of viral entry into host cell;GO:0045071 transmembrane protein 1 negative regulation of viral genome replication DTX3L deltex E3 ubiquitin ligase 3L GO:1902966 positive regulation of protein localization to early endosome;GO:2001034 positive regulation of double-strand break repair via nonhomologous end joining;GO:1902965 regulation of protein localization to early endosome IFIT3 interferon induced protein with GO:0035457 cellular response to interferon-alpha;GO:0060337 type I interferon signaling pathway;GO:0035455 response to tetratricopeptide repeats 3 interferon-alpha APOBEC3D apolipoprotein B mRNA editing GO:0045869 negative regulation of single stranded viral RNA replication via double stranded DNA intermediate;GO:0045091 enzyme catalytic subunit 3D regulation of single stranded viral RNA replication via double stranded DNA intermediate;GO:0039692 single stranded viral RNA replication via double stranded DNA intermediate SLAMF6 SLAM family member 6 GO:0072540 T-helper 17 cell lineage commitment;GO:0002295 T-helper cell lineage commitment;GO:0072539 T-helper 17 cell differentiation TESC tescalcin GO:0032417 positive regulation of sodium:proton antiporter activity;GO:0032415 regulation of sodium:proton antiporter activity;GO:0030854 positive regulation of granulocyte differentiation KBTBD7 kelch repeat and BTB domain GO:0043687 post-translational protein modification;GO:0000165 MAPK cascade;GO:0016567 protein ubiquitination containing 7 HECTD3 HECT domain E3 ubiquitin GO:0043161 proteasome-mediated ubiquitin-dependent protein catabolic process;GO:0006511 ubiquitin-dependent protein protein ligase 3 catabolic process;GO:0010498 proteasomal protein catabolic process DHRS9 dehydrogenase/reductase 9 GO:0042904 9-cis-retinoic acid biosynthetic process;GO:0042905 9-cis-retinoic acid metabolic process;GO:0002138 retinoic acid biosynthetic process PDE6G phosphodiesterase 6G GO:0022400 regulation of rhodopsin mediated signaling pathway;GO:0016056 rhodopsin mediated signaling pathway;GO:0045742 positive regulation of epidermal growth factor receptor signaling pathway DFFB DNA fragmentation factor GO:0030263 apoptotic chromosome condensation;GO:0006309 apoptotic DNA fragmentation;GO:0030262 apoptotic nuclear subunit beta changes OMA1 OMA1 zinc metallopeptidase GO:0010637 negative regulation of mitochondrial fusion;GO:0002024 diet induced thermogenesis;GO:0010635 regulation of mitochondrial fusion NETO2 neuropilin and tolloid like 2 GO:2000312 regulation of kainate selective glutamate receptor activity;GO:1901016 regulation of potassium ion transmembrane transporter activity;GO:2000649 regulation of sodium ion transmembrane transporter activity KCNE3 potassium voltage-gated GO:1905025 negative regulation of membrane repolarization during ventricular cardiac muscle cell action potential;GO:1903765 channel subfamily E regulatory negative regulation of potassium ion export across plasma membrane;GO:1903946 negative regulation of ventricular cardiac subunit 3 muscle cell action potential PTH2R parathyroid hormone 2 GO:0007186 G-protein coupled receptor signaling pathway;GO:0007166 cell surface receptor signaling pathway;GO:0007165 receptor signal transduction GPA33 glycoprotein A33 GO:0007165 signal transduction;GO:0023052 signaling;GO:0007154 cell communication MAL mal, T cell differentiation GO:0001766 membrane raft polarization;GO:0031580 membrane raft distribution;GO:1902043 positive regulation of extrinsic protein apoptotic signaling pathway via death domain receptors FRRS1 ferric chelate reductase 1 GO:0055114 oxidation-reduction process;GO:0008152 metabolic process;GO:0008150 biological_process AKR1E2 aldo-keto reductase family 1 GO:0055114 oxidation-reduction process;GO:0008152 metabolic process;GO:0008150 biological_process member E2 ZMAT3 zinc finger matrin-type 3 GO:0043065 positive regulation of apoptotic process;GO:0043068 positive regulation of programmed cell death;GO:0010942 positive regulation of cell death PARS2 prolyl-tRNA synthetase 2, GO:0006433 prolyl-tRNA aminoacylation;GO:0006418 tRNA aminoacylation for protein translation;GO:0043039 tRNA mitochondrial aminoacylation TTLL11 tubulin tyrosine ligase like 11 GO:0018095 protein polyglutamylation;GO:0051013 microtubule severing;GO:0018200 peptidyl-glutamic acid modification
89 Chapter 3
SLA Src like adaptor GO:0038083 peptidyl-tyrosine autophosphorylation;GO:0018108 peptidyl-tyrosine phosphorylation;GO:0046777 protein autophosphorylation TNFAIP8L2 TNF alpha induced protein GO:0050868 negative regulation of T cell activation;GO:1903038 negative regulation of leukocyte cell-cell adhesion;GO:0051250 8 like 2 negative regulation of lymphocyte activation FRAT2 FRAT2, WNT signaling pathway GO:1904886 beta-catenin destruction complex disassembly;GO:0060070 canonical Wnt signaling pathway;GO:0032984 protein- regulator containing complex disassembly CD82 CD82 molecule GO:0007166 cell surface receptor signaling pathway;GO:0007165 signal transduction;GO:0023052 signaling LY6E lymphocyte antigen 6 family GO:0007166 cell surface receptor signaling pathway;GO:0007165 signal transduction;GO:0023052 signaling member E SEMA4B semaphorin 4B GO:0048843 negative regulation of axon extension involved in axon guidance;GO:0048841 regulation of axon extension involved in axon guidance;GO:1902668 negative regulation of axon guidance CPEB4 cytoplasmic polyadenylation GO:2000766 negative regulation of cytoplasmic translation;GO:0042149 cellular response to glucose starvation;GO:0035235 element binding protein 4 ionotropic glutamate receptor signaling pathway ZFP64 ZFP64 zinc finger protein GO:0031060 regulation of histone methylation;GO:0031056 regulation of histone modification;GO:0016571 histone methylation FANCF FA complementation group F GO:0036297 interstrand cross-link repair;GO:0006281 DNA repair;GO:0006974 cellular response to DNA damage stimulus DCP1B decapping mRNA 1B GO:0031087 deadenylation-independent decapping of nuclear-transcribed mRNA;GO:0000290 deadenylation-dependent decapping of nuclear-transcribed mRNA;GO:0031086 nuclear-transcribed mRNA catabolic process, deadenylation-independent decay 3
90 Ringsideroblasts in acute myeloid leukemia
Supplementary figures
r enes ons s mR R R R R R R R R R R R 3 R R R R R Supplementary Figure 1. SNP array results overview all individual patients Image represents screenshot taken following analysis using Nexus software
R s deacetylate istones rotein ubi uitination R ubi uitin ligases ubi uitinate target roteins interleukin mediated signaling at ay myeloid cell omeostasis R etallo rotease s negati e regulation o rotein modi ication rocess istone modi ication R ryt rocytes take u o ygen and release carbon dio ide double strand break re air demet ylation regulation o cellular catabolic rocess myeloid cell de elo ment regulation o rotein com le disassembly regulation o small ase mediated signal transduction R t com le regulation o ase acti ity leukocyte di erentiation de os orylation canonical nt signaling at ay log R ell ycle R etabolism o R re lication R ell ycle eck oints R ranslation c romosome segregation R ase sa rotein e ort negati e regulation o cell cycle R ignaling by nterleukins in lammatory res onse R e citric acid cycle and res iratory electron trans ort re air symbiont rocess con ormation c ange cytokine roduction R strand elongation mitoc ondrion organi ation R R ositi e regulation o cell cycle log Supplementary figure 2 Functional annotation of upregulated genes (n=1196 top panel) and downregulated genes (n=1309 bottom panel) in RS- AML CD34+ vs NBM CD34+
91 Chapter 3
luster n R ell ycle R etabolism o R c romosome segregation R ranslation R Re lication R e citric acid cycle and res iratory electron trans ort re lication mitotic cell cycle ase transition R R de endent cotranslational rotein targeting to membrane R rocessing o a ed ntron ontaining re mR R n ectious disease re air ositi e regulation o metabolic rocess R locali ation R strand elongation con ormation c ange regulation o c romosome segregation biosynt etic rocess sa ell cycle ositi e regulation o cell cycle log luster n 3 ossi ication negati e regulation o cell roli eration sa signaling at ay ositi e regulation o a o totic rocess skeletal system de elo ment cardio ascular system de elo ment cellular res onse to li id res onse to li o olysacc aride regulation o rotein kinase acti ity at cell di erentiation negati e regulation o rotein modi ication rocess sa ranscri tional misregulation in cancer r yt mic rocess R regulation o neurogenesis leukocyte di erentiation re roducti e structure de elo ment res onse to gro t actor R al a ka a signaling com le R R R R log luster n cell acti ation cytokine mediated signaling at ay leukocyte acti ation in ol ed in immune res onse regulation o innate immune res onse cytokine roduction res onse to inter eron gamma regulation o small ase mediated signal transduction R ositi e regulation o ydrolase acti ity actin cytoskeleton organi ation cell selection R mmunoregulatory interactions bet een a ym oid and a non ym oid cell endocytosis R eneration o second messenger molecules regulation o leukocyte mediated immunity transmembrane rece tor rotein tyrosine kinase signaling at ay iral rocess c emota is sa atural killer cell mediated cytoto icity log luster n asculature de elo ment R nterleukin signaling cytokine roduction e tracellular matri organi ation leukocyte migration regulation o cascade regulation o cell ad esion sa ytokine cytokine rece tor interaction in lammatory res onse regulated e ocytosis sa luid s ear stress and at erosclerosis res onse to ounding sa emato oietic cell lineage R R ignaling by Rece tor yrosine inases R R ignaling by endot elial cell de elo ment regulation o ion trans ort kidney de elo ment log
92 Ringsideroblasts in acute myeloid leukemia
luster n R cti ated stimulates transcri tion o R androgen rece tor regulated genes and R eubi uitination rotein ubi uitination R ubi uitin ligases ubi uitinate target roteins interleukin mediated signaling at ay istone modi ication double strand break re air regulation o cellular catabolic rocess negati e regulation o rotein modi ication rocess ositi e regulation o ase acti ity de os orylation establis ment o cell olarity R cti ation o gene e ression by R R regulation o small ase mediated signal transduction R t com le R R acti ates gene e ression R etabolism o li ids rotein locali ation to cytoskeleton c ordate embryonic de elo ment log
Supplementary figure 3 3 Functional annotation of different clusters indicated in figure 6A.
SF3B1 PRPF8 no a e− no a
ns ns ns ns ns ns r r
R eneral R eneral mut mut mut mut Supplementary figure 4 A) Expression of SF3B1 in indicated groups, B) expression of PRPF8 in indicated groups. Abbreviations: NBM: normal bone marrow CD34+ cells, RS-AML: AML with ringsideroblast phenotype. One-way anova followed by pairwise t-test was used to determine statistical significance, * p = < 0.05; ** p = < 0.01.
93