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Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction.Significance:This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT.See related commentary by Licht, p. 1794.This article is highlighted in the In This Issue feature, p. 1775

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Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations, and molecular profiles. The contribution of the cell-of-origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte-precursor-like cells (OPC). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1+/SHH-dependent brainstem OPCs, while H3.3K27M gliomas frequently resemble dorsal PAX3+/BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, while other epigenetic changes are mainly contingent on the cell-of-origin chromatin state and cycling rate.

Deregulation of the epigenome underlies oncogenesis in numerous primary brain tumours in children and young adults. In this review, we describe how recurrent mutations in isocitrate dehydrogenases or histone 3 variants (oncohistones) in gliomas, expression of the oncohistone mimic enhancer of Zeste homologs inhibiting protein (EZHIP) in a subgroup of ependymoma, and epigenetic alterations in other embryonal tumours promote oncogenicity. We review the proposed mechanisms of cellular transformation, current tumorigenesis models and their link to development. We further stress the narrow developmental windows permissive to their oncogenic potential and how this may stem from converging effects deregulating polycomb repressive complex (PRC)2 function and targets. As altered chromatin states may be reversible, improved understanding of aberrant cancer epigenomes could orient the design of effective therapies.

Enzymes that catalyse CpG methylation in DNA, including the DNA methyltransferases 1 (DNMT1), 3A (DNMT3A) and 3B (DNMT3B), are indispensable for mammalian tissue development and homeostasis1–4. They are also implicated in human developmental disorders and cancers5–8, supporting the critical role of DNA methylation in the specification and maintenance of cell fate. Previous studies have suggested that post-translational modifications of histones are involved in specifying patterns of DNA methyltransferase localization and DNA methylation at promoters and actively transcribed gene bodies9–11. However, the mechanisms that control the establishment and maintenance of intergenic DNA methylation remain poorly understood. Tatton–Brown–Rahman syndrome (TBRS) is a childhood overgrowth disorder that is defined by germline mutations in DNMT3A. TBRS shares clinical features with Sotos syndrome (which is caused by haploinsufficiency of NSD1, a histone methyltransferase that catalyses the dimethylation of histone H3 at K36 (H3K36me2)8,12,13), which suggests that there is a mechanistic link between these two diseases. Here we report that NSD1-mediated H3K36me2 is required for the recruitment of DNMT3A and maintenance of DNA methylation at intergenic regions. Genome-wide analysis shows that the binding and activity of DNMT3A colocalize with H3K36me2 at non-coding regions of euchromatin. Genetic ablation of Nsd1 and its paralogue Nsd2 in mouse cells results in a redistribution of DNMT3A to H3K36me3-modified gene bodies and a reduction in the methylation of intergenic DNA. Blood samples from patients with Sotos syndrome and NSD1-mutant tumours also exhibit hypomethylation of intergenic DNA. The PWWP domain of DNMT3A shows dual recognition of H3K36me2 and H3K36me3 in vitro, with a higher binding affinity towards H3K36me2 that is abrogated by TBRS-derived missense mutations. Together, our study reveals a trans-chromatin regulatory pathway that connects aberrant intergenic CpG methylation to human neoplastic and developmental overgrowth. H3K36me2 targets DNMT3A to intergenic regions and this process, together with H3K36me3-mediated recruitment of DNMT3B, has a key role in establishing and maintaining genomic DNA methylation landscapes.

Summary Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. We show that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon de-repression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. Our results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks.

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction. Significance: This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT. See related commentary by Licht, p. 1794. This article is highlighted in the In This Issue feature, p. 1775

Diffuse midline gliomas and posterior fossa type-A ependymomas contain the highly recurrent histone H3 K27M mutation and the H3 K27M-mimic EZHIP, respectively. In vitro, H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3K27me3 levels, however residual peaks of H3K27me3 remain at CpG islands through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with an allosterically activated form of PRC2 in vivo. The formation of H3 K27M- and EZHIP-PRC2 complexes occurs at CpG islands containing H3K27me3 and impedes PRC2 and H3K27me3 spreading. While EZHIP is not found outside of placental mammals, we find that expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved molecular mechanism. Our results highlight the mechanistic similarities between EZHIP and H3 K27M in vivo and provide mechanistic insight for the retention of residual H3K27me3 in tumors driven by these oncogenes.

BACKGROUND Pediatric High-Grade Gliomas (pHGGs) show recurrent high-level amplifications around the oncogenes MET, MYCN and EGFR. However what drives expression of the oncogenes from these amplicons remains unclear. We aim to discover enhancers on these amplicons that are responsible for oncogene expressions and the core regulatory transcription factors (TFs) they bind. METHOD Using RNA-seq from 12 pHGG cell lines, we identified groups of high and low-expressing pHGG lines for MET, MYCN and EGFR. We then compared the H3K27Ac ChIP-seq between the two groups using diffbind. This allowed us to identify statistically significant peaks that are differentially activated in the oncogene-high v.s. oncogene-low expressing groups. Additionally, we overlapped the positions of these candidate oncogene enhancers with the regions that are recurrently incorporated into high-level amplicons based on published whole genome sequencing data. Using a previously defined set of core regulatory TFs we determined which TF binds the amplified oncogene enhancers and could be driving oncogenic expressions of MET, MYCN and EGFR in pHGGs. RESULTS We identify 3 cell lines for both the high- and low-expressing groups for each oncogene. Cell lines with high expression of the oncogene showed distinct enhancers with significant enrichment in H3K27Ac compared to the cell lines with low expression for each oncogene. Of all enhancers with enrichment high oncogene expression groups those with binding sites for known pHGG core regulatory circuit TF were preferentially incorporated into the high-level amplicons of the oncogene. We also identified core TFs that bind enhancers for MYCN, EGFR and MET as well as core TFs that are unique to a single oncogene. CONCLUSION We identified candidate core transcription factor that drives expression of multiple oncogenes in pHGG. These could serve as a potential novel therapeutic target for pHGGs with addiction to MYCN or RTK signaling.

The discovery of H3K27M mutations in pediatric gliomas marked a new chapter in cancer epigenetics. Numerous studies have investigated the effect of this mutation on H3K27 trimethylation, but only recently have we started to realize its additional effects on the epigenome. Here, we use isogenic glioma H3K27M(+/-) cell lines to investigate H3K27 methylation and its interaction with H3K36 and H3K9 modifications. We describe a “Step Down” effect of H3K27M on the distribution of H3K27 methylation: me3 is reduced to me2, me2 is reduced to me1, while H3K36me2/3 delineate the boundaries for the spread of H3K27me marks. We also observe a replacement of H3K27me2/3 silencing by H3K9me3. Using a computational simulation, we explain our observations by reduced effectiveness of PRC2 and constraints imposed on deposition of H3K27me by antagonistic H3K36 modifications. Our work further elucidates the effect of H3K27M in gliomas, as well as general principles of deposition of H3K27 methylation.

BACKGROUND Driver single nucleotide variants (SNV) and somatic copy number aberrations (SCNA) of pediatric high-grade glioma (pHGGs), including Diffuse Midline Gliomas (DMGs) are characterized. However, structural variants (SVs) in pHGGs and the mechanisms through which they contribute to glioma formation have not been systematically analyzed genome-wide. METHODS Using SvABA for SVs as well as the latest pipelines for SCNAs and SNVs we analyzed whole-genome sequencing from 174 patients. This includes 60 previously unpublished samples, 43 of which are DMGs. Signature analysis allowed us to define pHGG groups with shared SV characteristics. Significantly recurring SV breakpoints and juxtapositions were identified with algorithms we recently developed and the findings were correlated with RNAseq and H3K27ac ChIPseq. RESULTS The SV characteristics in pHGG showed three groups defined by either complex, intermediate or simple signature activities. These associated with distinct combinations of known driver oncogenes. Our statistical analysis revealed recurring SVs in the topologically associating domains of MYCN, MYC, EGFR, PDGFRA & MET. These correlated with increased mRNA expression and amplification of H3K27ac peaks. Complex recurring amplifications showed characteristics of extrachromosomal amplicons and were enriched in coding SVs splitting protein regulatory from effector domains. Integrative analysis of all SCNAs, SNVs & SVs revealed patterns of characteristic combinations between potential drivers and signatures. This included two distinct groups of H3K27M DMGs with either complex or simple signatures and different combinations of associated variants. CONCLUSION Recurrent SVs associate with signatures shaped by an underlying process, which can lead to distinct mechanisms to activate the same oncogene.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here, we show that 50% of G34R/V-tumours (n=95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. While considered gliomas, G34R/V-tumours actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V-mutations impair neuronal differentiation. The lineage-of-origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V-tumours harbour dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell-fate specification. G34R/V may become dispensable for tumour maintenance, while mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumours. G34R/V-gliomas are neuronal malignancies, where interneuron progenitors are stalled in differentiation by G34R/V-mutations, and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signalling.

High-grade gliomas (HGG) defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3

The C allele of the rs2736100 single nucleotide polymorphism located in the second intron of the TERT gene has recently been identified as a susceptibility factor for myeloproliferative neoplasms (MPN) in the Icelandic population. Here, we evaluate the role of TERT rs2736100_C in sporadic and familial MPN in the context of the previously identified JAK2 GGCC predisposition haplotype. We have confirmed the TERT rs2736100_C association in a large cohort of Italian sporadic MPN patients. The risk conferred by TERT rs2736100_C is present in all molecular and diagnostic MPN subtypes. TERT rs2736100_C and JAK2 GGCC are independently predisposing to MPN and have an additive effect on disease risk, together explaining a large fraction of the population attributable fraction (PAF = 73.06%). We found TERT rs2736100_C significantly enriched (P = 0.0090) in familial MPN compared to sporadic MPN, suggesting that low-penetrance variants may be responsible for a substantial part of familial clustering in MPN. Am. J. Hematol. 89:1107–1110, 2014. © 2014 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.

Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.

To the editor: Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis, have in most instances a sporadic occurrence, but familial clustering of MPNs has been reported and familial cases are about 7% to 8% of all MPN patients

The study aimed to identify genetic lesions associated with secondary acute myeloid leukemia (sAML) in comparison with AML arising de novo (dnAML) and assess their impact on patients’ overall survival (OS). High-resolution genotyping and loss of heterozygosity mapping was performed on DNA samples from 86 sAML and 117 dnAML patients, using Affymetrix Genome-Wide Human SNP 6.0 arrays. Genes TP53, RUNX1, CBL, IDH1/2, NRAS, NPM1, and FLT3 were analyzed for mutations in all patients. We identified 36 recurrent cytogenetic aberrations (more than five events). Mutations in TP53, 9pUPD, and del7q (targeting CUX1 locus) were significantly associated with sAML, while NPM1 and FLT3 mutations associated with dnAML. Patients with sAML carrying TP53 mutations demonstrated lower 1-year OS rate than those with wild-type TP53 (14.3% ± 9.4% vs. 35.4% ± 7.2%; P 5 0.002), while complex karyotype, del7q (CUX1) and del7p (IKZF1) showed no significant effect on OS. Multivariate analysis confirmed that mutant TP53 was the only independent adverse prognostic factor for OS in sAML (hazard ratio 2.67; 95% CI: 1.33–5.37; P 5 0.006). Patients with dnAML and complex karyotype carried sAML-associated defects (TP53 defects in 54.5%, deletions targeting FOXP1 and ETV6 loci in 45.4% of the cases). We identified several co-occurring lesions associated with either sAML or dnAML diagnosis. Our data suggest that distinct genetic lesions drive leukemogenesis in sAML. High karyotype complexity of sAML patients does not influence OS. Somatic mutations in TP53 are the only independent adverse prognostic factor in sAML. Patients with dnAML and complex karyotype show genetic features associated with sAML and myeloproliferative neoplasms. Am. J. Hematol. 87:1010–1016, 2012. V C 2012 Wiley Periodicals, Inc.

To identify rare causal variants in late-onset Parkinson disease (PD), we investigated an Austrian family with 16 affected individuals by exome sequencing. We found a missense mutation, c.1858G>A (p.Asp620Asn), in the VPS35 gene in all seven affected family members who are alive. By screening additional PD cases, we saw the same variant cosegregating with the disease in an autosomal-dominant mode with high but incomplete penetrance in two further families with five and ten affected members, respectively. The mean age of onset in the affected individuals was 53 years. Genotyping showed that the shared haplotype extends across 65 kilobases around VPS35. Screening the entire VPS35 coding sequence in an additional 860 cases and 1014 controls revealed six further nonsynonymous missense variants. Three were only present in cases, two were only present in controls, and one was present in cases and controls. The familial mutation p.Asp620Asn and a further variant, c.1570C>T (p.Arg524Trp), detected in a sporadic PD case were predicted to be damaging by sequence-based and molecular-dynamics analyses. VPS35 is a component of the retromer complex and mediates retrograde transport between endosomes and the trans-Golgi network, and it has recently been found to be involved in Alzheimer disease.

Background Myeloproliferative neoplasms constitute a group of diverse chronic myeloid malignancies that share pathogenic features such as acquired mutations in the JAK2, TET2, CBL and MPL genes. There are recent reports that a JAK2 gene haplotype (GGCC or 46/1) confers susceptibility to JAK2 mutation-positive myeloproliferative neoplasms. The aim of this study was to examine the role of the JAK2 GGCC haplotype and germline mutations of TET2, CBL and MPL in familial myeloproliferative neoplasms. Design and Methods We investigated patients with familial (n=88) or sporadic (n=684) myeloproliferative neoplasms, and a control population (n=203) from the same demographic area in Italy. Association analysis was performed using tagged single nucleotide polymorphisms (rs10974944 and rs12343867) of the JAK2 haplotype. Sequence analysis of TET2, CBL and MPL was conducted in the 88 patients with familial myeloproliferative neoplasms. Results Association analysis revealed no difference in haplotype frequency between familial and sporadic cases of myeloproliferative neoplasms (P=0.6529). No germline mutations in TET2, CBL or MPL that segregate with the disease phenotype were identified. As we observed variability in somatic mutations in the affected members of a pedigree with myeloproliferative neoplasms, we postulated that somatic mutagenesis is increased in familial myeloproliferative neoplasms. Accordingly, we compared the incidence of malignant disorders between sporadic and familial patients. Although the overall incidence of malignant disorders did not differ significantly between cases of familial and sporadic myeloproliferative neoplasms, malignancies were more frequent in patients with familial disease aged between 50 to 70 years (P=0.0198) than in patients in the same age range with sporadic myeloproliferative neoplasms. Conclusions We conclude that the JAK2 GGCC haplotype and germline mutations of TET2, CBL or MPL do not explain familial clustering of myeloproliferative neoplasms. As we observed an increased frequency of malignant disorders in patients with familial myeloproliferative neoplasms, we hypothesize that the germline genetic lesions that underlie familial clustering of myeloproliferative neoplasms predispose to somatic mutagenesis that is not restricted to myeloid hematopoietic cells but cause an increase in overall carcinogenesis.

Background: Glioblastoma is a grade IV malignant brain tumor with poor prognosis and rapid disease progression. Recurrent somatic mutations in histone H3 genes have been identified in the majority of pediatric glioblastoma cases. The K27M mutation in H3.1 and H3.3 histones globally inhibits lysine methylation at the K27 position, whereas H3.3 G34R/V possibly affects histone lysine methylation at the K36 position. H3 K27M mutation has been shown to dramatically decrease the total levels of H3K27me3 and H3K27me2 marks and increase H3K27ac levels. However, the effect of H3 K27M on global epigenomic changes is not fully characterized. Furthermore, standard profiling of histone marks by chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) is not quantitative, a significant caveat when global levels of histone marks change so drastically. Methods: We assembled a collection of H3 K27M mutant and wild-type cell lines derived from the glioblastoma patients. The epigenomes of these cell lines were comprehensively characterized by profiling for six histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3, H3K36me2, H3K36me3) using ChIP-seq. In addition, we derived isogenic cell lines overexpressing H3.3 K27M, as well as cell lines with knockin or knockout of the K27M mutation using the CRISPR/Cas9 genome editing system. These cell lines were profiled for H3K27me3 mark by ChIP-seq. We used a modified ChIP-seq protocol, chromatin immunoprecipitation with exogenous reference genome (ChIP-Rx), which allows quantitation of histone mark abundance by normalization to proportions of added Drosophila chromatin in the ChIP reaction. RNA sequencing was performed on both primary and isogenic cell lines. Results: The most striking difference we observed between H3 K27M and wild-type cells was in H3K27me3 mark. Using ChIP-Rx, we observe significantly lower levels of H3K27me3 mark in H3 K27M cell lines, both in primary cells and isogenic contexts. Despite very low total levels of H3K27me3 mark, K27M mutant cells display enrichment of the mark in certain regions, at comparable levels to wild-type cell lines. Using our isogenic cell line models, we show that K27M mutation is indeed responsible for those genome-wide changes in the epigenome. Correlating H3K27me3 distribution with transcriptome data, we show that expression changes mainly among the genes that are lowly expressed in these cells. Pathway analysis of differentially expressed genes shows enrichment for neural development and differentiation that suggests links to disease pathogenesis. Conclusions: Despite the fact that primary cell lines have different origins and a variety of additional driver mutations, their epigenomes appears to be remarkably similar, due to being shaped predominantly by the effects of histone mutations, as demonstrated in isogenic cell line systems. Global changes in H3K27me3 levels and distribution in H3 K27M mutant cells lead to specific changes in gene expression. The changes induced by K27M mutations also appear to be specific to the cell type and/or developmental context of origin. This may help better understand the effect they have in reshaping the epigenome to promote oncogenesis. Citation Format: Ashot S. Harutyunyan, Brian Krug, Simon Papillon-Cavanagh, Haifen Chen, Shriya Deshmukh, Warren A. Cheung, Rui Li, Jad Belle, Denise Bechet, Nicolas De Jay, Michele Zeinieh, Tenzin Gayden, Caterina Russo, Leonie Mikael, Damien Faury, Claudia Kleinman, Tomi Pastinen, Jacek Majewski, Nada Jabado. Identification of epigenomic changes induced by H3 K27M mutation in glioblastoma using patient-derived and CRISPR/Cas9 edited cell lines [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B44.

Introduction: Pediatric glioblastomas (pGBM) are malignant brain tumors associated with a dismal prognosis. A subset of pGBMs carry mutations of either the Lysine 27 or Glycine 34 (G34) amino acid residues of histone 3 variant genes. The same G34 residue is also mutated in 85-95% of giant cell tumors of bone (GCTs), albeit to Tryptophan (G34W) in GCTs rather than to Arginine or Valine (G34R/V) as in pGBMs. The G34 mutation is predicted to impede access of histone methyltransferases like SetD2 to the nearby Lysine 36 residue, thereby altering the epigenome and transcriptome. Methods: To elucidate the tumorigenic effect of G34 mutations, we used the gene-editing technology CRISPR/Cas9 to correct the G34W mutation to wild-type in 2 GCT cell lines. We then investigated CRISPR-edited cell lines using functional assays, proteomic, epigenomic, and transcriptomic analyses. Results: Correction of the G34W mutation to wild-type in CRISPR-edited GCT cells results in phenotypic and functional changes suggestive of reduced tumorigenicity. By mass spectrometry, G34W-mutant GCT cell lines display decreased level of Lysine 36 trimethylation (H3K36me3) on the mutant G34-peptide, similar to G34-mutated pGBM cell lines. However, unlike pGBMs, GCTs display increased levels of Lysine 36 dimethylation (H3K36me2) on the mutant G34W-peptide. Ongoing Experiments and Analyses: We are currently comparing the level and distribution of multiple histone marks by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) of CRISPR-edited wild-type clones relative to the parent G34W-mutant GCT line. We are also assessing differential gene expression by RNA-Seq and characterizing the methylation signature by 850K DNA methylation array of G34W-mutant GCTs and edited clones. Conclusion: The G34W-mutation clearly has an impact on tumorigenic potential, as evidenced by in vitro functional assays. The G34W-mutant peptide of GCT cell lines features a distinct profile of post-translational histone modifications compared to G34R- or G34V-mutant peptides of pGBM cell lines. Further investigation could elucidate the epigenetic mechanism(s) through which the G34W mutation confers its tumorigenic properties. Citation Format: Shriya Deshmukh, Sima Khazaei, Dylan Marchione, Ashot Harutyunyan, Benjamin Garcia, Nada Jabado. Characterizing the epigenetic effects of the histone 3.3 G34W mutation in giant cell tumors of bone [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A39.

In this review, some principal population genetic features of familial Mediterranean fever (FMF) are considered. These relate to the time and the place of founder mutations' origins, the role of ancient migrations and contacts between populations in the spatial spreading of the disorder, the influence of environmental factors and cultural traditions on the rate of FMF incidence, and possible selective advantage in carriers of FMF causing gene (MEFV) mutations.

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.

To the editor: Transformation of hematopoietic cells depends on the acquisition of genetic events leading to cytokine independence, typically associated with acquisition of an autocrine cytokine loop or/and increased expression or/and mutation of JAK genes.[1][1] Rearrangement of the JAK2 gene

Rare germline variants in regions of loss of heterozygosity may influence clinical course of hematological malignancies

To the Editor: Myeloproliferative neoplasms have an inherent tendency toward leukemic transformation. The genetic mechanisms of transformation remain largely unknown. We analyzed biopsy specimens of myeloproliferative neoplastic tissue from 330 patients for chromosomal aberrations associated with leukemic transformation (the analysis was performed with the use of Genome-Wide Human SNP [single-nucleotide polymorphism] Array, Affymetrix). Of those patients, 308 had chronic-phase myeloproliferative neoplasms and 22 had postmyeloproliferative-phase neoplasm secondary acute myeloid leukemia (AML). Among these 22 patients, 1 carried the myeloproliferative leukemia virus oncogene (MPL) W515L and all others carried the Janus kinase 2 gene (JAK2) V617F mutation. . . .

Somatic mutations in the calreticulin (CALR) gene were recently discovered in patients with sporadic essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations. We studied CALR mutation status in familial cases of myeloproliferative neoplasm. In a cohort of 127 patients, CALR indels were identified in 6 of 55 (11%) subjects with ET and in 6 of 20 (30%) with PMF, whereas 52 cases of polycythemia vera had nonmutated CALR. All CALR mutations were somatic, found in granulocytes but not in T lymphocytes. Patients with CALR-mutated ET showed a higher platelet count (P = .017) and a lower cumulative incidence of thrombosis (P = .036) and of disease progression (P = .047) compared with those with JAK2 (V617F). In conclusion, a significant proportion of familial ET and PMF nonmutated for JAK2 carry a somatic mutation of CALR.

The main molecular basis of essential thrombocythemia and hereditary thrombocytosis is acquired, and germ-line-activating mutations affect the thrombopoietin signaling axis. We have identified 2 families with hereditary thrombocytosis presenting novel heterozygous germ-line mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, whereas the other presents 2 JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of Janus kinase 2 (JAK2) R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-thrombopoietin receptor (MPL) but not of Ba/F3-human receptor of erythropoietin cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed thrombopoietin-independent phosphorylation of signal transducer and activator of transcription 1. The JAK2 R867Q and S755R/R938Q proteins had significantly longer half-lives compared with JAK2 V617F. The longer half-lives correlated with increased binding to the heat shock protein 90 (HSP90) chaperone and with higher MPL cell-surface expression. Moreover, these mutants were less sensitive to JAK2 and HSP90 inhibitors than JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL while inducing a decreased sensitivity to clinically available JAK2 inhibitors.

Broséus, J., Lippert, E., Harutyunyan, A.S., Jeromin, S., Zipperer, E., Florensa, L., Milosevic, J.D., Haferlach, T., Germing, U., Luño, E., Schnittger, S., Kralovics, R., Girodon, F.

BACKGROUND Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients. METHODS We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms. RESULTS Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. CONCLUSIONS Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.)

We investigated a cohort of 200 patients diagnosed with essential thrombocythemia (ET, N=115) or prefibrotic primary myelofibrosis (prePMF, N=85). All diagnoses were performed by strictly adhering to the 2016 WHO criteria. Investigations into the molecular genetic markup of these patients were performed using next generation sequencing methods for the main genes involved in myeloproliferative neoplasms, JAK2, CALR and MPL. Additionally, four more genes considered as the most commonly mutations associated with an elevated risk for early death or leukemic transformation in PMF, comprising of ASXL1, EZH1, IDH1/2 and SRSF2 were analyzed. The median age at diagnosis in the cohort was 58,8 years, 56,4 years for ET patients and 60,7 years for prePMF patients. Median observation time was 8 years, 8,5 years in the ET group and 7,4 years in the prePMF group. 120 (60%) patients were JAK2V617F positive, 75 (65,2%) ET and 45 (52,9%) prePMF. 54 (27%) patients showed CALR positivity, 21 (18,3%) ET and 33 (38,8%) prePMF. Only a few patients were MPL positive, 8 (4%) in the whole cohort, 4 (3,5%9 ET and 4 (4,7%) prePMF. Triple negative patients, showing none of the above three mutations were 18 (9%), 15 ET (13%) and 3 prePMF (3,5%). We investigated, whether the presence or number of high molecular risk mutations between the two diagnostic groups had an effect on overall survival. Each group was divided into two categories: patients with no high molecular risk mutation and patients with one or more high molecular risk mutations. Among the ET patients, 86 (74,8%) showed no such mutation, whereas 29 (25,2%) had one or more. In the prePMF group, 73 (85,9%) displayed no mutation and 12 (14,1%) having one or more. The difference between ET and prePMF regarding the number of high molecular risk mutations was not statistically significant (p=0,075678). Additionally, we examined the difference within each diagnostic group (Figure 1). Here, we divided each portion of the cohort into three categories: patients with no high molecular risk mutation, patients with one high molecular risk mutation and patients with two or more high molecular risk mutations. Of the 29 ET patients with one or more high molecular risk mutations, 7 had two or more, of the 12 prePMF patients 4 respectively. Comparing the overall survival between ET and prePMF patients, a clear significant difference favoring ET patients could be observed (Figure 2A). No significant difference regarding overall survival was detected between patients with or without one or more high molecular risk mutations (Figure 2B). Looking at differences in overall survival within each diagnostic group with regards to the number of high molecular risk mutations, neither in the ET nor in the prePMF cohort a significant difference could be found (Figure 2C, D). In conclusion, our investigation shows that the most common high molecular risk mutations have no significant impact on overall survival in WHO-defined ET and prePMF patients. Figure 1 Proportions of high molecular risk mutations (HRM) in ET and prePMF patients Figure 1. Proportions of high molecular risk mutations (HRM) in ET and prePMF patients Figure 2 Overall survival of (A) ET versus prePMF patients, (B) patients without versus patients with one or more high molecular risk mutations, (C) ET patients with and without high molecular risk mutations and (D) prePMF patients with and without high molecular risk mutations. Figure 2. Overall survival of (A) ET versus prePMF patients, (B) patients without versus patients with one or more high molecular risk mutations, (C) ET patients with and without high molecular risk mutations and (D) prePMF patients with and without high molecular risk mutations. Disclosures Gisslinger: AOP Orphan: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Baxalta: Consultancy, Honoraria. Thiele:Novartis: Consultancy, Honoraria; Incyte Corporation: Consultancy, Honoraria. Kralovics:Qiagen: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Research Funding.

It is thought that myeloproliferative neoplasms (MPNs) are driven by somatic mutations, although hereditary factors also play a prominent role in the pathogenesis of the disease. Hereditary thrombocytosis and erythrocytosis are not malignant disorders but are clinically similar to MPNs. Several mutations have been found that explain a proportion of hereditary thrombocytosis and hereditary erythrocytosis. Germline variants can influence the risk of leukemic transformation in MPNs and the course of the disease through interaction with acquired chromosomal aberrations. Overall, it has been shown that germline factors play an important part in MPN pathogenesis.

Chronic myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) have an inherent tendency to progress to acute myeloid leukemia (AML). Using high-resolution SNP microarrays, we studied a total of 517 MPN and MDS patients in different disease stages, including 77 AML cases with previous history of MPN (N = 46) or MDS (N = 31). Frequent chromosomal deletions of variable sizes were detected, allowing the mapping of putative tumor suppressor genes involved in the leukemic transformation process. We detected frequent deletions on the short arm of chromosome 6 (del6p). The common deleted region on 6p mapped to a 1.1-Mb region and contained only the JARID2 gene--member of the polycomb repressive complex 2 (PRC2). When we compared the frequency of del6p between chronic and leukemic phase, we observed a strong association of del6p with leukemic transformation (P = 0.0033). Subsequently, analysis of deletion profiles of other PRC2 members revealed frequent losses of genes such as EZH2, AEBP2, and SUZ12; however, the deletions targeting these genes were large. We also identified two patients with homozygous losses of JARID2 and AEBP2. We observed frequent codeletion of AEBP2 and ETV6, and similarly, SUZ12 and NF1. Using next generation exome sequencing of 40 patients, we identified only one somatic mutation in the PRC2 complex member SUZ12. As the frequency of point mutations in PRC2 members was found to be low, deletions were the main type of lesions targeting PRC2 complex members. Our study suggests an essential role of the PRC2 complex in the leukemic transformation of chronic myeloid disorders.

Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs) are clonal myeloid disorders with increased production of terminally differentiated cells. The disease course is generally chronic, but some patients show disease progression (secondary myelofibrosis or accelerated phase) and/or leukemic transformation. We investigated chromosomal aberrations in 408 MPN samples using high-resolution single-nucleotide polymorphism microarrays to identify disease-associated somatic lesions. Of 408 samples, 37.5% had a wild-type karyotype and 62.5% harbored at least 1 chromosomal aberration. We identified 25 recurrent aberrations that were found in 3 or more samples. An increased number of chromosomal lesions was significantly associated with patient age, as well as with disease progression and leukemic transformation, but no association was observed with MPN subtypes, Janus kinase 2 (JAK2) mutational status, or disease duration. Aberrations of chromosomes 1q and 9p were positively associated with disease progression to secondary myelofibrosis or accelerated phase. Changes of chromosomes 1q, 7q, 5q, 6p, 7p, 19q, 22q, and 3q were positively associated with post-MPN acute myeloid leukemia. We mapped commonly affected regions to single target genes on chromosomes 3p (forkhead box P1 [FOXP1]), 4q (tet oncogene family member 2 [TET2]), 7p (IKAROS family zinc finger 1 [IKZF1]), 7q (cut-like homeobox 1 [CUX1]), 12p (ets variant 6 [ETV6]), and 21q (runt-related transcription factor 1 [RUNX1]). Our data provide insight into the genetic complexity of MPNs and implicate new genes involved in disease progression.

Myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These disorders may undergo phenotypic shifts, and may specifically evolve into secondary myelofibrosis (MF) or acute myeloid leukemia (AML). We studied genomic changes associated with these transformations in 29 patients who had serial samples collected in different phases of disease. Genomic DNA from granulocytes, i.e., the myeloproliferative genome, was processed and hybridized to genome-wide human SNP 6.0 arrays. Most patients in chronic phase had chromosomal regions with uniparental disomy (UPD) and/or copy number changes. Disease progression to secondary MF or AML was associated with the acquisition of additional chromosomal aberrations in granulocytes (P = 0.002). A close relationship was observed between aberrations of chromosome 9p (UPD and/or gain) and progression from PV to post-PV MF (P = 0.002). The acquisition of one or more aberrations involving chromosome 5, 7, or 17p was specifically associated with progression to AML (OR 5.9, 95% CI 1.2-27.7, P = 0.006), and significantly affected overall survival (HR 18, 95% CI 1.9-164, P = 0.01). These observations indicate that disease progression from chronic-phase MPN to secondary MF or AML is associated with specific chromosomal aberrations that can be detected by means of high-resolution SNP array analysis of granulocyte DNA.

Transformation to acute leukemia is a major complication of myeloproliferative neoplasms (MPNs), however, the genetic changes leading to transformation remain largely unknown. We screened nine patients with post-MPN leukemia for chromosomal aberrations using microarray karyotyping. Deletions on the short arm of chromosome 7 (del7p) emerged as a recurrent defect. We mapped the common deleted region to the IKZF1 gene, which encodes the transcription factor Ikaros. We further examined the frequency of IKZF1 deletions in a total of 29 post-MPN leukemia and 526 MPN patients without transformation and observed a strong association of IKZF1 deletions with post-MPN leukemia in two independent cohorts. Patients with IKZF1 loss showed complex karyotypes, and del7p was a late event in the genetic evolution of the MPN clone. IKZF1 deletions were observed in both undifferentiated and differentiated myeloid cell types, indicating that IKZF1 loss does not cause differentiation arrest but rather renders progenitors susceptible to transformation, most likely through chromosomal instability. Induced Ikzf1 haploinsufficiency in primary murine progenitors resulted in elevated Stat5 phosphorylation and increased cytokine-dependent growth, suggesting that reduced expression of IKZF1 is sufficient to perturb growth regulation. Thus, IKZF1 loss is an important step in the leukemic transformation of a subpopulation of MPN patients.

Inherent tendency for thrombosis is a major complication in myeloproliferative neoplasms (MPN). The molecular basis of thrombosis in MPN is not well understood, however, genetic factors have been proposed to play a role. To agnostically investigate the role of common germline variation in MPN thrombophilia, we performed genome-wide association studies (GWAS) in MPN patient cohorts characterized for arterial thrombosis (AT) events. In our discovery cohort (n=383) from Vienna, Austria, 18% of patients have suffered from AT after MPN diagnosis, and 33% of patients showed records for AT events at any time. Of this discovery cohort, a subset of patients (n=302) selected independently from thrombotic status was genotyped on the Affymetrix Genome-Wide SNP 6.0 array platform. After assessment of case-control setup and genotyping quality as standardly implemented in GWAS, confidently genotyped single nucleotide polymorphisms (SNPs) were tested for allelic association with occurrence of AT after MPN diagnosis. We observed an association signal beyond genome-wide statistical significance (P 0.3) spans ~300 kilobases, covering intergenic sequence as well as promoter and 5' exons of the TULP4 gene. To test for the possibility of a rare coding variant in TULP4 or other more distant genes underlying the association through long-range linkage disequilibrium, we used genotype imputation in conjunction with the 1000 genomes reference panel to infer genotypes on all untyped variants (MAF>1%) in a 5 megabase region centered on the core haplotype. We did not detect any coding variants reflecting the association, and the tag-SNPs from the initial GWAS remained the most strongly associated variants. Causative non-coding genetic variation identified in GWAS is thought to exert its function through differential regulation of specific target genes. Therefore, we evaluated a potential influence of the 6q25.3 risk haplotype on TULP4 gene expression in peripheral blood (The Cancer Genome Atlas (TCGA) LAML dataset, RNA-Seq on 173 acute myeloid leukemia patients). Indeed we detected significantly decreased TULP4 expression in risk haplotype carriers (P =0.029), providing indirect evidence for reduced TULP4 transcript levels impacting on elevated risk for AT in MPN. Further studies will be required to functionally assess the potential role of TULP4 in MPN-related AT. Disclosures Gisslinger: Novartis: Honoraria, Research Funding, Speakers Bureau; Geron: Consultancy; Sanofi Aventis: Consultancy; Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

The genomic landscape of myeloproliferative neoplasms (MPNs) has altered dramatically due to the recent discovery of somatic mutations of calreticulin (CALR). This discovery enables for the first time molecular information, in addition to JAK2V617F, to be used in the majority of MPN patients as an affirmative variable to discriminate MPNs from reactive myeloid proliferations. The clinical course of essential thrombocythemia (ET) or primary myelofibrosis (PMF) in patients carrying the CALR mutation was reported to be more indolent than in JAK2 positive patients and was associated with increased survival. Our aim was to investigate whether the impact of CALR expression on prognosis and clinical outcome is different in prefibrotic/early PMF (prePMF) compared to WHO-ET. In a cohort of 348 adult patients with the clinical diagnosis of either ET or PMF mutational analysis for CALR was available. Eligibility criteria for the study included: availability of mutation analysis for JAK2, MPL and CALR; availability of representative, treatment-naive bone marrow biopsy (BM); availability of a histological and clinical consensus on the diagnosis; complete long-term documentation of clinical data and outcome. Consenting clinico-pathological findings in our cohort were consistent with 115 cases showing WHO-ET and 85 patients with prePMF. In comparison to WHO-ET, prePMF revealed minor/borderline age- and gender-matched anemia, slight increase in serum LDH level and leukocyte count, minor to slight splenomegaly, and an occasional left shift in granulo- and erythropoiesis with occurrence of a few myelo-and/or erythroblasts (table 1). An accurate differentiation between both MPN entities was shown to exert a significant difference in terms of overall and relative survival and hematologic transformation into overt PMF and AL. The present study revealed a different CALR mutation frequency in ET in contrast to most of the investigations published recently. We observed CALR mutations in 18% of WHO-ET; JAK2, MPL and CALR wildtype (wt) was observed in 13% of WHO-ET. The discrepancy in the frequencies of CALR positivity in our ET cohort to most of the recently published studies may be due to our strict adherence to the WHO criteria for diagnosis of ET. Regarding prePMF, we observed CALR mutations in 39% of the patients. 92% of the JAK2 and MPL wt subgroup carried the CALR mutation, with JAK2, MPL and CALR wt being observed in only 3% of prePMF. The most remarkable differences between WHO-ET and prePMF were seen in the comparison of the overall survival (figure 1). While the CALR mutation did not have any beneficial influence on survival in WHO-ET, it was associated with a superior overall survival in prePMF. Such a striking difference was not seen at the time of transformation into overt myelofibrosis, and there was only a slightly shorter time to progression to fibrosis in CALR wt prePMFs. There was a trend showing that CALR mutated prePMF patients have shorter thrombosis-free survival compared to CALR wt prePMF patients. There was no impact of the CALR mutations on thrombosis-free survival in WHO-ET. The present data confirm that WHO-ET and prePMF are biologically different sub-entities of MPNs. In prePMF, almost 100% of patients are now associated with a known disease-causing mutation. Our data support the classical clinical approach in the diagnosis of thrombocytosis, using BM histology to differentiate WHO-ET from prePMF and to estimate the outcome of the disease more accurately. Table 1:Total cohort (N=200)WHO-ET (N=115)prePMF (N=85)PAge at diagnosis, years0,04median58,8556,460,7range19-8819-8427-88Sex0,587male784335female1227250Hb, g/dL Figure 1 Figure 1. Disclosures Thiele: AOP Orphan Pharmaceuticals: Consultancy, Honoraria; Incyte Corporation: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria.

The main molecular basis of essential thrombocytemia and hereditary thrombocytosis are acquired and germline activating mutations affecting the thrombopoietin signaling axis, respectively. We have identified two families with hereditary thrombocytosis presenting novel heterozygous germline mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, while the other presents two JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of JAK2 R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-MPL but not of Ba/F3-EPOR cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed a TPO-independent phosphorylation of STAT1. JAK2 R867Q and S755R/R938Q, compared to JAK2 V617F, had significantly longer half-lives correlating with an increased MPL cell surface expression. Moreover, these mutations conferred a resistance to JAK2 and HSP90 inhibitors compared to JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL, while inducing resistance to clinically available JAK2 inhibitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 3755 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by excessive production of myeloid cells and the presence of the BCR-ABL fusion oncogene resulting from the t(9;22) reciprocal translocation. The leukemic clone in CML often accumulates other somatic lesions that may collaborate with BCR-ABL oncogenicity or confer resistance to tyrosine kinase inhibitors. The chromosomal instability in CML is believed to be caused by the BCR-ABL oncogene. We hypothesized that the accumulation of cytogenetic lesions in CML are acquired after the t(9;22). Thus, imatinib therapy should not only cause the molecular remission of BCR-ABL positivity but also the remission of most cytogenetic lesions. If cytogenetic lesions preceded t(9;22) acquisition, a residual clone should be detectable after remission of BCR-ABL positivity. In order to test these predictions, we determined high-resolution karyotypes of CML patients at diagnosis and at time points with variable durations of imatinib therapy. At the same time, we aimed to characterize the overall cytogenetic aberration profile of CML at different disease stages. The copy number abnormalities and loss of heterozygosity coupled with acquired uniparental disomy (UPD) were determined using Affymetrix SNP 6.0 arrays. Overall 62 patients were included in the study. For 13 patients paired DNA samples were available, the first one taken at diagnosis and the second after treatment with imatinib. These samples were used to evaluate the cytogenetic remission after treatment. In 7 out of 13 patients we could detect an additional chromosomal aberration at diagnosis. Interestingly, the clone size assessment based on copy number signal intensity data was concordant with BCR-ABL burden. Analysis of the follow-up samples revealed that 6 patients with additional cytogenetic aberrations at diagnosis showed complete cytogenetic remission. In one patient imatinib therapy led to significant reduction of the BCR-ABL burden but a residual BCR-ABL negative clone persisted. This residual clone exhibiting del6p, del10q and del13q was detectable as a minor clone at diagnosis and it fully replaced the BCR-ABL positive clone after 8 months of imatinib therapy. This data indicates that the BCR-ABL fusion was either acquired through the clonal progression of the initial clone carrying described cytogenetic aberrations, or two distinct clones were present at diagnosis. In order to define the cytogenetic profile of CML samples we analyzed an additional number of 12 patient samples taken at diagnosis, as well as 37 samples taken after treatment with tyrosine kinase inhibitors. In 35.5% of the patients we could detect cytogenetic lesions, consisting of 28 deletions, 9 gains and 1 UPD. The most recurrent deletions were in the breakpoint region of the BCR (n=3) and ABL (n=5). Deletions of 13q (4 events) defined two common deleted regions (CDR) overlapping with previously defined CDR in other myeloproliferative neoplasms and chronic lymphoid leukemia. Other set of deletions clustered on chromosome 7p targeting 5 genes, among which was ABCB5, a member of ATP-binding cassette transporter family. One of the patients was also found to be a carrier of 11pUPD. In order to characterize the target gene within the UPD region we performed whole exome next generation sequencing of this sample and found 9 candidate mutations which are currently evaluated. Compared to other myeloproliferative neoplasms, CML exhibits low cytogenetic complexity. This is demonstrated by the fact that only 35.5% of patients have chromosomal aberrations in addition to t(9;22). Our data indicates that the majority of aberrations in CML occur after t(9;22) during the clonal evolution of the leukemic cells. Most of these aberrations disappear after treatment with tyrosine kinase inhibitors. However in a proportion of patients clonal hematopoiesis persists, even after successful targeting of BCR-ABL positive cells. In these patients imatinib therapy does not restore polyclonal hematopoiesis despite the BCR-ABL remission. The persistence of a clone prone to accumulation of other mutations can potentially lead to induction of other hematological phenotypes. Disclosures: No relevant conflicts of interest to declare.

Abstract 4099 Deletions on chromosome 20q (del20q) are among the most frequent cytogenetic aberrations in myeloid disorders. Mapping of common deleted regions (CDRs) has the potential to link often large deletions to single gene defects. In the past years 20q CDRs from several patient cohorts have been reported, most of them combining patients with different myeloid malignancies, such as myeloproliferative neoplasms (MPN), chronic myeloid leukemia and myelodysplastic syndrome. However, it is not clear whether the same genes within del20q are relevant for pathogenesis in the different myeloid diseases. We aimed to delineate a common deleted region exclusively based on a substantial number of MPN patients. Using a copy number assay, we screened for del20q in a total of 822 MPN patients combined from three independent MPN patient cohorts. Del20q was present in granulocyte DNA from 11 patients (1.1%). We consequently mapped the 11 deletions using Affymetrix 6.0 microarray karyotyping, resulting in a common deleted region of 6.4 Mb size, comprising 82 transcribed genes. In a next step, we aimed to investigate the remaining undeleted allele for the presence of somatic mutations. We applied a next generation sequencing approach and sequenced 774 coding exons within our del20q CDR in 11 patients with del20q. We prepared a DNA pool containing equal amounts of granulocyte DNA from each patient and amplified 774 exons in separate PCR reactions. The PCR reactions were pooled, concatemerized by ligation, refragmented, and prepared for a single-end 36bp sequencing read on a Genome Analyzer IIx (Illumina). The single-end read delivered a coverage between 1000- and 5000-fold per exonic base. We identified five putative variants in the genes SGK2, SEMG1, SEMG2, WFDC9 and SLC13A3 that were non-synonymous and were not annotated in any of the public SNP databases. Further validation in the single patients using classical capillary sequencing identified heterozygous calls in SGK2, SEMG1 and SEMG2 as false positives. The putative variants in WFDC9 (position chr20:43670771; G/A; NCBI36/hg18) and SLC13A3 (position chr20:44654462; C/G; NCBI36/hg18) could be confirmed in granulocyte DNA from a subset of the del20q patients. However, the variants could also be detected in DNA from a control non-myeloid tissue indicating their germ line origin. The variants were present in healthy individuals at variable frequencies, identifying them as novel SNPs presumably not involved in MPN pathogenesis. From the absence of mutations on the undeleted allele in patients with del20q we can conclude that haploinsufficiency is the most likely functional consequence of tumor suppressor inactivation within the del20q CDR in myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

Abstract 998 Chromosomal deletions are frequent cytogenetic defects in hematological malignancies. Common deleted regions (CDRs) defined by the minimal physical overlap of all deletion events are thought to harbor tumor suppressors relevant for pathogenesis. Haploinsufficiency is likely to be a common consequence of chromosomal deletions affecting the function of tumor suppressors within the deleted locus. The use of RNA interference (RNAi) offers a unique opportunity to mimick haploinsufficiency by partial knock-down of the gene transcripts. Since deletions are usually large containing a high number of candidate genes, we aimed to develop a screening method targeting several candidates at once and assaying for tumor suppressor features in a pool of knock-downs. As a model for our approach we selected a CDR on chromosome 20q frequently found clonal in myeloid diseases. We established a screen for knock-downs causing cytokine hypersensitivity. This CDR (physical position chr20:38.7- 42.2) spans 3.5Mb and contains 16 genes (MAFB-JPH2). We cloned 3 short hairpin RNAs (shRNAs) for each mouse homologue of the 16 target genes into the pLKO.2 lentiviral vector. The 48 shRNA constructs were “bar-coded” with different, unique 24bp DNA bar-codes, that can be identified and quantified using the microbead-based xMAP technology (Luminex). We lentivirally delivered the constructs independently into the erythropoietin (Epo) dependent murine cell line Baf3/EpoR, pooled the individual knock-downs in equal amounts and assayed for cytokine hypersensitivity based proliferation advantage under stringent Epo concentrations. Applying a scoring system based on the relative increase/decrease of the individual bar-codes in the pool over time, we could identify the knock-down of topoisomerase 1 (Top1) to induce Epo-concentration dependent outgrowth. Two different Top1 shRNA constructs succeeded in consistently mediating proliferative advantage in three biological replicates. We set up a validation experiment pooling Top1 knock-down cells with control cells (bar-coded, no shRNA) in a 1:1 ratio and could observe significant dominance of the Top1 knock-down cells establishing after 10–15 days in culture and increasing over time (p Disclosures: No relevant conflicts of interest to declare.