Supplementary Figure 1

Transcription

1 Supplementary Figure 1 Platelets from affected patients are comparable to controls on electron micrograph. Thin-section transmission electron micrographs of representative platelets from a normal control (a) and an affected patient (b). The ultrastructure of patient-derived platelets is comparable to that of control cells, with occasional elongated -granules seen in the former. Scale bars, 500 nm.

2 Supplementary Figure 2 The c.1252a>g change is a splice-site mutation. RT-PCR of the transcript encoded by exons 6 8 of ETV6 for individuals I-2 and II-3 of family 3 with the c.1252a>g mutation. (a) Two transcripts are visible in the gel when this region is amplified: the expected 386- bp transcript as seen in the unaffected control (C+) and a smaller, 285-bp transcript, which by reverse sequencing, demonstrate skipping of exon 7 and a peak for a G substitution, indicating leakage of the missense mutation (b,c). (d) The sequence of the cloned product confirms the presence of the G nucleotide in

3 exon 7.

4 Supplementary Figure 3 Immunoblots of ETV6 protein expression in transfected HEK293T cells. Plasmids containing DDK-tagged cdna encoding either WT, p.p214l, p.r418g (missense mutation acquired by c.1252a>g) or p.385_418del (deletion mutation acquired by c.1252a>g) ETV6 were transiently transfected into HEK293T cells. Whole-cell lysates were separated by 7.5% SDS-PAGE and probed with an anti-ddk antibody. Blots show equal expression of WT (lane 1), p.p214l (lane 2), p.r418g (lane 3) and p.385_418del (lane 4). A smaller protein is seen when p.385_418del is expressed.

5 Supplementary Figure 4 Immunoblot analysis of ETV6 protein content in patient-derived and control platelets. Reduced whole-cell lysates from the equivalent of 10 7 platelets were loaded in each lane, with equal loading confirmed by probing for GAPDH. Subjects were as described in the Figure 1 pedigrees with the addition of two unrelated normal controls. ETV6 protein size and platelet levels were similar for affected individuals (family 1, II-1 and III-1; family 3, I-2 and II-3), an unaffected relative (family 1, III-2) and controls.

6 Supplementary Figure 5 Dimerization of mutants with WT ETV6. Cell lysates of HEK293T cells transfected with WT ETV6-Myc/DDK or WT ETV6-His alone or cotransfected with WT ETV6-His in addition to Myc/DDK-tagged WT ETV6, c.641c>t (p.p214l) ETV6, c.1252a>g (p.r418g) ETV6 or c.1153_1253del (p.385_418del) ETV6 were incubated with anti-myc antibody conjugated beads, and protein complexes were isolated. Eluate was probed for DDK (top) and His (bottom). Lanes 1 and 2 show that WT ETV6-Myc/DDK was pulled down by the beads, but WT ETV6-His was not. Cotransfection experiments (lanes 3 6) show that both the indicated Myc/DDK-tagged protein and the His-tagged WT ETV6 protein were pulled down, indicating that the WT ETV6-His protein was complexed with the corresponding Myc/DDK-tagged protein and that dimerization occurred.

7 Supplementary Figure 6 Subcellular distribution of protein produced by transduced wild-type and mutant ETV6 alleles in maturing (>15- m) day 12 cultured megakaryocytes. Protein produced by transduced alleles was detected via staining for Myc tag (green), while size and stage were determined from nuclear morphology (DNA, blue) and staining for megakaryocyte-specific Von Willebrand factor (VWF, red) and CD61 (magenta). Transduced cells showed differing subcellular distribution patterns of Myc-tagged ETV6, with ETV6 P214L and ETV6 R418G showing a largely cytoplasmic distribution, in contrast to the nuclear localization observed for ETV6 WT. Confocal z sections; scale bars, 5 m.

8 Supplementary Figure 7 Transcriptional changes induced by mutant ETV6. (a) Relationship of RNA-seq transcript profiles in platelets between two affected individuals (P214L mutation), two unaffected relatives and three unrelated controls. Shown is the sample-to-sample distance matrix, with hierarchical clustering using rlog-transformed read counts, of 15,865 detected transcripts. (b) Hierarchical clustering and heat map analysis of the relative expression of 351 transcripts involved in platelet biogenesis or function. Expression values are DESeq2 normalized and rlog transformed.

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10 Supplementary Figure 8 Heat map analysis of the relative expression of 351 transcripts involved in platelet biogenesis or function. Heat map showing higher-resolution hierarchical clustering of all transcripts associated with platelet function or platelet biogenesis. Expression values are DESeq2 normalized and rlog transformed. This list of transcripts was curated from the Reactome, and Gene Ontology databases and from transcripts enriched in platelets compared to all other tissues in Illumina s Human Body Map 2.0.

11 Supplementary Note Phenotypic Characterization of the Families Several individuals from the three families presented here exhibit the same blood phenotype consisting of mild to moderate thrombocytopenia (44, ,000/ µl) and elevated red cell mean corpuscular volume (MCV) ( fl). The transmission appears to be autosomal dominant. All other parameters of the complete blood counts are within normal range. Please see Supplemental Table 1 for detailed list of blood counts in the three families. The mean platelet volume is normal in all individuals with thrombocytopenia, and ultrastructure of platelets by Electron Microscopy shows normal distribution and size of organelles (Supplemental Fig 1). Platelet aggregation studies in the two affected members of Family 3 (I-2 and II-3) showed normal aggregation to all agonists. Platelet aggregation studies in two affected members of Family 1 (III-1 and III-10) showed decreased aggregation with ADP (15% and 2% vs. 80%) and arachidonic acid (4% and 1% vs. 94%) when compared to a family control. In these patients platelet receptor density for the common glycoproteins GPIb, GPIIb, GPIIIa and GPIX was assessed by flow cytometry and observed to be normal in both individuals when compared to their family control (data not shown). Bone marrow aspirates from 3 individuals while not affected by leukemia (III-1, III-9 and III-10) showed very similar morphological abnormalities, including mild megakaryocytic hyperplasia with hyperchromatic forms as well as hypolobated megakaryocytes. They also showed signs of mild dyserythropoiesis (abnormal red cell development) such as nuclear cytoplasmatic dys-synchrony (Fig 1). Bone marrow examination of 3 members of the Family 3 (I-2, II-3 and II-4) showed normal number of megakaryocytes but also dyserythropoiesis. Additionally, there were three cases of acute leukemia reported among a total of 10 individuals affected with the phenotype. Two individuals from family 1 developed acute lymphoblastic leukemia (ALL) classified as B precursor ALL by morphology and flow cytometry. No major cytogenetic abnormalities were detected by conventional karyotyping. Fluorescence in situ hybridization (FISH) for the common translocation ETV6-RUNX1 (also known as TEL-AML) was negative in both cases. Individual III-1 developed leukemia at 45 months of age and was successfully treated with conventional 1

12 chemotherapy. She achieved remission but had an isolated CNS relapse 1 year after completion of treatment and was treated again with chemotherapy. This girl has been free of leukemia for over 8 years. Individual II-7 also developed B precursor ALL with no cytogenetic abnormalities at 37 years of age. She was treated with conventional chemotherapy and has been free of disease for over 6 years. Individual I-2 from family 2 developed B-cell leukemia at age 14 and was treated with conventional chemotherapy. She has been free of disease for more than a decade. 2

13 Sequence alignment of ETV6 mutations p.p214 Human ETV6 I R R L S P A E R A Q Rhesus ETV6 I R R L S P A E R A Q Mouse Etv6 S R R L S P V E K A Q Dog ETV6 I R R L S P A E R A Q Elephant ETV6 I R R L S P A E R A Q Chicken ETV6 V R M L S P A D R V P X tropicalis Str.6936 L R R P S P A D R A P Zebrafish etv6 I Y H R P T S A E D P p.r418g Human ETV6 Q R L L F R F M K T P Rhesus ETV6 Q R L L F R F M K T P Mouse Etv6 Q R L L F R F M K T P Dog ETV6 Q R L L F R F M K T P Elephant ETV6 Q R L L F R F M K T P Chicken ETV6 Q R L L F R F M K T P X tropicalis Str.6936 Q R L L F Zebrafish etv6 Q R L L F R F L K T P Expression of ETV6 in platelets Western blot analysis of platelet lysates from two affected individuals with the p.p214l mutation (II-1 and III-1, Family 1) and the p.r418g mutation (I-2 and II-3, Family 3) showed the presence of ETV6 in all affected individuals (Supplemental Figure 3) Exome and RNA sequencing of the Leukemia Sample Genetic variants identified in the leukemia sample were filtered to include only those in exons and resulting in non-synonymous amino acid changes or deletion. The COSMIC database (accessed online November 25, 2014) was searched for previously identified variants at those loci. Three of these loci have been previously reported as mutated in 3

14 cancer cells. Three mutations have been found in KCNQ5, including an identical 451G>A mutation, that was confirmed to be somatic, as well as G>T and G>C changes. An identical 2161G>C mutation has been found in ANKRD12, but it was not confirmed to be somatic. The mutation previously found in PCNX was a 3731C>A change. Four of these genes are reported to be altered in leukemia. ATF7IP has been found as a fusion partner with PDGFRB and JAK2 in ALL, and to be recurrently deleted or mutated in ETV-RUNX1 + ALL 1-3. EPOR, which encodes the erythropoietin receptor, may cooperate with ETV6-RUNX1 during leukemogenesis 4-6, and is also recurrently translocated in ALL 3,7 ; however, no point mutations in this gene have been reported after sequencing over 340 cases of ALL 1,3,7,8. STAG2 has been found to be translocated, deleted and mutated in samples of ALL 1,3,8. To our knowledge, SBF1 is not implicated in the development of precursor B cell ALL, but has been found in a translocation in T cell large granular lymphocytic leukemia 9. Sequencing of RNA from the leukemia revealed a novel fusion of PAX5 and SHB genes. PAX5 somatic mutations are found in approximately 30% of ALL cases, including those with ETV6-RUNX1 translocation. 10 PAX5 is occasionally involved in translocations with ETV6 11 and PAX5 germline mutations have recently been described in families with predisposition to leukemia. 12 The functional consequence of and the extent to which any of the mutations observed in this patient contributed to leukemogenesis remain to be determined. Of note, the P214L and R418G mutations (found in the germline in our families) have also been described in the COSMIC database as somatic mutations found in a case of gastric cancer and acute myeloblastic leukemia (AML) respectively. Platelet Transcriptome RNA seq of the platelet transcriptome was performed in two affected members from family 1 (II-1 and II-7) and compared to two non-affected individuals from the same family (II-3 and II-5) and 3 unrelated individuals. Bioinformatics analyses of platelet RNAseq data showed that a large number of transcripts were differentially expressed in the affected individuals, demonstrating concordance of expression patterns, when 4

15 compared to controls (Supp Fig 7a). The RNA-seq data suggests that ETV6 directly affects a limited number of transcripts, which potentiate transcriptome-wide alterations in expression during megakaryocyte/platelet development. In particular, pronounced expression changes in transcripts involved in platelet production and function were observed in patients with the p.p214l mutation (Supp Fig 7b and Supp Fig 8). Transcript levels were both increased and decreased, suggesting that ETV6 may indirectly influence the expression of several genes, independently of a direct repressive effect. However, of the platelet specific transcripts that were significantly altered, the majority was decreased by the ETV6 mutation. Altered transcripts related to platelet biogenesis included transcripts such as Myosin,Light Chain 9 (MYL9), Aquaporin 10 (AQP10), Caspase 9 (CASP9), Filamin A (FLNA), Talin 1 (TLN1), Arachidonate 12-Lipoxygenase (ALOX12), Von Willebrand Factor (VWF), Glycoprotein 9 (GP9), as well as groups of genes with similar function, such as cytoskeletal genes (i.e. FLNA, MYL9 and DYNC1I1) The fold changes and significance values of all 351 platelet related transcripts can be found in the supplementary dataset. 5

16 References 1. Papaemmanuil, E. et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nat Genet 46, (2014). 2. Kobayashi, K. et al. ATF7IP as a novel PDGFRB fusion partner in acute lymphoblastic leukaemia in children. Br J Haematol 165, (2014). 3. Roberts, K.G. et al. Targetable kinase-activating lesions in Ph-like acute lymphoblastic leukemia. N Engl J Med 371, (2014). 4. Inthal, A. et al. Role of the erythropoietin receptor in ETV6/RUNX1-positive acute lymphoblastic leukemia. Clin Cancer Res 14, (2008). 5. Torrano, V., Procter, J., Cardus, P., Greaves, M. & Ford, A.M. ETV6-RUNX1 promotes survival of early B lineage progenitor cells via a dysregulated erythropoietin receptor. Blood 118, (2011). 6. van der Weyden, L. et al. Modeling the evolution of ETV6-RUNX1-induced B- cell precursor acute lymphoblastic leukemia in mice. Blood 118, (2011). 7. Roberts, K.G. et al. Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia. Cancer Cell 22, (2012). 8. Holmfeldt, L. et al. The genomic landscape of hypodiploid acute lymphoblastic leukemia. Nat Genet 45, (2013). 9. Izykowska, K. et al. Submicroscopic genomic rearrangements change gene expression in T-cell large granular lymphocyte leukemia. Eur J Haematol 93, (2014). 10. Mullighan, C.G. et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 446, (2007). 11. Strehl, S., Konig, M., Dworzak, M.N., Kalwak, K. & Haas, O.A. PAX5/ETV6 fusion defines cytogenetic entity dic(9;12)(p13;p13). Leukemia 17, (2003). 6

17 12. Shah, S. et al. A recurrent germline PAX5 mutation confers susceptibility to pre-b cell acute lymphoblastic leukemia. Nat Genet 45, (2013). 13. Fenrick, R. et al. TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1. Mol Cell Biol 20, (2000). 14. Jalagadugula, G. et al. Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency. Blood 116, (2010). 15. Patel, S.R. et al. Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes. Blood 106, (2005). Videos 1-4. Aberrant Localization of ETV6. All show 3D volume renders (maximum intensity) prepared from confocal laser fluorescence microscopy z-sections of representative Day 12 cultured megakaryocytes stained for ETV6 (green), DNA (blue) and tubulin (magenta) via Imaris 7.6. The nuclear concentration of ETV6 observed for endogenously-expressed protein (Video 1: control non-transduced megakaryocyte) is also seen in a cell transduced with wild-type ETV6 (Video 2), while cells expressing ETV6 P214L (Video 3) or ETV6 R418G (Video 4) show extensive cytoplasmic ETV6 staining. Supplementary Data Set. 351 Platelet Specific Transcripts. Significant values and Fold Changes of the 351 platelet specific targets described in Supplementary Figures 7 and 8. 7

18 Supplementary Table 1. Complete Blood Counts (CBC) for families 1, 2 and 3 Family/Individuals WBC HGB HCT MCV MCH MCHC RDW PLT MPV F1 II F1 II F1 II F1 II F1 II F1 II F1 III F1 III F1 III F1 III F1 III F1 III F1 III F1 III F1 III F2 I-1 N/A N/A N/A N/A N/A N/A N/A N/A N/A F2 I F2 II F2 II F3 I-1 N/A N/A N/A N/A N/A N/A N/A N/A N/A F3 I N/A F3 II N/A 8

19 Supplementary Table 2. Somatic variants identified by exome sequencing in diagnostic leukemia sample in individual III-1, Family 1. Gene Variant type Nucleotide change AA change Present in COSMIC Implicated in leukemogenesis VPS13D nonsyn SNV c.c10442a p.s3481x No No MAP4K3 nonsyn SNV c.g1547c p.r516t No No KCNQ5 nonsyn SNV c.g451a p.e151k Yes (3) No JAZF1 nonsyn SNV c.c12g p.i4m No No RARRES2 nonsyn SNV c.c232t p.r78w No No PRRC2B nonsyn SNV c.g6329a p.r2110q No No MKI67 nonsyn SNV c.g1747a p.e583k No No ZNF259 nonsyn SNV c.c71t p.p24l No No ATF7IP nonsyn SNV c.g604a p.g202s No Yes PCNX nonsyn SNV c.c3731t p.s1244f Yes (1) No MED24 nonsyn SNV c.g607a p.e203k No No DBF4B nonsyn SNV c.c1832g p.a611g No No ANKRD12 nonsyn SNV c.g2161c p.e721q Yes (1) No HMHA1 nonsyn SNV c.c1399t p.r467c No No EPOR nonsyn SNV c.c1223g p.s408c No Yes EPOR nonsyn SNV c.c1202g p.s401x No Yes SBF1 nonsyn SNV c.c1117t p.r373w No Yes STAG2 frameshift del c.838delg p.e280fs No Yes STAG2 nonsyn SNV c.g1924t p.e642x No Yes 9