EVI1 Overexpression Research Articles

Abstract A45: EVI1 – a novel oncogene in breast carcinoma

Abstract Ecotropic viral integration site 1 (EVI1) is a nuclear transcription factor essential for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is found in ~10% of acute myeloid leukemia, where it associates with particularly aggressive disease. EVI1 is also expressed in solid tumors, but remains largely unstudied in this setting. A recent analysis of primary breast carcinoma (BC) samples suggests that enhanced EVI1 expression indicates poor outcome specifically in estrogen receptor (ER) negative tumors (Patel et al., 2011). Here, we explored expression, functional roles and molecular targets of EVI1 in human BC. EVI1 expression was analyzed by qRT-PCR and immunoblot analysis in 8 human BC cell lines and by immunohistochemistry in a cohort of 373 primary BC patient samples collected at the University Hospital Zurich. EVI1 was found to be expressed in most BC samples independently of their ER status. Long-term survival was influenced by EVI1 in ER-, but not ER+ patients, confirming previous results. To investigate the function of EVI1 in BC, cell lines with high and low endogenous EVI1 expression were transduced with EVI1 shRNA knockdown or inducible EVI1-expressing lentiviral particles. Growth, proliferation and cell cycle assays demonstrated that EVI1 promoted BC cell growth by accelerating cell cycle transition. Consistently, EVI1 knockdown BC cells showed reduced in vivo tumor growth compared to control cells in two xenotransplantation models (NSG mice and zebrafish embryo). Interestingly, estrogen supplementation rescued proliferation and tumorigenicity of EVI1-knockdown ER+ but not ER- BC cells, indicating that signaling through the ER pathway might override EVI1-dependent effects on proliferation. Conversely, EVI1-induced proliferation might be particularly important for tumors that are not regulated via the ER pathway, which is in line with its documented prognostic significance in such tumor types. In addition to its effects on proliferation, EVI1 was also found to inhibit BC cell apoptosis in response to both intrinsic and extrinsic stimuli, as previously shown by our group in acute lymphoblastic leukemia cells (Konantz et al, 2013). Moreover, in vitro migration assays showed impaired migration in EVI1 knockdown MDAMB231 cells suggesting that EVI1 regulates BC biology via different mechanisms. Currently, we are exploring the molecular mechanisms by which EVI1 mediates these different effects. Microarray analysis revealed molecules of the MAPK pathways, pro-apoptotic genes and, most interestingly, genes involved in GPCR signaling pathway (e.g. KISS1) as most significantly regulated by EVI1. Analysis of the KISS1 promoter revealed several potential EVI1-binding sites, and a direct association of EVI1 protein with one of these could be confirmed by CHIP. We are currently further exploring the relevance of this binding site for EVI1-mediated activation of KISS1 using luciferase KISS1 reporter assays and site-directed-mutagenesis. In ER- BC, KISS1 expression was reported to enhance cell motility and invasiveness and to reduce cell adhesion. Interestingly, the impaired migration observed in EVI1 knockdown MDAMB231 cells could be indeed rescued by stimulation with Kisspeptin. Neither EVI1 overexpression nor stimulation with Kisspeptin could influence the poorly migrating ER+ MCF7 cells. Taken together, our data identify EVI1 as a novel oncogene particularly important for ER- BC biology. Next to stimulation of BC cell proliferation and reduction of apoptosis sensitivity, we show that EVI1 stimulates ER- BC cell migration via activation of its direct molecular target KISS1. Citation Format: Hui Wang, Thorsten Schäfer, Martina Konantz, Selina Reich, Martin Braun, Sven Perner, Zsuzsanna Varga, Holger Moch, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. EVI1 – a novel oncogene in breast carcinoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A45.

A Combination of EVI1 and PRDM16 Expression Clarified the Clinical Features of Intermediate/High Risk Patients in Pediatric Acute Myeloid Leukemia

BackgroundSeveral molecular markers, such as FLT3-internal tandem duplication (ITD), NPM1, CEBPA are well known to correlate with mortality in patients with acute myeloid leukemia (AML). Recently, a number of gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT and FLT3. However, DNMT3A, IDH1/2, and TET2 are rare in pediatric patients with AML, thus accurate risk evaluation remained challenging even after incorporating these molecular markers.On the other hand, overexpression of the EVI1 gene is reported to be associated with adverse outcome in pediatric AML. Moreover, we have previously reported that measuring of PRDM16 gene expression was a powerful tool to predict the prognosis of pediatric AML. PRDM16 gene is highly homologous to the MDS1/EVI1 gene, which is an alternatively spliced transcript of the EVI1 gene. In this study, we investigated EVI1 gene expression to verify the prognosis of EVI1 gene expression and the relationship between EVI1 and PRDM16 gene expression.MethodsBetween 2006 and 2010, 485 de novo pediatric AML patients participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 116 patients were excluded from the study because of misdiagnosis and unavailability of their RNA samples. Therefore, 369 patients were analyzed. Quantitative RT-PCR analysis was performed in these patients using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to EVI1 and PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between these gene expressions and other genetic alterations, and clarified the prognostic impact of EVI1 and association between EVI1 and PRDM16 genes.ResultsA total of 58 of 369 patients (15.7%) showed high expression of EVI1 gene. Overexpression of EVI1 gene was strongly associated with dismal prognosis; low risk (LR; 1 of 123 patients, or 0.8%); intermediate risk (IR; 38 of 147 patients, or 25.9%); high risk (HR; 6 of 50 patients, or 12%); and non-complete remission (Non-CR; 13 of 49 patients, or 26.5%), (P < 0.001). Overexpression of EVI1 correlated with the following characteristics: younger age at diagnosis; M4, M5, and M7 subtype; higher coincidence of MLL-rearrangement; and lower coincidence of t(8;21), and inv(16). EVI1 overexpression was very frequent among patients with de novo pediatric AML and IR/non-CR groups. Furthermore, more than half of patients in M6 (5 of 8 patients, or 62.5%) were EVI1 high expression. Interestingly, no patients with EVI1 high expression in M7 had a fusion of CBFA2T3-GLIS2. Patients with EVI1 overexpression also more frequently harbored a complex karyotype and monosomy 7. The frequencies of patients with high or low EVI1 expression differed widely with respect to each genetic alteration, as follows: t(8;21), 1% vs 99%, P < 0.001; inv(16), 0% vs 100%, P < 0.001; NUP98-JARID1A, 83% vs 17%, P < 0.001; OTT-MAL, 100% vs 0%, P = 0.02; and KIT, 5% vs 95%, P = 0.003. The overall survival (OS) and event-free survival (EFS) among patients with EVI1 high expressions were significantly lower than that among patients without such gene aberrant expression (3-year OS 54% vs. 77%, P=0.008G3-year EFS: 34% vs 58%, P<0.001), respectively.On the other hand, a total of 84 of 369 patients (22.8%) showed high expression of PRDM16 gene. The OS and EFS among PRDM16 overexpressing patients were significantly worse than those among low expression group (3-year OS: 51% vs 81%, P<0.001; 3-year EFS: 32% vs 64%, P<0.001), respectively. Remarkably, concerning 125 patients with high EVI1 and/or PRDM16 expression, their prognosis was much worse than that of patients without these high expression (3-year OS: 54% vs 84%, P<0.001; 3-year EFS: 32% vs 68%, P<0.001) , respectively.ConclusionsWe investigated EVI1 and PRDM16 gene expression in de novo pediatric AML patients, and their high expression was associated with inferior survival, respectively. We suggest that high EVI1 and/or PRDM16 expression is useful marker for adverse outcome. On the other hand, low EVI1 and PRDM16 expressions are useful to elucidate low risk patients. DisclosuresNo relevant conflicts of interest to declare.

Correlation Between Bone Marrow Dysplasia and Genomic Profile in De Novo Acute Myeloid Leukemia (AML): A Study By the ALFA Group

Introduction: AML with multilineage dysplasia (MLD) are included in the WHO subset of "AML with myelodysplasia-related changes" (AML-MRC), together with AML arising from previous MDS or AML with MDS-related cytogenetic abnormalities. In the WHO classification, MLD is defined by dysplasia in at least 50% of the cells in at least two bone marrow (BM) myeloid cell lines. On the other hand, some genetically defined AML subgroups are specifically associated with morphologic changes, but close correlations do not exist for most of these entities. We searched for correlations between BM dysplasia and molecular aberrations in de novo AML patients included in 2 ALFA clinical trialsMethods: BM cytomorphology was retrospectively reassessed in 192 patients with de novo AML (excluding CBF-AML), aged 18 to 70 enrolled in ALFA-0702 (n=123) and ALFA-0701 (n=69) clinical trials in 5 centers. 4 distinct morphologists performed the analysis from BM smears. Dysmegakaryopoiesis (DM), dyserythropoiesis (DE) and dysgranulopoiesis (DG) were quantified (respectively on 30, 200 and 200 cells) using 22 criteria designed by GFHC, which allow better evaluation of cytoplasmic and nuclear dysplasia in all BM lineages. Dysplasia was also evaluated using WHO criteria. NPM1, FLT3, MLL, CEBPA, IDH1, IDH2, WT1, DNMT3A, RUNX1, TET2 and ASXL1 gene mutations and EVI1 gene overexpression were detected by standard methods, as previously published (Renneville et al. Oncotarget 2014).Results: In the 192 patients analyzed, the incidence of molecular abnormalities was: MLL-PTD 5% (8/155), NPM1 31% (52/170), FLT3-TKD 9% (15/171), FLT3-ITD 19% (34/171), CEBPA double mutated (CEBPA-dm)11% (17/152), EVI1 overexpression 11% (17/152), IDH1 R132 9% (14/146), IDH2 R140 6% (10/159), IDH2 R172 2% (2/92), RUNX1 8% (6/67), DNMT3A 26% (11/43), TET2 12% (5/43) and ASXL1 7% (4/62). DG, DE and DM was evaluable in 59%, 83% and 85% of the patients, respectively. WHO-MLD was identified in 43/192 (22%) patients, and was not significantly associated with any genetic marker, even in AML with normal karyotype (Table 1). On the other hand, when using GFHC criteria, we observed in NPM1 mutated patients a higher % of bi-tri or multi nucleated megakaryocytes (25% vs 10%, p=0.03), of cytoplasmic DG (74% vs 58%, p=0.03); and more dysplasia in other cell lines including eosinophils, basophils, mastocytes, monocytes (p=0.008). In CEBPA-dm patients, lower % of global DG (21% vs 54%, p=0.04) was seen. In EVI1 overexpressing patients, we found a higher % of global DM, of micromegacaryocytes and of hypolobulated megacaryocytes (80% vs 31%, p=0.01; 18% vs 2%, p=0.01 and 19% vs 6%, p=0.001 respectively). In DNMT3A mutated patients, we observed a lower % of bi-tri or multi nucleated megakaryocytes (2% vs 28%, p=0.01) and a higher % of nuclear and cytoplasmic DG (21% vs 2%, p=0.005 and 1.2% vs 0%, p=0.03, respectively). In TET2 mutated patients, we observed less defects in nuclear segmentation and a higher % of abnormal chromatin condensation in granulocytes (1% vs 9%, p=0.02 and 6% vs 0%, p=0.008, respectively).Conclusion: Presence of WHO-MLD was not significantly correlated with any genetic subgroup. The 22 BM dysplasia parameters designed by the GFHC were evaluable in a majority of patients, and allowed us to find some specific cytomorphologic features in de novo AML with NPM1, CEBPA-DM, DNMT3A, TET2 mutation, or EVI1 overexpression. Those findings suggest that the definition of MLD may be refined by using more in depth quantification of dysplasia, especially with GFCH parameters. This study will be expanded with the inclusion of whole exome sequencing data (ongoing).Table 1Correlation between MLD, normal karyotype and molecular abnormalities% AML-MLD% AML-MLD in AML with normal karyotypeMLL-PTD37,5%29%NPM125%28%FLT3-TKD27%33%FLT3-ITD27%25%CEBPA-dm0%0%IDH1 R13221%18%IDH2 R14010%0%IDH2 R1720%0%RUNX140%40%DNMT3A0%0%TET20%0%ASXL125%50%EVI124%0% DisclosuresFenaux:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Clinical Outcome, Molecular and Gene Expression Analysis of 20 MLL-Rearranged AML from a Single Institution

BackgroundRearrangements of the mixed lineage leukemia (MLL) gene at 11q23 (due to translocation, partial tandem duplication or gene amplification) are frequent in AML. Translocations give rise to fusion genes with AF9, AF6, MLLT10 and ENL, among others; partial tandem duplications or self-fusion fuses the 5' region of an MLL into the other MLL gene; and MLL amplification results in several copies of the wild-type gene.In this work we report the molecular analysis, gene expression profiling and clinical outcome for a cohort of MLL- rearrangedAML.Patients and MethodsHerein we include 20 MLL-rearrangement harboring patients, diagnosed in our institution of AML.MLL gene fusion was detected by cytogenetics; FISH with LSI MLL dual color, Break Apart probe, and chromosome painting probes; 10 cases could be verified by reverse transcription PCR (RT-PCR) and sequencing. Gene expression was analyzed in ten cases by real-time PCR in a reaction with specific primers and SybrGreen. Relative quantification was performed by 2-DDCt method.Survival analysis was carried-out by Kaplan-Meyer method. Overall survival was considered as the time from diagnostics to exitus.ResultsIn a time course from March 2004 to June 2015, the systematic screening for genetic rearrangements in AML patients at diagnosis, revealed 20 patients harboring a rearranged MLL. Fourteen of these were translocations, and in nine of them we could demonstrate the genetic fusion at the mRNA sequence level. There were three amplifications of the MLL gene in double minute (dmin), homogeneously stained region (hsr) and in several marker chromosomes.MLL rearrangement harboring patients had a median overall survival of 284±119 (N=18) days and only 25% of them survive at 1.7 years.Gene expression profiling of AML relevant genes (HOXA5, FLT3, EVI1, MEIS1 and RUNX1) showed a general upregulation of HOXA5, and a heterogenic regulation of FLT3. EVI1 and MEIS1 expression, undetectable in health control's peripheral blood, were upregulated only in some cases, interestingly the only two survivors in the series had undetectable EVI1 expression (Fig 1A). RUNX1 had a downregulation in MLL amplification cases, especially patient with 15-20 MLL copies in HSR in hyperdiploid cells that showed 7.4*107±2.5*107 fold downregulation (p<0.01), almost undetectable by real-time PCR (Fig 1B). Also was significatively (p<0.05) downregulated in two of the four MLL-ENL harboring patients and the only included MLL-AF6 harboring patient.ConclusionsIn this work we show that MLL-rearranged AML have a poor outcome, and most of them should not be included in intermediate-risk karyotype. Accordingly, the classification of MLL cases by the genetic partner or gene expression profile may provide a more precise prognostics. In this sense, overexpression of EVI1 has been previously associated to a worst outcome, but there are other genes that could be useful in prognostics such as MEIS1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantification of EVI1 transcript levels in acute myeloid leukemia by RT-qPCR analysis: A study by the ALFA Group

EVI1 overexpression confers poor prognosis in acute myeloid leukemia (AML). Quantification of EVI1 expression has been mainly assessed by real-time quantitative PCR (RT-qPCR) based on relative quantification of EVI1-1D splice variant. In this study, we developed a RT-qPCR assay to perform quantification of EVI1 expression covering the different splice variants. A sequence localized in EVI1 exons 14 and 15 was cloned into plasmids that were used to establish RT-qPCR standard curves. Threshold values to define EVI1 overexpression were determined using 17 bone marrow (BM) and 31 peripheral blood (PB) control samples and were set at 1% in BM and 0.5% in PB. Samples from 64 AML patients overexpressing EVI1 included in the ALFA-0701 or -0702 trials were collected at diagnosis and during follow-up (n=152). Median EVI1 expression at AML diagnosis was 23.3% in BM and 3.6% in PB. EVI1 expression levels significantly decreased between diagnostic and post-induction samples, with an average variation from 21.6% to 3.56% in BM and from 4.0% to 0.22% in PB, but did not exceed 1 log10 reduction. Our study demonstrates that the magnitude of reduction in EVI1 expression levels between AML diagnosis and follow-up is not sufficient to allow sensitive detection of minimal residual disease.

Abstract 1990: The key role of MECOM complex locus and its products in high-grade ovarian carcinoma pathogenesis and clinical outcomes

Abstract The ecotropic virus integration site 1(Evi1) transcription factor, encoded in the MECOM complex locus, is implicated in several cancers, including ovarian cancer(OC). High-grade serous OC(HG-SOC) represents its most common and aggressive form. The pathobiological and clinical roles of MECOM and its products in HG-SOC are poorly understood. Here, across all HG-SOC stages and grades, our meta-analysis demonstrated MECOM amplification in about 80% and EVI1 overexpression in 100% HG-SOC samples, relative to normal controls. We observed that Evi1 directly promotes double-strand DNA breaks repair via binding to DNA recombination/repair protein complexes, including a novel Rad50 isoform. To elucidate the molecular basis and clinical significance of these findings, we studied genome-wide genome-wide Evi1-DNA binding, DNA copy number alterations, gene expression and proteome-scale Evi1 interactions, combining cell line and clinical meta-data and network/pathway analyses. Our experiments revealed major, previously unknown Evi1-binding DNA motifs specifying expression of 309 target genes, whose protein products were classified into eight functional and prognosis-significant modules implicated in embryogenesis, EMT, RNA metabolism, cancer cell survival, retinoic acid, anti-viral and therapeutic responses. The Evi1 pathway modules can collectively stratify the HG-SOC patients into four high-confidence post-surgery survival subgroups(P&amp;lt;1.0×10-50), over-performing previously reported HG-SOC predictors and clinical markers. This tumor stratification correlates with chemotherapy sensitivity and treatment outcomes. Starting from early stages of HG-SOC, MECOM/Evi1 amplifies genomic instability and activates the Evi1 pathway that promotes most cancer hallmarks. Thus, MECOM/Evi1 and the Evi1 pathway provide mechanistic means for HG-SOC re-classification, introduces high-confidence early diagnostic and prognostic biomarkers and perspective therapeutic targets. Citation Format: Arsen O. Batagov, Surya Pavan Yenamandra, Piroon Jenjaroenpun, Anna V. Ivshina, Vladimir A. Kuznetsov. The key role of MECOM complex locus and its products in high-grade ovarian carcinoma pathogenesis and clinical outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1990. doi:10.1158/1538-7445.AM2015-1990

Azacitidine treatment for patients with myelodysplastic syndrome and acute myeloid leukemia with chromosome 3q abnormalities

Acute Myeloid Leukemia (AML) and myelodysplasia (MDS) with chromosome 3q abnormalities have a dismal outcome either untreated or with conventional treatments. Azacitidine (AZA) is now considered as the standard of care in high-risk MDS and oligoblastic AML patients. The objective of this study was to evaluate the impact of azacitine treatment in this cytogenetic subgroup. We report here a multicentre retrospective study of 157 patients treated with AZA for AML/MDS with chromosome 3q abnormalities and 27 patients with isolated EVI-1 overexpression. Median age was 65 years, 40 patients (25%) had inv(3)(q21q26.2) or t(3;3)(q21;q26.2), 36 patients (23%) had other balanced 3q26 rearrangements, 8 patients (5%) had balanced 3q21 rearrangements and 73 patients (46%) had other 3q abnormalities. The overall response rate was 50% (29% CR). Median overall survival was 10.6 months. By multivariate analysis, patients with lower bone marrow blast counts, higher platelet counts, non-complex cytogenetics, and absence of prior treatment with intensive chemotherapy had a better outcome. 27 patients were allo-transplanted and achieved a 21-month median OS. Balanced 3q21 translocations were associated with a better response rate and overall survival. Outcome of patients with isolated EVI-1 overexpression was comparable to that of patients with chromosome 3q lesions. Thus, AML/MDS patients with 3q abnormalities appear to be a heterogeneous group in their response to AZA, and AZA may represent a suitable option in particular as a bridge to allogeneic transplantation.

Chronic neutrophilic leukemia with overexpression of EVI-1, and concurrent CSF3R and SETBP1 mutations: A case report.

Chronic neutrophilic leukemia (CNL) is a rare type of myeloproliferative neoplasm, characterized by sustained neutrophilia, splenomegaly, bone marrow granulocytic hyperplasia (without evidence of dysplasia) and an absence of the Philadelphia chromosome. Thus far, ~150 cases of CNL have been described in the literature; however, none have demonstrated overexpression of the ecotropic viral integration site-1 (EVI-1, also known as MECOM) gene. The present study describes a case that fulfilled the World Health Organization diagnostic criteria for CNL, and was associated with overexpression of EVI-1, as well as novel concurrent mutations of colony stimulating factor 3 receptor (CSF3R) and SET binding protein-1 (SETBP1). In addition, the current study briefly reviewed the relevant literature regarding novel genetic findings associated with the diagnosis and treatment of CNL. To the best of our knowledge, this is the first case report of CNL with associated EVI-1 overexpression, and concurrent CSF3R and SETBP1 mutations.

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1+DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

3q26/EVI1 rearrangements in myeloid hemopathies: a cytogenetic review.

The EVI1 gene, located in chromosomal band 3q26, is a transcription factor that has stem cell-specific expression pattern and is essential for the regulation of self-renewal of hematopoietic stem cells. It is now recognized as one of the dominant oncogenes associated with myeloid leukemia. EVI1 overexpression is associated with minimal to no response to chemotherapy and poor clinical outcome. Several chromosomal rearrangements involving band 3q26 are known to induce EVI1 overexpression. They are mainly found in acute myeloid leukemia and blastic phase of Philadelphia chromosome-positive chronic myeloid leukemia, more rarely in myelodysplastic syndromes. They include inv(3)(q21q26), t(3;3)(q21;q26), t(3;21)(q26;q22), t(3;12)(q26;p13) and t(2;3)(p15-23;q26). However, many other chromosomal rearrangements involving 3q26/EVI1 have been identified. The precise molecular event has not been elucidated in the majority of these chromosomal abnormalities and most gene partners remain unknown.

Molecular Prognostic Factors in Acute Myeloid Leukemia (AML) Patients Receiving First Line Therapy with Azacytidine (AZA)

▪ BACKGROUNDAML in elderly patients, secondary AML and/or AML with complex karyotype respond poorly to conventional intensive chemotherapy (CT) and have a poor prognosis. In those patients, however, AZA can bring some benefit, especially in the absence of adverse cytogenetics, if WBC < 15 × 10⁹/L and performance status (PS) < 2 (Thepot, Am J Hemat, 2014; Pleyer Ann Hematol, 2014). Whether molecular findings can predict outcome in AML receiving first line AZA (as they do in AML receiving CT) is however unknown. PATIENTS AND METHODS96 AML patients who received between 2007 and 2012 first line treatment with AZA (75mg/m2/d x 7 d/4 weeks SC for a median of 7 cycles, range 1-33) in 8 French centers were retrospectively analyzed. Initial response was evaluated based on blood and marrow examination after 4 -6 cycles of AZA. We analyzed the impact on overall survival (OS) of conventional clinico-biological parameters, and molecular biology parameters including EVI1 expression, MLL-PTD, and the mutational status of 16 genes analyzed by next-generation sequencing (NGS) and Sanger sequencing: ASXL1, CEBPA, DNMT3A, EZH2, FLT3, IDH1, IDH2, NPM1, NRAS, KRAS, RUNX1, SF3B1, SRSF2, U2AF1, TET2, and TP53. RESULTSMedian age was 73 (range, 44-88). Median PS was 1 (range, 0-3). 55% patients had secondary AML, including post MDS or MPN AML (N=39), and therapy related AML (N=14). 64% of patients had high risk cytogenetics and only 14 normal karyotype. Median WBC was 3.87 G/L and 28 patients had WBC >10 G/L. Median platelets and Hb were 61 G/L and 9.25 g/dL respectively. 41 and 55 patients had marrow blasts < 30% and ≥ 30% respectively, and median circulating blast % was 15% (range, 0-95). The median number of somatic mutations per patient was 2 (range, 0-5). The most frequently mutated genes were TP53 (40%), TET2 (22%) and SRSF2 (19%), then DNMT3A (14%), ASXL1 (13.5%), IDH2 (11%) and RUNX1 (11%). EVI1 overexpression (defined stringently in this study as DD CT ≤ 8 EVI1/ABL) was found in 12 patients. Overall response rate was 29% (14% CR, 8% PR and 7% Cri), and median response duration was 9.4 months. Median OS was 10.9 months. In univariate analysis, the following factors were significantly associated with shorter OS: platelets and Hb below median (p<0.0001 and p=0.0007 respectively), PS≥2 (p=0.008), and circulating blasts as continuous variable (p=0.03) while WBC and cytogenetics had no significant impact.OS was significantly worse in patients with TP53 gene mutation (median 8.9 vs 12.3 months in unmutated cases, p=0.01). It tended to be worse for SRSF2 gene mutation (median 8.4 vs 11.5 months, p=0.16) and TET2 gene mutation (median 8.8 vs 11.7 months, p=0.20) and tended to be better in patients with EVI1 overexpression (median 16.1 vs 9.4 months in other patients, p=0.17), while the mutational status of ASXL1, DNMT3A, IDH2 and RUNX1 genes had no impact on OS.Median survival of patients with at least one mutation of TP53, TET2, SRSF2 genes, was 9.1 vs 14.8 months if the 3 mutations were absent (p=0.0008, figure A). In multivariate analysis (logistic regression according to the Wald method), higher PS (p=0.0061), lower Hb (p=0.0014), lower platelets (p=0.0139) higher % circulating blasts (p=0.0145) and presence of TP53, TET2 or SRSF2 mutation (p=0.0035) were associated with poorer OS. Due to the low number of patients with EVI1 overexpression, this parameter was not included in the multivariate analysis. However, the 7 patients with EVI1 overexpression without any poor prognostic mutation had a median OS of 25.1 months ,versus 8.8 in patients carrying TP53, TET2 and/or SRSF2 mutations and 12.6 months in remaining patients without EVI overexpression (p=0.022) (figure B). [Display omitted] [Display omitted] CONCLUSIONIn this first analysis (to our knowledge) of the prognostic value of molecular factors in poor risk AML (55% secondary cases, 64% unfavorable karyotype) receiving first line treatment with AZA, mutations of the TP53 and to a lesser extent TET2 or SRSF2 genes were adverse prognostic factors, and added prognostic value to conventional clinical and hematological parameters. For TET2, the effect seemed different from the favorable prognosis observed in MDS treated with AZA (Itzykson, Leukemia, 2011; Sekeres, Blood, 2012). Surprisingly also, EVI1 overexpression, a poor prognostic factor in AML, seemed associated with better outcome with AZA treatment in the absence of any poor prognostic mutation Those findings will require confirmation in larger prospective series. DisclosuresNo relevant conflicts of interest to declare.

The Comparative Value of Hematopoietic Stem Cell Transplantation and Chemotherapy in Cytogenetically Normal AML Subclassified By NPM1 Mutation Status and FLT3-ITD Allelic Ratio

Post-remission treatment (PRT) in patients with cytogenetically normal (CN) acute myeloid leukemia (AML) in first complete remission (CR1) is a subject of continued debate. Molecular diagnostic analyses provide additional prognostic information that may be used for a risk adapted approach. While FLT3 internal tandem duplications (FLT3-ITD), particularly FLT3-ITD with a high mutant to wild-type ratio are associated with an unfavorable prognosis, NPM1 mutations in the absence of FLT3-ITD are associated with relatively favorable outcome. Allogeneic hematopoietic stem cell transplantation (alloHSCT) is generally not applied in patients with NPM1 mutations without FLT3-ITD, while the role of alloHSCT and autologous HSCT (autoHSCT) in patients with FLT3-ITD is not settled. In the present study, we compared outcome of PRT with alloHSCT and autoHSCT versus chemotherapy by time-dependent analysis in patients with CN AML in CR1, according to molecular subtype.A total number of 488 patients were included with newly diagnosed, CN AML, who were treated in HOVON-SAKK phase III trials or in Marseille, France between 1995 and 2013. Patients were excluded if molecular information was not available or if EVI1 overexpression was present. The ratio of FLT3-ITD mutant to wild-type was available for 91% of the patients with FLT3-ITD positive AML. PRT consisted of alloHSCT (n=184) following reduced intensity conditioning (RIC, n=94) or myeloablative conditioning (MAC, n=90), autoHSCT (n=117), or chemotherapy (n=187). Endpoints of this study were 5 year overall survival (OS), relapse-free survival (RFS), relapse and non-relapse mortality (NRM) measured from start of consolidation. All reported estimates of outcome are at 5 years after PRT. To perform a time-dependent statistical analysis of PRTs, a multivariable cox regression model with time-dependent covariates autoHSCT and alloHSCT was applied with adjustment for FLT3-ITD and/or NPM1 mutational status, white blood cell count at diagnosis, late CR, age, and sex.The median age of all patients was 49 (range: 18-65) years, which was not different by type of PRT. A significantly higher proportion of patients that needed two cycles of chemotherapy to reach a CR received an alloHSCT. Both alloHSCT and autoHSCT were more frequently applied in the recent years. Sex, white blood cell count at diagnosis, and FLT3-ITD mutant to wild-type ratio were not significantly different between the types of PRT. Figure 1A/B depicts OS and RFS by molecular subcategory taking the FLT3-ITD allelic ratio into account. Favorable OS was found for patients with NPM1 mutated FLT3-ITD negative AMLs (72±4%), with similar OS for recipients of alloHSCT, autoHSCT, and chemotherapy (69±6%, 77±7%, and, 70±8%, respectively, p=0.56). Outcome in patients with a FLT3-ITD mutant to wild-type ratio of > 0.60 appeared to be very poor with OS and RFS of 17±8% and 4±4%, respectively. In these high risk patients, recipients of chemotherapy (n=13) and autoHSCT (n=3) all relapsed, while 2/5 patients receiving alloHSCT relapsed. Patients with NPM1 negative/FLT3-ITD negative AML or AMLs with a low allelic burden of FLT3-ITD were considered as an intermediate risk group because of similar OS (53±4% and 47±4%, respectively) and RFS (43±4% and 40±4%, respectively). In this intermediate risk group, OS following alloHSCT was 55±4% as compared with 39±5% following chemotherapy (not significant) and RFS was 51±4% vs 29±5%, p=0.004, Figure 1C/D. Multivariable analysis with adjustment for covariates showed better OS (HR 0.69, p=0.04), and better RFS (HR 0.57, p=0.001) following alloHSCT as compared to chemotherapy in this intermediate risk group, with similar results for RIC and MAC. AutoHSCT was associated with a trend towards improved OS (56±6% vs 39±5%, respectively p=0.07, Figure 1C) and significantly improved RFS (41±6% vs 29±5%, p=0.04, Figure 1D) as compared to chemotherapy.Collectively, these results suggest that alloHSCT may provide better survival than chemotherapeutic PRT in patients with CN, NPM1 negative/FLT3-ITD negative or FLT3-ITD positive AML with a low allelic burden. AutoHSCT may still be an alternative if no donor is available in CR1. Given the poor prognosis in patients with a FLT3-ITD mutant to wild-type ratio of >0.60, we would recommend that particular subgroup for PRT by alloHSCT using sibling or alternative donors early following CR. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Generation and Analysis of a Novel Mouse Model for Chronic Myelomonocytic Leukemia (CMML) with Acquired Expression of c-CBLQ367P

Myelodysplastic syndromes (MDS) are disorders originated from hematopoietic stem cells (HSCs), which are characterized by ineffective hematopoiesis, dysplasia mainly in the myeloid lineage, and high progression ratio to acute myeloid leukemia (AML). Recently, we identified mutations of the c-CBL (Casitas B cell lymphoma, a cellular homologue of v-CBL) gene in patients with MDS and myeloproliferative neoplasms (MPN). The mutations are detected in about 8% of the patients with the highest frequency in chronic myelomonocytic leukemia (CMML) cases with acquired uniparental disomy (aUPD) at 11q. c-CBL encodes a RING finger-based E3 ubiquitin ligase that negatively regulates receptor-mediated intracellular signaling. c-CBL is highly expressed in HSCs, strongly suggesting that it functions as a fine regulator of hematopoietic homeostasis. In fact, c-CBL knockout (KO) mice showed a myeloproliferative phenotype, owing to the hyper-responsiveness of HSCs to cytokine stimulation and subsequent augmented hematopoietic progenitor pool. In addition, c-CBL knockin (KI) mice harboring a mutation in the RING finger domain in one allele and a null mutation in the other allele exhibit an MPD-like disease and eventually progress to AML. These findings indicate that dysfunction of c-CBL perturbs normal hematopoietic development and contributes to hematopoietic abnormalities, but the precise leukemogenic mechanism(s) remains elusive. To gain insights into this issue and to create a novel animal model for mutated c-CBL-harboring leukemia, we generated conditional knock-in (cKI) mice that express wild-type c-CBL at steady state and inducibly express c-CBLQ367P, which was identified in patients with chronic myelomonocytic leukemia (CMML). After induced expression of c-CBLQ367P, the cKI mice exhibited a rapid and sustained increase in myelomonocytic cells with dysplasia in the peripheral blood and splenic enlargement with proliferation of myeloid cells, which closely resemble to the phenotype of human CMML. The bone marrow (BM) was hypercellular with predominance of myeloid cells, and increased number of HSC subpopulations and early myeloid progenitors were observed. In addition, phosphorylation of AKT, STAT3 and STAT5 was detected in long-term hematopoietic stem cells (LT-HSCs) of c-CBLQ367P cKI mice, indicating that PI3K/AKT and JAK/STAT signaling pathways are activated in c-CBLQ367P LT-HSCs. Moreover, competitive repopulation assays revealed that mice transplanted with c-CBLQ367P LT-HSCs showed significantly higher donor-derived chimerism than those transplanted with control LT-HSCs and displayed expansion of myelomonocytic cells as observed in c-CBLQ367P cKI mice, indicating that c-CBLQ367P conferred a proliferative advantage to LT-HSCs and that the phenotypes observed in c-CBLQ367P cKI mice were hematopoietic cell-intrinsic. CMML is known to progress to AML, possibly with additional genetic aberrations. To investigate the mechanism(s) underlying the disease evolution, we performed retroviral insertional mutagenesis using MOL4070A, a derivative of Moloney murine leukemia virus capable of inducing myeloid diseases. Almost all MOL4070A-infected c-CBLQ367P cKI mice developed AML, while no disease was observed in virus-injected control mice. Inverse PCR method identified Evi1 gene as a common integration site in the diseased mice and high Evi1 expression was detected in Evi1-integrated tumors. Mice transplanted with Evi1-transduced c-CBLQ367P cKI c-kit-positive BM cells developed AML at a high frequency and in a shortened period as compared to those transplanted with Evi1-transduced control cells. Taken together, we demonstrated that acquired expression of c-CBLQ367P plays a causative role in the development of CMML by activating PI3K/AKT and JAK/STAT pathways in HSCs and found that Evi1 overexpression cooperates with c-CBLQ367P to develop AML. Our mouse model provides a powerful tool for understanding of the pathogenesis of CMML and for developing novel therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

Mice Carrying a Hypomorphic Evi1 Allele Are Embryonic Viable but Exhibit Severe Congenital Heart Defects

The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1's critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1fl3) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1δex3/δex3 mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1fl3/fl3 mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1δex3/δex3 knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1δex3/δex3 mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.

Clinical impact of gene mutations and lesions detected by SNP-array karyotyping in acute myeloid leukemia patients in the context of gemtuzumab ozogamicin treatment: Results of the ALFA-0701 trial

We recently showed that the addition of fractionated doses of gemtuzumab ozogamicin (GO) to standard chemotherapy improves clinical outcome of acute myeloid leukemia (AML) patients. In the present study, we performed mutational analysis of 11 genes (FLT3, NPM1, CEBPA, MLL, WT1, IDH1/2, RUNX1, ASXL1, TET2, DNMT3A), EVI1 overexpression screening, and 6.0 single-nucleotide polymorphism array (SNP-A) analysis in diagnostic samples of the 278 AML patients enrolled in the ALFA-0701 trial. In cytogenetically normal (CN) AML (n=146), 38% of the patients had at least 1 SNP-A lesion and 89% of the patients had at least 1 molecular alteration. In multivariate analysis, the independent predictors of higher cumulative incidence of relapse were unfavorable karyotype (P = 0.013) and randomization in the control arm (P = 0.007) in the whole cohort, and MLL partial tandem duplications (P = 0.014) and DNMT3A mutations (P = 0.010) in CN-AML. The independent predictors of shorter overall survival (OS) were unfavorable karyotype (P <0.001) and SNP-A lesion(s) (P = 0.001) in the whole cohort, and SNP-A lesion(s) (P = 0.006), DNMT3A mutations (P = 0.042) and randomization in the control arm (P = 0.043) in CN-AML. Interestingly, CN-AML patients benefited preferentially more from GO treatment as compared to AML patients with abnormal cytogenetics (hazard ratio for death, 0.52 versus 1.14; test for interaction, P = 0.04). Although the interaction test was not statistically significant, the OS benefit associated with GO treatment appeared also more pronounced in FLT3 internal tandem duplication positive than in negative patients.

Azacitidine Treatment For Patients With Myelodysplastic Syndromes and Acute Myeloid Leukemia Harboring Chromosome 3q Abnormalities

BackgroundKaryotype is one of the key prognosis factors in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Chromosome 3q abnormalities, including aberrant expression of EVI-1, represent a small subset of patients and are associated with a poor outcome in both MDS and AML patients treated with conventional treatment. Azacitidine is the current standard of care for high-risk MDS and is also active in AML. Given that AZA may provide responses in patients with poor chances of response to conventional treatment as patients with monosomy 7 and given the strong epigenetic modifications related to EVI-1 dysregulation, AZA may represent an interesting option for patients with 3q abnormalities. Therefore, we analyzed the outcome of patients with Chromosome 3q abnormalities treated with Azacitidine. Patients and methodsThis is a retrospective multicenter study. Patients were included if 1/ Diagnosis of MDS, CMML, or AML, including therapy related neoplasms 2/ harbored chromosome 3q abnormalities and/or EVI-1 overexpression by PCR 3/ received at least one cycle of azacitidine for either frontline or relapse treatment. Response was evaluated according IWG 2006 criteria. This cohort of patients was compared to 406 high risk MDS and AML patients without 3q abnormalities included in the French AZA compassional program. ResultsA total of 184 patients were analyzed. Median age was 64 years. According to WHO classification, patients presented RA/RCMD/RAEB-1 28% of cases, RAEB-2/CMML-2 31% of cases, and AML 41% of cases. Median bone marrow blast count was 15%. Cytogenetics were inv(3)/t(3;3) 22%, del(3q)/monosomy 3 26%, t(3q26;x) 20%, EVI-1 overexpression without 3q abnormality 14%, t(3q21;x) 4% and other 3q abnormalities 14%. 58% of patients had complex karyotype. Azacitidine was used frontline for 67% of patients, 33% being previously treated with intensive chemotherapy. Median number of cycle of azacitidine was 5 (1-33). Overall response rate was 47% (Complete or Partial response 27%). 31 patients (25% of patients younger than 70y) bridged to allogeneic transplantation. With a median follow-up of 16 months, median overall survival was 10.5 months. Younger age (below 65y, 13m vs 10m, p=0.052), low bone marrow blast count (below 20%, 13m vs 8m, p=0.001), presence of t(3q21;x) (74 vs 10 months, p=0.008), few cytogenetic abnormalities (below 3, 14m vs 9m, p=0.003), and use of Azacitidine frontline (13m vs 8 m, p=0.029) were associated with a better survival. Long term survival remains limited with a 3 years survival probability below 10%. Interestingly, baseline characteristics and prognosis of patients with EVI-1 overexpression without 3q cytogenetic abnormality were comparable with patients with 3q26 cytogenetic abberation. Then, we compared our cohort with MDS/AML patients without 3q abnormalities, their overall response rate was 30% (41% for HR MDS, 15% for AML, including 11% CR/PR), and median OS was 10.5 months (15 months for HR MDS and 7 months for AML). When we merged the 2 cohorts and performed multivariate analyses, presence of 3q abnormality had no prognosis impact on either response or survival. ConclusionAzacitidine seems to limit the dismal outcome associated with 3q abnormalities and thus represents a good alternative to conventional chemotherapy including for patients eligible for allogeneic transplantation. Moreover, our data showed that 3q group is heterogeneous with a favorable outcome for the small subset of patients with 3q21 abnormalities. Disclosures:Prebet:CELGENE: Honoraria. Off Label Use: Use of azacitidine in acute myeloid leukemia.

Epigenetic aberrations in myeloid malignancies (Review)

The development of novel technologies, such as massively parallel DNA sequencing, has led to the identification of several novel recurrent gene mutations, such as DNA methyltransferase (Dnmt)3a, ten-eleven-translocation oncogene family member 2 (TET2), isocitrate dehydrogenase (IDH)1/2, additional sex comb-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2) and ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX) mutations in acute myeloid leukemia (AML) and other myeloid malignancies. These findings strongly suggest a link between recurrent genetic alterations and aberrant epigenetic regulations, resulting from an abnormal DNA methylation and histone modification status. This review focuses on the current findings of aberrant epigenetic signatures by these newly described genetic alterations. Moreover, epigenetic aberrations resulting from transcription factor aberrations, such as mixed lineage leukemia (MLL) rearrangement, ecotropic viral integration site 1 (Evi1) overexpression, chromosomal translocations and the downregulation of PU.1 are also described.

Monitoring Response and Resistance to the Novel Arsenical Darinaparsin in an AML Patient

Acute myeloid leukemia (AML) with inversion of chromosome 3 is characterized by overexpression of EVI1 and carries a dismal prognosis. Arsenic-containing compounds have been described to be efficacious in malignancies overexpressing EVI1. Here, we describe a case of AML with inv(3)(q21q26.2) treated with the organic arsenical darinaparsin. Using a “personalized medicine approach,” two different arsenicals were screened for anti-leukemic effect against the patient’s cells ex vivo. The most promising compound, darinaparsin, was selected for in vivo treatment. Clinical effect was almost immediate, with a normalization of temperature, a stabilization of white blood cell (WBC) counts and an increased quality of life. Longitudinal monitoring of patient response and resistance incorporating significant correlative studies on patient-derived blood samples over the two cycles of darinaparsin given to this patient allowed us to evaluate potential mechanisms of response and resistance. The anti-leukemic effects of darinaparsin correlated with inhibition of the alternative NF-κB pathway and production of the inflammatory cytokine IL-8. Emergence of resistance was suspected during treatment cycle 2 and supported by xenograft studies in nude mice. Darinaparsin resistance correlated with an attenuation of the effect of treatment on the alternative NF-κB pathway. The results from this patient indicate that darinaparsin may be a good treatment option for inv(3) AML and that inhibition of the alternative NF-κB pathway may be predictive of response. Longitudinal monitoring of disease response as well as several correlative parameters allowed for the generation of novel correlations and predictors of response to experimental therapy in a heavily pretreated patient.

Deregulated Expression ofEVI1Defines a Poor Prognostic Subset ofMLL-Rearranged Acute Myeloid Leukemias: A Study of the German-Austrian Acute Myeloid Leukemia Study Group and the Dutch-Belgian-Swiss HOVON/SAKK Cooperative Group

To evaluate the prognostic value of ecotropic viral integration 1 gene (EVI1) overexpression in acute myeloid leukemia (AML) with MLL gene rearrangements. We identified 286 patients with AML with t(11q23) enrolled onto German-Austrian Acute Myeloid Leukemia Study Group and Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group prospective treatment trials. Material was available from 177 AML patients for EVI1 expression analysis. We divided 286 MLL-rearranged AMLs into three subgroups: t(9;11)(p22;q23) (44.8%), t(6;11)(q27;q23) (14.7%), and t(v;11q23) (40.5%). EVI1 overexpression (EVI1(+)) was found in 45.8% of all patients with t(11q23), with t(6;11) showing the highest frequency (83.9%), followed by t(9;11) at 40.0%, and t(v;11q23) at 34.8%. Concurrent gene mutations were rare or absent in all three subgroups. Within all t(11q23) AMLs, EVI1(+) was the sole prognostic factor, predicting for inferior overall survival (OS; hazard ratio [HR], 2.06; P = .003), relapse-free survival (HR, 2.28; P = .002), and event-free survival (HR, 1.79; P = .009). EVI1(+) AMLs with t(11q23) in first complete remission (CR) had a significantly better outcome after allogeneic transplantation compared with other consolidation therapies (5-year OS, 54.7% v 0%; Mantel-Byar, P = .0006). EVI1(-) t(9;11) AMLs had lower WBC counts, more commonly FAB M5 morphology, and frequently had additional trisomy 8 (39.6%; P < .001). Among t(9;11) AMLs, EVI1(+) again was the sole independent adverse prognostic factor for survival. Deregulated EVI1 expression defines poor prognostic subsets among AML with t(11q23) and AML with t(9;11)(p22;q23). Patients with EVI1(+) MLL-rearranged AML seem to benefit from allogeneic transplantation in first CR.

RUNX1/AML1 Mutants Collaborate with BMI1 in the Development of Myelodysplastic Syndromes (MDS) / Acute Myeloid Leukemia (AML) in a Mouse BMT Model.

Abstract 2820RUNX1/AML1 mutations have been frequently detected in patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). RUNX1 mutations are rarely detected in lower-risk MDS, whereas approximately 20% patients with higher-risk MDS (H-MDS) have the mutations. The mutations were distributed throughout the RUNX1 protein, and replacement of D171 amino acid in runt homology domain was the most frequent target of mutations in the RUNX1 gene. The D171N mutant showed a loss of normal RUNX1 trans-activation potential and dominant-negative suppression. In mouse transplantation systems D171N-transduced mice exhibited AML with multilineage dysplasia in collaboration with Evi1 overexpression. However, EVI1 overexpression was very rare in patients. Instead, most of H-MDS patients with RUNX1 mutations displayed a high expression of BMI1.RUNX1 D171N mutant showed an increased self-renewal capacity, differentiation block, dysplasia in all 3 lineages, slightly increased immature cells and no proliferation ability using enforced expression in human CD34+ cells, and the D171N-transduced cells showed low expression level of BMI1. Both D171N and BMI1 transduced cells displayed long-term proliferation ability. When BMI1 transduced later into D171N cells, the cells expanded with a retained CD34+ cell fraction, suggesting that BMI1 have a potential to boost the D171N cells to H-MDS.To confirm the collaboration of BMI1 overexpression with D171N mutant in vivo, we performed mouse BMT using BM cells transduced with both D171N and BMI1. Ly-5.1 murine BM mononuclear cells infected with retrovirus harboring D171N/BMI1 or control vectors were transplanted into sublethally irradiated syngeneic Ly-5.2 mice.Mice that received transplants of BMI1-transduced cells remained healthy over the observation period (n=12/12), as well as those that were transplanted with empty vectors-transduced cells (n=4/4). Most of the mice that received transplants of D171N -transduced cells developed MDS/AML mainly 6–8 months after transplantation (n=6/11, P<0.0001), as observed in the previous report. Of note, mice that received transplants of BM cells expressing D171N/BMI1 developed MDS/AML with significantly shorter latencies (mainly 3–5 months) compared with the D171N group (n=12/12, P=0.001). Morbid mice with D171N or D171N/BMI1 exhibited similar phenotypes, characterized by leukocytosis, anemia, and marked splenomegaly, while the mice with BMI1 or empty vectors, sacrificed 8 months after BMT, showed none of these phenotypes. In the leukemic mice with D171N or D171N/BMI1, BM and spleen were occupied by immature myeloid cells including myeloid blasts. More myeloblasts in BM were observed in D171N/BMI1 mice than in D171N ones. The leukemic cells displayed similar morphological abnormalities and surface markers: leukemic cells were CD11blow to high, Gr-1low, B220low and c-kitlow to high. The normal structure of the spleen was completely destroyed with massive blast and immature myeloid cell infiltration, and these cells also invaded into the hepatic portal areas in the liver. Meanwhile, BMI1-transduced BM cells did not become dominant in vivo and myeloid cells showed normal differentiation. Collectively, BMI1 overexpression has a strong potential to induce MDS/AML in concert with D171N in a mouse BMT model, although BMI1 overexpression by itself does not result in maturation block or leukemogenesis.BMI1 is well-known to be essential for self-renewal of HSCs, in part via repression of genes involved in senescence, and self-renewal of HSCs is enhanced by BMI1 expression. To address the mechanisms by which BMI1 would contribute to MDS/AML development, we analyzed gene expressions involved in BMI1 downstream signaling pathways. It is suggested that BMI1 overexpression may act as one of the partner abnormalities collaborating with master gene mutations for MDS-genesis.RUNX1 D171N mutant showed no proliferation ability using enforced expression in human CD34+ cells, and D171N-transduced mice exhibited MDS/AML in collaboration with Evi1 overexpression. In addition, co-transduction of D171N and BMI1 into BM cells resulted in faster induction of MDS/AML in BMT mice. Taken together, the RUNX1 mutant may require collaborating genes such as EVI1 and BMI1 to develop MDS/AML. We confirmed that BMI1 have a potential to boost the D171N cells to MDS/AML in vivo. Disclosures:No relevant conflicts of interest to declare.