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The transcription factor C/EBPa initiates the neutrophil gene expression program in the bone marrow (BM). Knockouts of the Cebpa gene or its +37kb enhancer in mice show 2 major findings: (1) neutropenia in BM and blood; (2) decrease in long-term hematopoietic stem cell (LT-HSC) numbers. Whether the latter finding is cell-autonomous (intrinsic) to the LT-HSCs or an extrinsic event exerted on the stem cell compartment remained an open question. Flow cytometric analysis of the Cebpa +37kb enhancer knockout model revealed that the reduction in LT-HSC numbers observed was proportional to the degree of neutropenia. Single-cell transcriptomics of wild-type (WT) mouse BM showed that Cebpa is predominantly expressed in early myeloid-biased progenitors but not in LT-HSCs. These observations suggest that the negative effect on LT-HSCs is an extrinsic event caused by neutropenia. We transplanted whole BMs from +37kb enhancer-deleted mice and found that 40% of the recipient mice acquired full-blown neutropenia with severe dysplasia and a significant reduction in the total LT-HSC population. The other 60% showed initial signs of myeloid differentiation defects and dysplasia when they were sacrificed, suggesting they were in an early stage of the same pathological process. This phenotype was not seen in mice transplanted with WT BM. Altogether, these results indicate that Cebpa enhancer deletion causes cell-autonomous neutropenia, which reprograms and disturbs the quiescence of HSCs, leading to a systemic impairment of the hematopoietic process.

Acute myeloid leukemia (AML) with inv(3)/t(3;3)(q21q26) is a distinct World Health Organization recognized entity, characterized by its aggressive course and poor prognosis. In this subtype of AML, the translocation of a GATA2 enhancer (3q21) to MECOM (3q26) results in overexpression of the MECOM isoform EVI1 and monoallelic expression of GATA2 from the unaffected allele. The full-length MECOM transcript, MDS1-EVI1, is not expressed as the result of the 3q26 rearrangement. Besides the classical inv(3)/t(3;3), a number of other 3q26/MECOM rearrangements with poor treatment response have been reported in AML. Here, we demonstrate, in a group of 33 AML patients with atypical 3q26 rearrangements, MECOM involvement with EVI1 overexpression but no or low MDS1-EVI1 levels. Moreover, the 3q26 translocations in these AML patients often involve superenhancers of genes active in myeloid development (eg, CD164, PROM1, CDK6, or MYC). In >50% of these cases, allele-specific GATA2 expression was observed, either by copy-number loss or by an unexplained allelic imbalance. Altogether, atypical 3q26 recapitulate the main leukemic mechanism of inv(3)/t(3;3) AML, namely EVI1 overexpression driven by enhancer hijacking, absent MDS1-EVI1 expression and potential GATA2 involvement. Therefore, we conclude that both atypical 3q26/MECOM and inv(3)/t(3;3) can be classified as a single entity of 3q26-rearranged AMLs. Routine analyses determining MECOM rearrangements and EVI1 and MDS1-EVI1 expression are required to recognize 3q-rearranged AML cases.

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or β CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFβR3 and aberrant TGFβ1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFβ pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.

Purpose: Detection of leptomeningeal metastasis is hampered by limited sensitivities of currently used techniques: MRI and cytology of cerebrospinal fluid (CSF). Detection of cell-free tumor DNA in CSF has been proposed as a tumor-specific candidate to detect leptomeningeal metastasis at an earlier stage. The aim of this study was to investigate mutation and aneuploidy status in CSF-derived cell-free DNA (cfDNA) of patients with breast cancer with a clinical suspicion of leptomeningeal metastasis. Experimental Design: cfDNA was isolated from stored remnant CSF and analyzed by targeted next-generation sequencing (NGS; n = 30) and the modified fast aneuploidy screening test-sequencing system (mFAST-SeqS; n = 121). The latter method employs selective amplification of long interspaced nuclear elements sequences that are present throughout the genome and allow for fast and cheap detection of aneuploidy. We compared these results with the gold standard to diagnose leptomeningeal metastasis: cytology. Results: Leptomeningeal metastasis was cytology proven in 13 of 121 patients. Low DNA yields resulted in insufficient molecular coverage of NGS for the majority of samples (success rate, 8/30). The mFAST-SeqS method, successful in 112 of 121 (93%) samples, detected genome-wide aneuploidy in 24 patients. Ten of these patients had cytology-proven leptomeningeal metastasis; 8 additional patients were either concurrently diagnosed with central nervous system metastases by radiological means or developed these soon after the lumbar puncture. The remaining six cases were suspected of leptomeningeal metastasis, but could not be confirmed by cytology or imaging. Aneuploidy was associated with development of leptomeningeal metastasis and significantly worse overall survival. Conclusions: Aneuploidy in CSF-derived cfDNA may provide a promising biomarker to improve timely detection of leptomeningeal metastasis.

In acute myeloid leukemia (AML) with inv(3)(q21;q26) or t(3;3)(q21;q26), a translocated GATA2 enhancer drives oncogenic expression of EVI1. We generated an EVI1-GFP AML model and applied an unbiased CRISPR/Cas9 enhancer scan to uncover sequence motifs essential for EVI1 transcription. Using this approach, we pinpointed a single regulatory element in the translocated GATA2 enhancer that is critically required for aberrant EVI1 expression. This element contained a DNA-binding motif for the transcription factor MYB, which specifically occupied this site at the translocated allele and was dispensable for GATA2 expression. MYB knockout as well as peptidomimetic blockade of CBP/p300-dependent MYB functions resulted in downregulation of EVI1 but not of GATA2. Targeting MYB or mutating its DNA-binding motif within the GATA2 enhancer resulted in myeloid differentiation and cell death, suggesting that interference with MYB-driven EVI1 transcription provides a potential entry point for therapy of inv(3)/t(3;3) AMLs. Significance: We show a novel paradigm in which chromosomal aberrations reveal critical regulatory elements that are nonfunctional at their endogenous locus. This knowledge provides a rationale to develop new compounds to selectively interfere with oncogenic enhancer activity. This article is highlighted in the In This Issue feature, p. 2659

Myocardial infarction is a leading cause of mortality. While advances in the acute treatment have been made, the late-stage mortality is still high, driven by an incomplete understanding of cardiac remodeling processes1,2. Here we used single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of different physiological zones and timepoints of human myocardial infarction and human control myocardium to generate an integrative high-resolution map of cardiac remodeling. This approach allowed us to increase spatial resolution of cell-type composition and provide spatially resolved insights into the cardiac transcriptome and epigenome with identification of distinct cellular zones of injury, repair and remodeling. We here identified and validated mechanisms of fibroblast to myofibroblast differentiation that drive cardiac fibrosis. Our study provides an integrative molecular map of human myocardial infarction and represents a reference to advance mechanistic and therapeutic studies of cardiac disease.

Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.

Summary Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN., Graphical Abstract, Highlights Combinatorial CSF3R and RUNX1 mutations seen in SCN-AML do not result in AML in mice An additional mutation in Cxxc4 causes AML development in CSF3R/RUNX1 mutant mice Mutant CXXC4 protein is more stable than wild-type and reduces TET2 protein levels CXXC4 mutations are also found in de novo AML patients, Olofsen et al. show that acquisition of a mutation in Cxxc4 results in increased CXXC4 protein levels, reduced TET2 protein, increased inflammatory signaling, and leukemic progression of a CSF3R/RUNX1 mutant mouse model of severe congenital neutropenia (SCN).

The tendency of 5-methylcytosine (5mC) to undergo spontaneous deamination has had a major role in shaping the human genome, and this methylation damage remains the primary source of somatic mutations that accumulate with age. How 5mC deamination contributes to cancer risk in different tissues remains unclear. Genomic profiling of 3 early-onset acute myeloid leukemias (AMLs) identified germ line loss of MBD4 as an initiator of 5mC-dependent hypermutation. MBD4-deficient AMLs display a 33-fold higher mutation burden than AML generally, with >95% being C>T in the context of a CG dinucleotide. This distinctive signature was also observed in sporadic cancers that acquired biallelic mutations in MBD4 and in Mbd4 knockout mice. Sequential sampling of germ line cases demonstrated repeated expansion of blood cell progenitors with pathogenic mutations in DNMT3A, a key driver gene for both clonal hematopoiesis and AML. Our findings reveal genetic and epigenetic factors that shape the mutagenic influence of 5mC. Within blood cells, this links methylation damage to the driver landscape of clonal hematopoiesis and reveals a conserved path to leukemia. Germ line MBD4 deficiency enhances cancer susceptibility and predisposes to AML.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic–stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

Bone marrow fibrosis (BMF) develops in various hematological and non-hematological conditions and is a central pathological feature of myelofibrosis. Effective cell-targeted therapeutics are needed, but the cellular origin of BMF remains elusive. Here, we show using genetic fate tracing in two murine models of BMF that Gli1(+) mesenchymal stromal cells (MSCs) are recruited from the endosteal and perivascular niche to become fibrosis-driving myofibroblasts in the bone marrow. Genetic ablation of Gli1(+) cells abolished BMF and rescued bone marrow failure. Pharmacological targeting of Gli proteins with GANT61 inhibited Gli1(+) cell expansion and myofibroblast differentiation, and attenuated fibrosis severity. The same pathway is also active in human BMF and Gli1 expression in BMF significantly correlates with the severity of the disease. In addition, GANT61 treatment reduced the myofibroblastic phenotype of human MSCs isolated from patients with BMF, suggesting that targeting of Gli proteins could be a relevant therapeutic strategy.

Introduction Transcriptional deregulation is a central event in the development of acute myeloid leukemia (AML), with most mutations occurring in genes related to transcription, chromatin regulation and DNA methylation. Furthermore, alterations involving cis-regulatory elements have been shown to play a critical role in aberrant gene expression in AML. Genetic variation in cis-regulatory regions usually involves a single allele, which results in differential expression of the two alleles. This phenomenon, termed allele-specific expression (ASE), is therefore an accurate marker for cis-regulatory variation (Pastinen, 2010). We propose that a systematic study of genes with aberrant ASE in AML may uncover aberrantly expressed genes caused by abnormalities in cis-regulatory elements. Therefore we aim to 1) chart the landscape of ASE in AML, 2) establish a link between relevant ASE events and AML subtypes, and 3) investigate the mechanisms driving ASE. Methods We performed whole exome sequencing (WES) and RNA-seq on leukemic blasts from 168 de novo AML patients, representing all major subtypes of the disease. Combining both datasets, we assessed ASE in every gene with informative (non-homozygous) single nucleotide variants (SNVs). Results Patients had a median of 37 genes with ASE, several of which were recurrently detected across multiple patients. To shorten the gene list we selected for this study genes known to be involved either in cancer or in myeloid development. The gene most commonly found to show ASE (53/140 cases with SNVs) was GATA2, which encodes a transcription factor crucial for proliferation and maintenance of hematopoietic stem cells with a known involvement in AML. Interestingly, integration with molecularly defined classification of AML revealed that all cases (n=17) with biallelic CEBPA mutations exhibited GATA2 ASE (p-value = 6.00·10-7, Fisher's test). Biallelic CEBPA mutations (CEBPA DM) identify an AML subtype with favorable clinical outcome and frequently co-occur with GATA2 mutations (Greif PA, 2012), pointing to a functional connection between these two genes. Indeed, 44% of the cases in our cohort exhibited a GATA2 mutation, and 27% carried a second, subclonal mutation in the same gene. Importantly, in cases where a GATA2 mutation was found, the mutant allele was always preferentially expressed. These findings were validated in the TCGA dataset, where all four CEBPA DM patients with informative SNVs in GATA2 exhibited GATA2 ASE. Although GATA2 ASE was present in other AML subtypes, none of these subtypes showed a significant association with this finding. Patients with a t(8;21) rearrangement (n=5), which represses CEBPA expression, did not exhibit GATA2 ASE, and we only observed GATA2 ASE in 4 out of 8 CEBPA silenced leukemias (Wouters BJ, 2007). Altogether, this demonstrates the uniqueness of the 1-to-1 relationship between CEBPA DM and GATA2 ASE, and excludes a causative role for inactive CEBPA protein in mediating mono-allelic expression of GATA2. The average expression of GATA2 in CEBPA DM patients was comparable to other AMLs, even in cases with monoallelic GATA2 expression. This suggests that a) ASE was achieved by repression of one allele rather than dramatically increased expression of the other, b) there was a compensation of the non-repressed allele. DNA methylation analysis of the GATA2 promoter did not reveal methylation-mediated gene silencing of the repressed allele. The long-distance +77 kb GATA2 enhancer appears to be involved in ASE, as RNA read-through levels at the enhancer were significantly different in CEBPA DM AMLs (p-value < 10-4, Wald test) in an allele-specific manner. The involvement of the enhancer was further confirmed by differences in H3K27ac levels between the two alleles. Conclusions An unbiased screen of 168 de novo AML cases revealed that all patients (n=17) with CEBPA biallelic mutations display GATA2 ASE. GATA2 mutations were found in 8 of the 17 cases, always in the allele that is preferentially expressed. Since GATA2 ASE is present in all CEBPA DM and GATA2 mutations only in a fraction, we hypothesize that GATA2 ASE is acquired first and mutations are only selected if they occur in the expressed allele. Moreover, given that other subgroups with CEBPA abnormalities do not show a similar pattern, we propose that ASE of GATA2 is not a consequence of CEBPA mutations, but rather a requirement for the development of AML in these patients. Disclosures No relevant conflicts of interest to declare.

The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)–rearranged leukemias are EVI1pos. High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1pos MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1neg MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1pos HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9–transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1neg. Moreover, shRNA-mediated knockdown of Evi1 in an Evi1pos MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1pos MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.

Introduction Acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21;q26) overexpress EVI1 and have a very poor prognosis. EVI1 is part of the MECOM (MDS1-EVI1-Combination) locus from which MDS1-EVI1 and EVI1 can be transcribed from two different promoters. Although EVI1 is expressed at high levels, MDS1-EVI1 is absent or expressed at very low levels in inv(3)/t(3;3)-AMLs. Aberrant EVI1 expression in these leukemias is driven by the long-distant GATA2 enhancer, translocated from 3q21 to EVI1 at 3q26 (Gröschel et al, 2014). As a result of this GATA2 enhancer hijack by EVI1, GATA2 is switched off on the rearranged allele, resulting in mono-allelic and low GATA2 expression. We hypothesize that leukemic transformation of inv(3)/t(3;3)-AMLs is driven by EVI1 overexpression and by low GATA2 and that these leukemias are marked by the absence of MDS1-EVI1 expression. We previously reported about a group of AML patients that presented with complex rearrangements of 3q26 (refer to as variant-3q26-AML) with frequent MECOM involvement and very poor survival (Lugthart et al, 2010). Here we address the questions if these variant-3q26-AMLs 1) overexpress EVI1 by enhancer hijacking, 2) are marked by absent MDS1-EVI1 and 3) express low levels of GATA2. Accordingly, the variant-3q26-AMLs should be classified as inv(3)/t(3;3)-AMLs. Results We identified 37 variant-3q26-AMLs with MECOM rearrangement as determined by Fluorescent in-situ hybridization (FISH). RNA-seq of these AMLs revealed EVI1 overexpression but also demonstrated the absence of MDS1-EVI1 in 90% of patient samples. Applying 3q-capture DNA-seq, we found that in 2 cases the patient cells harboured a "hidden" inv(3)(q21q26) with involvement of the GATA2 enhancer. In 7 cases recurrent 3q26/EVI1 translocations were identified, e.g. t(2;3)(p21;q26), t(3;8)(q26;q24), t(3;7)(q26;q11), involving the THADA, MYC or CDK6 loci respectively as previously described. Interestingly, we identified new translocations to the EVI1 locus in 13 AMLs, including a t(3;6)(q26;q21) and a t(3;4)(q26;p15), involving the CD164, and PROM1 loci respectively. In these samples we find clearly skewed expression of these genes to one allele, suggestively caused by the rearrangement and enhancer hijacking. CD164 plays a key role in adhesion, proliferation and migration of CD34+ hematopoietic progenitor cells (Watt et al, 2000). PROM1 (CD133) is expressed in human hematopoietic stem and progenitor cells and is thought to be involved in maintaining stem cell properties by suppressing differentiation (Bauer et al, 2008). We argue that EVI1 overexpression in these variant-3q26-AMLs is driven by hijacking enhancers of genes that are normally active in myeloid progenitors. In most of the patients the translocation breakpoints are in between the promoters of MDS1 and EVI1, explaining absence of MDS1-EVI1 expression. In addition, analysis of SNP-array data of these patients (N=33) showed Copy Number Loss (CNL) of the MDS1 exon(s) and not the EVI1 exons in at least 5 cases. Together these data suggest the importance of MDS1-EVI1 loss in 3q26-AMLs. Furthermore we wondered whether low GATA2 expression is an important event in variant-3q26-AMLs. Similar to inv(3)/t(3;3)-AMLs (Gröschel et al, 2014), RNA-seq revealed that the GATA2 expression was on average a two-fold lower in the variant-3q26-AMLs (N=37), compared to non-3q26 rearranged AMLs (N=114). Surprisingly, SNP-array analysis in 26 variant-3q26-AMLs revealed CNLs of GATA2 and/or its enhancer in 7 patients. Detailed SNP analysis in GATA2 exons by combined 3q-capture DNA-seq and RNA-seq uncovered another 7 cases with mono-allelic GATA2 expression or skewing to expression of one allele (allele_freq Conclusion Given their complex karyotype, variant-3q26-AMLs are often not recognised as 3q26/EVI1 AMLs. Although the exact mechanism remains elusive, the overall effect seems to be alike. EVI1 overexpression, potentially driven by enhancer hijacking of genes that are active in early myeloid progenitors, combined with absent MDS1-EVI1 and mono-allelic/low GATA2 expression results in AML with very poor survival. Given these data we believe variant-3q26-AMLs genocopy inv(3)/t(3;3)-AMLs and should be classified as such. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Myeloid malignancies bearing chromosomal inv(3)/t(3;3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3;3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3;3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3;3) acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome cases, suggesting recognition of inv(3)/t(3;3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signaling mutations may provide a target for a rational treatment strategy in this high-risk patient group.

Mutations in splice factor (SF) genes occur more frequently in myelodysplastic syndromes (MDS) than in acute myeloid leukemias (AML). We sequenced complementary DNA from bone marrow of 47 refractory anemia with excess blasts (RAEB) patients, 29 AML cases with low marrow blast cell count, and 325 other AML patients and determined the presence of SF-hotspot mutations in SF3B1, U2AF35, and SRSF2. SF mutations were found in 10 RAEB, 12 AML cases with low marrow blast cell count, and 25 other AML cases. Our study provides evidence that SF-mutant RAEB and SF-mutant AML are clinically, cytologically, and molecularly highly similar. An integrated analysis of genomewide messenger RNA (mRNA) expression profiling and DNA-methylation profiling data revealed 2 unique patient clusters highly enriched for SF-mutant RAEB/AML. The combined genomewide mRNA expression profiling/DNA-methylation profiling signatures revealed 1 SF-mutant patient cluster with an erythroid signature. The other SF-mutant patient cluster was enriched for NRAS/KRAS mutations and showed an inferior survival. We conclude that SF-mutant RAEB/AML constitutes a related disorder overriding the artificial separation between AML and MDS, and that SF-mutant RAEB/AML is composed of 2 molecularly and clinically distinct subgroups. We conclude that SF-mutant disorders should be considered as myeloid malignancies that transcend the boundaries of AML and MDS.

Background Deletion of chromosome 17p (del17p) is detected in 10% of multiple myeloma (MM) patients at diagnosis and is associated with both a dismal prognosis and increased prevalence after treatment. Even though this suggests that it might be a driver of disease progression, relatively little is known about the genomic landscape of these tumors. With this study, we aimed to identify recurrent aberrations in a large cohort of del17p MM patients and to assess their prognostic value within this patient group. Methods The study design consisted of a discovery phase and a validation phase. For the discovery phase, 44 newly diagnosed MM (NDMM) patients were included with a del17p in at least 50% of plasma cells, as detected with fluorescent in situ hybridization (FISH). From 12 del17p patients, 2 or 3 tumor samples were available to analyze clonal evolution during disease progression. DNA was isolated from peripheral blood and CD138+ enriched bone marrow mononuclear cells, followed by a custom capture of the whole exome, Chr17p and the IgH, Igk, IgL and MYC regions (SeqCap EZ Exome Plus, Nimblegen) and paired-end sequencing. Significantly mutated genes were determined with MutSigCV and significantly deleted or amplified genomic regions with GISTIC2. Single nucleotide variants (SNVs) in TP53, FAM46C, KRAS, NRAS, DIS3 and BRAF were validated using a custom amplicon panel (TruSeq Custom Amplicon Assay v1.5, Illumina), followed by deep sequencing on a MiSeq System. Findings were validated in a cohort of 463 NDMM patients of the UK Myeloma XI trial, from which whole exome sequencing data were available for paired tumor and germline DNA (Walker et al. - J Clin Oncol 2015). All samples in both the discovery and the validation cohort were reanalyzed with one bioinformatic pipeline, from alignment to variant calling. The copy number status of TP53 was determined with Sequenza, followed by selection of del17p samples after manual inspection of the results. From a third cohort of 233 MM patients with progressive disease (PDMM) treated at the University of Arkansas for Medical Sciences (UAMS), 406 cancer-related genes were paired-end sequenced from tumor DNA with the FoundationOne Heme assay (He et al. - Blood 2016). Results In the discovery cohort, we identified a commonly deleted region (CDR) on Chr17p of 235 kb, in which one or more somatic, nonsilent aberrations (SNSA) in TP53 were detected in 25/44 (57%) patients (adj. p In the UK Myeloma XI cohort, we detected a del17p in 32/463 (7%) of patients. Of these, 15/32 (47%) had an SNSA in the remaining allele. Follow-up data were available of 73/76 del17p NDMM patients from both cohorts (n=46 transplant-eligible (TE), n=27 nontransplant-eligible (NTE)), with a median follow-up time of 17 months. TP53 mutated patients had a worse overall survival (OS) than TP53 wildtype patients (p=0.02), of which the strongest effect on OS was seen of TP53 missense mutations (p We went on to look in the FoundationOne cohort of PDMM patients and detected a del17p in 37/233 (16%) of patients and an SNSA in TP53 in 25/37 (68%) of del17p PDMM patients, which is a higher rate than in the 2 NDMM cohorts. Conclusions (1) TP53 is somatically mutated in half of del17p MM patients at diagnosis, validated in 2 independent cohorts, and this percentage is higher in del17p MM patients at progression. (2) Particularly TP53 missense mutations have a significant, negative impact on both PFS and OS in del17p MM patients. (3) The rest of the genomic landscape of del17p MM is characterized by significant mutations in FAM46C and KRAS, as well as deletions of RB1, TRAF3 and FAM46C. Disclosures Ashby: University of Arkansas for Medical Sciences: Employment. He:Foundation Medicine, Inc: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Waage:Novartis, Amgen, Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Celgene: Consultancy, Honoraria. Avet-Loiseau:celgene: Consultancy; sanofi: Consultancy; janssen: Consultancy; amgen: Consultancy. Jackson:Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau. Morgan:Univ of AR for Medical Sciences: Employment; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Meyers: Consultancy, Honoraria. Sonneveld:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding.

DNA methylation patterns are frequently dysregulated in cancer, although little is known of the mechanisms through which specific gene sets become aberrantly methylated. The ecotropic viral integration site 1 (EVI1) locus encodes a DNA binding zinc-finger transcription factor that is aberrantly expressed in a subset of acute myeloid leukemia (AML) patients with poor outcome. We find that the promoter DNA methylation signature of EVI1 AML blast cells differs from those of normal CD34+ bone marrow cells and other AMLs. This signature contained 294 differentially methylated genes, of which 238 (81%) were coordinately hypermethylated. An unbiased motif analysis revealed an overrepresentation of EVI1 binding sites among these aberrantly hypermethylated loci. EVI1 was capable of binding to these promoters in 2 different EVI1-expressing cell lines, whereas no binding was observed in an EVI1-negative cell line. Furthermore, EVI1 was observed to interact with DNA methyl transferases 3A and 3B. Among the EVI1 AML cases, 2 subgroups were recognized, of which 1 contained AMLs with many more methylated genes, which was associated with significantly higher levels of EVI1 than in the cases of the other subgroup. Our data point to a role for EVI1 in directing aberrant promoter DNA methylation patterning in EVI1 AMLs.

Shwachman-Diamond Syndrome (SDS) is a congenital bone marrow failure disorder characterized by neutropenia and predisposition to leukemia. SDS is associated with loss-of-function mutations in the SBDSgene, involved in ribosome biogenesis, but the cellular and molecular events driving neutropenia in SDS remain poorly defined, largely due to a lack of mammalian disease models recapitulating the hematopoietic features of SDS. To achieve deletion of Sbds in early hematopoietic/myeloid progenitors, we generated Cebpacre/+Sbdsf/f R26 EYFP/+ mice (Sbds Δ/Δ), which were non-viable. Analysis of E14.5 embryos demonstrated global conservation of the hematopoietic hierarchy in Sbds Δ/Δ embryos compared to Cebpacre/+ R26 EYFP/+ controls (Sbds +/+). Functional relevance of Sbds deletion in Cebpa+progenitors was tested by transplanting fetal liver cells from E14.5 Sbds Δ/Δ or Sbds +/+ embryos into lethally irradiated B6.SJL mice. Deficiency of Sbds in different EYFP+ hematopoietic subsets was confirmed by qPCR (log2 fold change: LKS -2.25, CMP -9.42, GMP -9.1 and neutrophils -7.29). Mice transplanted with Sbds Δ/Δ cells developed profound neutropenia (log2FC: -2.56; p=0.002, n=7), which was stable during the time of analysis (16 weeks). FACS and morphological studies of bone marrow EYFP+ cells demonstrated increased frequencies of early progenitor populations, recapitulating the left-shifted hematopoiesis observed in human SDS patients (Dror et al., Ann N Y Acad Sci 2011), with a pronounced accumulation of cKitint Gr1low EYFP+ myelocytes-metamyelocytes (MC-MMs) (frequency of EYFP+ cells: Sbds +/+ 15.3±1.7 %, Sbds Δ/Δ 39.4±1.1 %; p= 8.7x10-8) and a marked decrease in Gr1+ Mac1+ cells (Sbds +/+: 54.5±7.5 %, Sbds Δ/Δ: 22.3±2.8 %; p= 1.6x10-4), indicating that neutropenia was caused by disrupted lineage progression from MC-MMs to neutrophils. In line with this, whole transcriptome analysis of prospectively isolated EYFP+ MC-MMs (RNA-seq, n=4) revealed enrichment for hematopoietic stem and progenitor cell signatures in Sbds Δ/Δ recipients, while myeloid signatures were enriched in Sbds +/+ mice (GSEA). Transcript analysis further showed reduced expression of granule components produced at the MC-MM stage, such as Ltf, Mmp8, Mmp9. As expected, reduced expression of Sbds (FDR=6.6x10-9, log2FC=-2.67) and dysregulation of ribosome proteins (RP) production were observed, with increased expression of over 60 RP genes in SbdsΔ/Δ MC-MMs. To investigate the molecular events underlying impaired lineage progression, we focused on p53 activation, a common mechanism of tissue failure in ribosomopathies. P53 protein was significantly and selectively accumulated in EYFP+ MC-MMs of Sbds Δ/Δ recipients as measured by FACS (Mean Fluorescence Intensity FC: 1.8; p=0.01), with increased expression of p53 targets such as Cdkn1a (p21), Bbc3 (PUMA) and Bax. This was accompanied by increased apoptosis (annexin V FACS analysis) in both MC-MMs and mature neutrophils (FC: 1.36; p=0.02 and FC=2.44; p=1.6x10-4, respectively). However, pharmacological inhibition of the p53 pathway by pifithrin-α (2 months, i.p.) failed to rescue neutropenia, suggesting that alternative mechanisms, such as disrupted expression of transcription factors, may contribute to neutropenia in SDS. Transcript analysis of Sbds-deficient MC-MMs revealed downregulation of Rara (Retinoic acid receptor α) and its target Cdkn1b (p27), implicated in cell cycle arrest and terminal granulopoiesis (Walkley et al., Blood 2004). Congruent with this finding, Ki67 staining showed that end-stage granulocytes from Sbds Δ/Δ recipients fail to exit cell cycle. In conclusion, we established a mouse model of neutropenia caused by Sbds deficiency in the hematopoietic system, providing experimental support for a direct causative link between Sbds deficiency and neutropenia in mammals. The data reveal a previously unanticipated, selective dependency of late myeloid cells on this ubiquitous protein, while its deficiency spares the function of rapid cycling hematopoietic progenitors. Mechanistically, disrupted expression transcription factors governing terminal myeloid differentiation may be implicated. We anticipate the mouse model to be a valuable tool in further dissecting the molecular pathogenesis of SDS and enabling preclinical studies for disease modulation. Disclosures No relevant conflicts of interest to declare.

Introduction: Acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21;q26) [inv(3)/t(3;3)] is associated with aberrant expression of the stem cell regulator EVI1 and dismal prognosis. Recently, we and others (Gröschel et al, Cell, 2014; Yamazaki et al, Cancer Cell, 2014) have shown, as a consequence of the inv(3)/t(3;3) rearrangements, that the proto-oncogene EVI1 is activated upon the structural repositioning of a distal GATA2 enhancer from 3q21 to EVI1, coinciding with loss of GATA2 expression from the rearranged allele. Notably, GATA2 deficiency has been shown to impair hematopoietic stem cell frequency and function (Lim et al, J. Clin. Invest., 2012) and Evi1 activation in inv(3) murine models is followed by leukemia onset after a long latency of 6 months (Yamazaki et al). We therefore hypothesize that additional cooperating genetic lesions, other than EVI1 activation and GATA2 deregulation, are required for full leukemic transformation. We sought to extend the molecular characterization of inv(3)/t(3;3) myeloid malignancies through next-generation sequencing. Methods: We selected 32 AML (including cell lines MUTZ-3 and UCSD-AML1), 4 CML-BC (including cell lines HNT-34 and MOLM-1), and 5 myelodysplastic syndrome (MDS) cases with inv(3)/t(3;3) rearrangements and performed whole transcriptome sequencing (n=40) as well as whole exome sequencing (WES, n=10) on the Illumina HiSeq 2500. For WES germline control we used DNA from cultured CD3+ T-cells from diagnostic bone marrow specimens. Variants were examined when recurrently detected in more than two patients or previously associated with leukemogenesis or cancer pathogenesis. Gene expression profiles (GEP) were constructed for 24 cases for subsequent analyses. Results: All examined variants, validated by Sanger sequencing, were assigned to mutational categories to discern patterns of mutations (Figure 1A). We observed that all 41 cases acquired at least one mutation belonging to the mutational categories reported as relevant for leukemia pathogenesis. Importantly, all AML and CML-BC patients, as well as 4 out of 5 MDS patients contained mutations in genes activating RAS/RTK signaling, amounting to 98% of all inv(3)/t(3;3) myeloid malignancies. An aggregate of 74% of all patients harbor mutually exclusive mutations in RAS-pathway associated genes; NRAS(27%), PTPN11(20%), KRAS(11%), and NF1(9%). Additionally, we found mutations in genes affecting signaling pathways involving RAS; FLT3(13%), BCR-ABL(11%), and KIT(2%) (Figure 1B). NPM1, CEBPA, and IDH1/2 mutations were absent in all cases. Furthermore, frequent mutations were found in transcription factor (32%), splice factor (29%), epigenetic modifier (29%), and tumor suppressor (10%) encoding genes (Figure 1A). GATA2 is the most commonly mutated transcription factor in inv(3)/t(3;3) myeloid malignancies (15%). As these mutations were found on the non-rearranged GATA2 allele, only mutant GATA2 is expressed due to GATA2 silencing mediated by the 3q21q26 rearrangement. Novel somatic truncating mutations and copy number loss were detected in the transcription factor FOXP1(10%). Additionally, we observed frequent mutations in the splice factor gene SF3B1(27%) in AML and MDS cases. Finally, the predominant monosomal karyotype within inv(3)/t(3;3) myeloid malignancies is contrasted by the low incidence of TP53 mutations (5%). No mutational pattern alluded to the high coincidence of monosomy 7 (68%), but inv(3)/t(3;3)/-7 cases were characterized by a strong down regulation of the transcription factor CUX1. In an effort to discriminate MDS and AML with inv(3)/t(3;3) we performed cluster and principle component analyses, revealing no cluster formation. Additionally, differential expression analysis was unable to differentiate AML from MDS, indicating strong homogeneity based on GEPs. Conclusion: Myeloid malignancies with inv(3)/t(3;3) harbor a common set of molecular alterations, i.e. EVI1 activation and GATA2 deregulation, conjugated by mutations activating signaling pathways. Mutational patterns and GEPs precluded the discrimination of MDS and AML with inv(3)/t(3;3), supporting the notion that these myeloid disorders should be categorized as AML irrespective of blast count. Exploiting signaling pathways therapeutically may prove a valuable adjunct to the scarce armamentarium of drugs effective in this malignancy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.