Stockholm, Sweden, June 13–16, 2013

Recently, advances in DNA sequencing have led to the identification of recur - Myeloid leukemogenesis rent somatic mutations in AML, but have also demonstrated that individual cas - es are driven by a small number of co-occurring mutations. Our ability to inter - pret these findings and address this important medical need relies on an S1126 improved understanding of the pathways corrupted by such mutations and par - ticularly the basis of the molecular synergy between them. Here, we report that A STUDY OF THE LEUKEMOGENIC INTERACTION BETWEEN MUTANT the two most commonly co-occurring somatic mutations in AML, namely NPM1 NPM1 AND NRASG12D IN CONDITIONAL KNOCK-IN MICE exon 12 mutations (NPM1c) and internal tandem duplications of FLT3 (FLT3- O Dovey 1,* , C Grove 1, J Cooper 1, A Mupo 1, P Wright 2, A Bradley 3, G Vassiliou 1 ITD), cooperate explosively to induce AML. Mice succumbed to AML after a 1Haematological Cancer Genetics, Wellcome Trust Sanger Institute, 2Depart - median of 49 days, and also many cases exhibited loss-of-heterozygosity for ment of Pathology, University of Cambridge, Addenbrooke’s Hospital, 3Mouse Flt3, as seen in human AML. In revealing this striking molecular synergy, our and Zebrafish genetics, Wellcome Trust Sanger Institute, Cambridge, United work offers a basis for the frequent co-occurrence of these two mutations and Kingdom provides a model for the detailed study of AML. To study the combined effects of NPM1c with FLT3-ITD we crossed Very recently, whole genome and targeted sequencing of indi - Aims: Background: conditional Npm1flox-cA/+ with constitutive Flt3ITD/+ to generate Npm1flox- vidual cases of AML have raised the possibility that in many instances as few cA/+; Flt3ITD/+ double heterozygous mice. as three specific mutated may be sufficient to promote leukaemia. Appre - Mice strains have been previously described. ciation of the biological synergy between the complement of leukaemogenic Methods: Mouse blood, femurs and spleens were stained with Gr1-PE and CD11b-FITC, mutations in driving disease progression is key to the development of new treat - B220-APC-Alexa750, CD3-PerCP-Cy5.5 and cKit-APC, run on a BD Fortessa ments. The largest subgroup of AML which constitutes 35% of all cases, name - cytometer and analysed with FlowJo 7.6.5. Bone marrow cells were plated in ly a karyotypically normal sub-group of AML, exhibits a mutation in the Npm1 M3434 and colonies counted after 7-10 days. For re-plating, 30,000 bone mar - (Npm1 c). A humanised conditional knock-in mouse model of the main form of Npm1 c mutations, Type A, has lately been described in which one third of row cells were plated and counted after 8 days. mice developed AML after a long latency (median17.5 months), suggesting a Results: In this study we observed universal and rapid emergence of AML in requirement for cooperating mutations. An insertional mutagenesis screen in Npm1c;FLT3ITD mice. All mice developed AML and became moribund aged 31- the same mice revealed rapid onset AML ~80% of mice (median days 99) with 68 days (Figure 1).Weekly blood showed a progressive increase in blood leuko - activating insertions in in Csf2 , Flt3 , Rasgrp1 or Kras , suggesting that activa - cyte counts in Npm1c/Flt3-ITD mice, to more than 25-fold that of control litter - tion of these downstream pathways are key in leukaemogenesis . Furthermore, mates, whilst hemoglobin and platelet counts were significantly reduced. Flow the aforementioned human sequencing studies, including the first AML genome analysis of blood samples demonstrated, in Npm1c/Flt3-ITD mice, a population to be sequenced, reveal commonly co-occurring mutations in Npm1 and Nras of blast cells and a large number of single Mac1+ precursors. Additionally, we also (Nras G12D , which results in a constitutively active form of the ). Notably, observed an increased number of mature myeloid (Gr1+/Mac1+) cells in only ~25% of mice genetically engineered to harbour the Nras G12D (NRAS LSL- Npm1c/Flt3-ITD mice indicating that any maturation block was incomplete.To G12D ) mutation develop AML. assay their self-renewal potential, bone marrow cells were studied in serial re-plat - ing assays. Npm1c/Flt3-ITD cells gave rise to significantly more colonies than any Aims: Comprehensive analysis of the molecular synergy between the leukae - mogenic mutations, Npm1 cA and NRAS G12D , in the development of AML. other genotype demonstrating their increased self-renewal potential. Interesting - Methods: To analyse the combined effects of Npm1 c with Nras G12D we crossed ly, Npm1c/Flt3-ITD littermates often progressed to AML at different rates or devel - conditional Npm1 flox-cA/+ ; Mx-1 Cre with NRAS LSL-G12D mice to generate oped more/less aggressive disease. To explain this we hypothesized that, as Npm1 flox-cA/+; NRAS LSL-G12D ; Mx-1 Cre double conditional heterozygous mice seen in human AML, LOH for Flt3 may be responsible. We found significant (RNMX mice). Mice were aged until moribund, tissue samples collected and spontaneous loss of the wild-type Flt3 allele in blood samples from Npm1c/Flt3- preserved for histopathology and nucleic acid extraction. Comparative analy - ITD mice and a tendency for higher blood leukocyte counts when LOH was pres - sis of haematopoietic cell compartments in younger mice ( i.e. 4 weeks post ent. LOH was also seen in bone marrow and spleen but not tail DNA, in keeping mutation induction) from combined and singular mutant cohorts was performed with somatic loss of the wild-type allele in leukemic cells. using standard FACS techniques. Similar comparative analyses were per - formed on bone marrow cell extracts on the Lineage –ve transcriptome and on self-renewal, using microarrays and serial re-plating assays respectively. Results: Histopathological analysis of RNMX mice revealed the development of AML with ~80% penetrance. These mice have a median survival of 89 days, much reduced when compared to NRAS G12D/+ (250 days) or Npm1 cA/+ mice (400 days). Peripheral blood counts of aged RNMX mice revealed significant - ly elevated leukocyte counts and reduced platelets. Cells isolated from dis - eased RNMX aged mice tested so far, were all transplantable. Subsequent EXOME analysis of RNMX AMLs so far, have revealed the accrual of yet fur - ther mutations including a particular instance of IDH1 R132Q , synonymous to the Idh1 R132H mutation associated, and known to co-occur with Npm1 cA and NRAS G12D , in human AML. Analysis of younger mice reveals Npm1 cA depend - ent skewing toward the myeloid lineage and a general increase in haematopoi - etic cells concomitant with the Nras G12D mutation. Combined, in RNMX mice, myeloid expansion is responsible for statistically significant increased haematopoietic cellularity. In vitro self-renewal properties of bone marrow cells are also enhanced in RNMX mice compared to other cohorts. Global transcrip - Figure 1. tome analysis has, so far, revealed a previously identified Npm1 cA signature of HOX gene overexpression, absent from NRAS mice, but persistent in the Summary and Conclusions: Recent studies have revealed that AML hetero - RNMX cohort. geneity is derived, to a large extent, from the specific combinations of somatic Summary and Conclusions: These observations in RNMX mice specifically driver mutations. Here, we show that the combination of Npm1c and Flt3-ITD, emphasise the role of combinatorial somatic driver mutations particular to AML is rapidly and universally leukemogenic in knock-in mice These findings are par - and provides a rationale for the co-occurrence of Npm1 cA and Nras G12D muta - ticularly striking in light of the fact that, in isolation, both Npm1c and Flt3-ITD tions (given that, in isolation, Npm1 cA or Nras G12D mutations have relatively mutations have relatively subtle effects on mouse haemopoiesis. subtle effects on mouse haemopoiesis and a prolonged latency/frequency of Our observations emphasize the remarkable complementarity between Npm1c AML). Combined with the observed spontaneous accrual of other known co- and Flt3-ITD. In the context of a stochastic model for AML pathogenesis, this occurring somatic mutations (IDH1), this model presents a valuable tool in potent molecular synergy goes some way towards explaining why NPM1c and studying the pathogenesis and treatment of AML. FLT3-ITD co-occur so frequently and makes the model described here a valu - able tool for the study of the pathogenesis and treatment of AML

S1127 S1128 MOLECULAR SYNERGY BETWEEN MUTANT NUCLEOPHOSMIN AND FLT3-ITD TO DRIVE ACUTE MYELOID LEUKEMIA IN MICE UNDERSTANDING THE PHYSIOLOGICAL FUNCTION OF THE TWO RNA- A Mupo 1,* , L Celani 2, O Dovey 1, J Cooper 1, C Grove 1, R Rad 1, P Sportoletti 2, BINDING MSI1 AND MSI2 INVOLVED IN HETAMOLOGICAL B Falini 2, A Bradley 1, G Vassiliou 1 MALIGNANCIES 1Wellcome Trust Sanger Institute, Hinxton, United Kingdom, 2University of J Hoell 1,* , S Duggimpudi 1, P Muench 2, K Hezaveh 1, C Tusche 2, A McHardy 2, Perugia, Perugia, Italy A Borkhardt 1 1Department of Pediatric Oncology, Hematology and Clinical Immunology, Cen - Background: Acute myeloid leukemia (AML) is the commonest myeloid malig - ter for Child and Adolescent Health, 2Department of Algorithmic Bioinformat - nancy, yet there has been little therapeutic progress for this disease in decades. ics, Heinrich Heine University Duesseldorf, Duesseldorf, Germany

haematologica | 2013; 98(s1) | 465 18 th Congress of the European Hematology Association

Background: Musashi homolog 2 (MSI2) is an RNA-binding protein (RBP) ing compared to MLL-AF9-transduced Hes1 +/+ cells. When infused into irradi - that was first associated with myeloid malignancies when it was shown to form ated syngenic mice, MLL-AF9/Hes1 -/- cells developed leukemia at shorter a fusion protein with HOXA9 in chronic myeloid leukemia (CML). Subsequent - latencies than MLL-AF9/ Hes1 +/+ cells (MLL-AF9/Hes1 -/- , 7-10 weeks, n=18 vs ly it was found that MSI2 expression is highly uP-regulated during CML pro - MLL-AF9/Hes1 +/+ , 10-14 weeks, n=18; P<0.001). Both MLL-AF9/Hes1 -/- and gression into blast crisis. Since then, high levels of MSI2 have been described MLL-AF9/Hes1 +/+ leukemia cells prepared from bone marrow of the first recip - in various malignancies including prostate cancer, pulmonary carcinomas and ients were transplantable and caused leukemia in the secondary recipients. AML, in which it is predictive of unfavorable outcome. When Hes1 was retrovirally re-expressed in MLL-AF9/Hes1 -/- cells, these cells Aims: in mammals is extensively controlled at the post-tran - developed leukemia in recipient mice at longer latencies than mock-transduced scriptional level by RBPs modulating all stages of the mRNA life cycle. This is MLL-AF9/Hes1 -/- cells (Hes1/MLL-AF9, 12 weeks, n=8 vs Mock/MLL-AF9, 5- achieved by covalent binding between RBPs and mRNAs depending on the 7 weeks, n=7 P<0.001). Taken together, these results indicate that Notch sig - unique RNA-recognition element (RRE) of each RBP. Neither the mechanism naling indeed suppresses MLL-AF9-triggered AML development and that Hes1 by which the overexpression of MSI2 contributes to leukemogenesis nor its is a definitive downstream mediator for this Notch function. MLL-AF9/Hes1 -/- physiological function is well understood. By identifying its transcriptome-wide and MLL-AF9/Hes1 +/+ leukemia cells were then compared for mRNA expres - mRNA targets we wish to uncover the physiological function of MSI2. This will sion by cDNA microarray. Among the genes with different expression levels allow us to understand the consequences of its deregulation in malignancies. between MLL-AF9/Hes1 -/- and MLL-AF9/Hes1 +/+ leukemia cells, Flt3 was Methods: We applied PAR-CLIP, a method that was developed to identify the expressed at significantly higher levels in MLL-AF9/Hes1 -/- leukemia cells. It transcriptome-wide RBP binding sites by incorporation of a photoreactive nucle - was also demonstrated that Flt3 was phosphorylated with Flt3 ligand stimula - oside into nascent mRNAs, to MSI2. Next-generation sequencing (NGS) then tion in the MLL-AF9-immortalized cells specifically with the Hes1 -/- background. allows the mapping of the exact crosslink sites by mapping thymidine to cyti - Summary and Conclusions: Canonical Notch signaling serves as a tumor dine transitions (T2C) in the cDNAs of PAR-CLIP libraries. To approach the sev - suppressor in MLL-AF9-induced AML through upregulation of Hes1.Hes1 is an eral tens of millions obtained NGS reads, extensive bioinformatic analysis was essential Notch signaling mediator for AML suppression. At least a part of Hes1 performed followed by biochemical methods to validate the findings. function might be explained by repression of Flt3. Results: Since RBPs within the same protein family often have overlapping mRNA targets, we included MSI1 into our study as it is closely related to and highly co-expressed with MSI2. The roughly 200-300 million NGS reads of S1130 each PAR-CLIP library gave rise to around12,000 single linkage clusters meet - ing stringent quality criteria: (1) more than 25 overlapping reads and (2) at least AML1-ETO COLLABORATES WITH THE TALE GENES one T2C transition in at least 20% of the cluster reads. We found that both MSI1 MEIS1 AND MEIS2 IN INDUCING AML 1,* 1 2 3 1 4 and MSI2 -despite their cytoplasmic localization- bind intronic, exonic and M Vegi , J Klappacher , F Oswald , R Claus , M Mulaw , A Mandoli , 2 4 1 5 6 7 3’UTR mRNAs at roughly equal distribution. We defined the RRE for MSI1 and V Thiel , J Martens , V Rawat , L Quintanilla-Fend , C Plass , K Döhner , 7 8 9 7 1 MSI2 using a motif discovery approach (MEME). In both proteins it consists of H Döhner , W Hiddemann , H Stunnenberg , M Feuring-Buske , C Buske 1 the six nucleotides TTTTAG . As expected -given the shared RRE- the target - Institute for Experimental Cancer Research, CCC and University Hospital of 2 ed genes of MSI1 and MSI2 overlap to a large degree (~75%). We recombi - Ulm, Department of Internal Medicine I, University Hospital of Ulm, Ulm, 3 4 nantly expressed MSI2 to perform biochemical validations. Department of Internal Medicine I, Freiburg, Germany, Department of Molec - analysis of both the RRE and the targeted mRNAs separately indicated a role ular Biology, Nijmegen Centre for Molecular Life Sciences, Nijmegen, Nether - 5 6 for MSI2 in MAPK and TGF-beta signaling. We also performed siRNA knock - lands, University of Tübingen, Institute of Pathology, Tübingen, Division of downs of both MSI1 and MSI2 and correlated the regulated genes with those Epigenomics and Cancer Risk Factors, German Cancer Research Center 7 carrying PAR-CLIP clusters. This resulted in 136/70 genes for MSI1/MSI2, (DKFZ), Heidelberg, Department of Internal Medicine III, University Hospital 8 respectively. These genes are currently subject to further functional analysis. of Ulm, Ulm, Department of Medicine III, Klinikum Grosshadern, LMU, Munich, 9 Summary and Conclusions: The RBP MSI2 is frequently overexpressed in Department of Molecular Biology, Nijmegen Centre for Molecular Life Sci - (hematological) malignancies. Its physiological function remains unclear since ences, Nijmegen, Germany its mRNA targets are unknown. By applying PAR-CLIP to MSI1 and MSI2 we defined their respective direct mRNA targets as well as their RRE. We includ - Background: AML1-ETO (AE) is the most frequent fusion gene in human ed MSI1 in our study since RBPs of the same protein family often target iden - AML. Previously, we and others have demonstrated that the fusion is not able tical mRNAs. Gene ontology analysis indicated a role for MSI2 in MAPK and to cause leukemia on its own in experimental murine models, but that it needs TGF-beta signaling pathways. Knockdown analysis revealed a total of 136/70 as a class II mutation collaborative partners such as FLT3-length mutation regulated genes for MSI1/MSI2, respectively. As predicted, the target genes (class I) to generate AML in the murine bone marrow transplantation model of MSI1 and MSI2 overlap to a large extent (~75%). So far, neither MSI1 (BMT model) (Schessl et al. , JCI 2005). expression nor mutational states in MSI2-related malignancies have been ana - Aims: We aimed at determining the functional role of the Hox co-factors Meis1 lyzed. However, this might be of interest, considering that overlapping target and Meis2 belonging to TALE family of homeodomain proteins. Meis1 is one genes are associated with similar biological functions. of the strongest known co-factors for Hox associated leukemogenesis, but was not linked to CBF leukemias so far. There are no data so far on the role of Meis2 in human AML. S1129 Methods: Taqmann analysis was done for AE positive samples to determine the expression of both MEIS genes. Expression of Meis genes and of AE were HES1 IS RESPONSIBLE FOR NOTCH SIGNALING-MEDIATED SUPPRES - achieved by retroviral gene transfer. To test the functional relevance of MEIS SION OF ACUTE MYELOID LEUKEMIA DEVELOPMENT genes in AE positive AML, lethally irradiated mice were transplanted with BM cells T Kato 1,* , M Sakata-Yanagimoto 1, Y Miyake 1, H Nishikii 1, H Muto 1, Y Yokoyama 1, solely expressing the fusion gene (n=8), EGFP (n=8, control) or with BM express - Y Asabe 1, K Suzukawa 1, N Obara 1, S Chiba 1 ing both genetic alterations AE+Meis1 (n=14) and AE+Meis2 (n=4). To test 1Department of Hematology, University of Tsukuba, Ibaraki, Japan, Tsukuba, dependence of human AE positive AML cell lines on MEIS expression lentiviral Japan based knock down of MEIS1 & 2 were performed. DNA binding of AE in human AML cell lines with or without MEIS depletion was assessed by ChIP-Seq. Background: The Hairy enhancer of split1 (Hes1) is well Results: Gene expression analysis revealed that MEIS1 is expressed at high characterized as a downstream target of Notch signaling. Hes1 is a basic helix- levels in a subgroup of AE positive AML patients. Furthermore, MEIS2 was looP-helix-type protein, and represses target gene expression. Notch signal - highly and aberrantly expressed virtually in all AE patients (n=70) compared to ing has been proposed to play both pro- and anti-tumorigenic roles; it promotes normal bone marrow. Expression patterns of both MEIS genes correlated with development of T-cell acute lymphoblastic leukemia (T-ALL), while serves as their promoter methylation in the t(8;21) patients and cell lines. Murine trans - a tumor suppressor for acute myeloid leukemia (AML). Meanwhile, Hes1 has plantation experiments showed that none of the mice in the AE as well as in been proven as an essential mediator of Notch signaling in T-ALL development, the control group developed disease. In contrast, mice transplanted with BM but its mediator for AML suppression remains to be elucidated. co-expressing AE+Meis1 and AE+Meis2 developed lethal disease after a medi - Aims: To explore whether Hes1 is responsible for Notch signaling-induced an latency of 102 and 255 days respectively. AE+Meis1 induced MPS in three, suppression of AML development. AML in seven and ALL in three cases, whereas AE+Meis2 induced AML in all Methods: Common myeloid progenitors (CMPs) purified from RBP-J f/f mouse cases. Functional relevance of MEIS expression was further confirmed in bone marrow (BM) were serially transduced with MLL-AF9 and Cre recombi - human AML cell lines as shRNA mediated depletion of MEIS1 as well as MEIS2 nase (iCre) using retroviral vectors, and transplanted into lethally irradiated impaired cell growth in AE positive AML cell lines and so far one primary AE syngenic mice. CMPs from Hes1 -/- mouse fetal liver were also retrovirally trans - AML sample. To understand the mechanisms of AE/MEIS collaboration co- duced with MLL-AF9 and transplanted after multiple rounds of replating, immunoprecipitation assays were performed documenting interaction of Meis1 Expression levels of downstream targets were evaluated by cDNA array and as well as Meis2 with AE. ChIP-Seq has been performed and data on changes quantitative RT-PCR. in DNA binding of AE in dependence of MEIS expression will be presented. Results: Mice transplanted with MLL-AF9/RBP-J -/- cells developed leukemia Summary and Conclusions: Our data demonstrate for the first time that at shorter latencies than those with MLL-AF9/RBPJ +/+ cells (MLL-AF9/RBPJ -/- AML1-ETO can collaborate with Meis1/2 and identify a novel collaborative part - , 3-8 weeks, n=14 vs MLL-AF9/RBPJ +/+ , 4-10 weeks, n=13; P<0.01). MLL- ner in t(8;21) positive AML. It furthermore shows that a class II mutation can AF9-transduced Hes1 -/- cells formed higher number of colonies at third replat - be converted into an overt oncogene by collaborating with its own class.

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