High EVI1 Expression Research Articles

Abstract 3040: Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer

Abstract Epithelial ovarian cancer (OC) is typically diagnosed at a late stage and, despite treatment with cytoreductive surgery and chemotherapy, 70-80% of patients relapse and eventually die due to the disease. Major challenges to improve patient outcomes are to overcome therapy resistance and to individually tailor therapy. Uncovering the molecular drivers of OC, especially in the most common subgroup high-grade serous (HGS), will help develop novel therapeutic strategies to meet these challenges. Using the Cancer Genome Atlas (TCGA) data, we observed that a locus in 3q26.2, near the EVI1 oncogene, was the most significantly amplified region in HGS OC, occurring in more than 80% of those tumors. We validated these findings in an OC dataset from the Hartwig Medical Foundation and in OC cell lines from the DepMap database. Furthermore, HGS OC had the highest mean copy number of the 3q26.2 locus co-occurring with high mean EVI1 expression among all tumor entities in the pan-cancer TCGA dataset. These EVI1 expression levels are significantly higher than in normal fallopian tube, from which HGS OC most likely arises. EVI1 overexpression promotes leukemogenesis in 3q26-rearranged acute myeloid leukemia (AML), as a result of chromosomal translocations that enable hijacking of hyperactive enhancers. Here, we hypothesize that EVI1 also plays a crucial role in OC development, but that its aberrant expression in this disease is driven by the amplification of yet undiscovered regulatory elements near EVI1. We showed that EVI1-positive OC cell lines, of which the majority are HGS, are EVI1-dependent and harbor a ~200 kb amplified region near EVI1. This region interacts with the EVI1 promoter (4C-Seq), is characterized by open chromatin (ATAC-Seq), and shows high levels of H3K27 acetylation (H3K27ac ChIP-Seq), suggesting the presence of an active enhancer. Furthermore, based upon H3K27ac signal, we identified within this region a super-enhancer of approximately 65 kb. In OC cell lines without detectable EVI1 levels or cell lines from other cancer subtypes, such as breast and colon cancer, this location had no active enhancer and did not loop to the EVI1 promoter. Altogether, these data point to the generation of a hyperactive super-enhancer driving EVI1 overexpression in OC. These findings emphasize the importance of epigenetic control in the regulation of EVI1 expression, as previously shown in 3q26-rearranged AML. Since OC tumor cell lines are strongly dependent on EVI1, interference with this super-enhancer, leading to loss of EVI1 expression, could be exploited to inhibit tumor growth. For this purpose, we generated GFP-tagged EVI1 cell line models (e.g. SKOV3), which will be used to discover small molecules to target the activity of this super-enhancer. In conclusion, we discovered a new mechanism of enhancer-mediated overexpression of the EVI1 oncogene as a molecular driver in ovarian cancer, with possible implications for therapy. Citation Format: Leonie Smeenk, Sabrina Kruse, Roger Mulet-Lazaro, Ingrid Boere, John Martens, Stefan Gröschel, Claudia Scholl, Stefan Fröhling, Ruud Delwel. Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3040.

The role and regulation of EVI1 in normal hematopoiesis and hematopoietic malignancies

EVI1 is a zinc finger transcription factor encoded by the MECOM locus and is essential for the development and maintenance of hematopoietic stem cells. However, overexpression of EVI1 in various myeloid malignancies is associated with aggressive clinical behavior and poor outcome. The locus encodes multiple isoforms that are differentially acting and independently regulated. EVI1 interacts with a variety of transcription and epigenetic factors via different domains. It also regulates cell survival, differentiation, and proliferation through a variety of mechanisms, including transcriptional activation and repression, regulation of other transcription factors' activity, and chromatin remodeling. While the mechanism by which 3q26 translocation leads to high EVI1 expression through enhancer hijacking of genes active in myeloid development is now better understood, regulation of EVI1 expression in the absence of chromosomal translocations and in normal hematopoiesis remains unclear. Recent studies have provided insight into the regulatory mechanisms of EVI1 expression and action, which may lead to development of targeted therapies in the near future.

High EVI1 and PARP1 expression as favourable prognostic markers in high-grade serous ovarian carcinoma

BackgroundMechanisms of development and progression of high-grade serous ovarian cancer (HGSOC) are poorly understood. EVI1 and PARP1, part of TGF-ß pathway, are upregulated in cancers with DNA repair deficiencies with DNA repair deficiencies and may influce disease progression and survival. Therefore we questioned the prognostic significance of protein expression of EVI1 alone and in combination with PARP1 and analyzed them in a cohort of patients with HGSOC.MethodsFor 562 HGSOC patients, we evaluated EVI1 and PARP1 expression by immunohistochemical staining on tissue microarrays with QuPath digital semi-automatic positive cell detection.ResultsHigh EVI1 expressing (> 30% positive tumor cells) HGSOC were associated with improved progression-free survival (PFS) (HR = 0.66, 95% CI: 0.504–0.852, p = 0.002) and overall survival (OS) (HR = 0.45, 95% CI: 0.352–0.563, p < 0.001), including multivariate analysis. Most interestingly, mutual high expression of both proteins identifies a group with particularly good prognosis. Our findings were proven technically and clinically using bioinformatical data sets for single-cell sequencing, copy number variation and gene as well as protein expression.ConclusionsEVI1 and PARP1 are robust prognostic biomarkers for favorable prognosis in HGSOC and imply further research with respect to their reciprocity.

S116: CELLULAR AND MOLECULAR MECHANISMS OF EVI1-EXPRESSING MLL-REARRANGED ACUTE MYELOID LEUKEMIA

Background: Expression of a doxycycline (DOX)-inducible acute myeloid leukaemia (AML)-associated iMLL-AF9 fusion transgene in long-term haematopoietic stem cells (LT-HSC) can lead to an invasive and chemo-resistant disease expressing the transcription factor EVI1. High EVI1 expression has been suggested as marker of poor outcome in AML patients even without rearrangements of the EVI1 locus at 3q26. Aims: We addressed the association between EVI1 expression, the cellular origin and poor disease outcome in AML driven by the iMLL-AF9 fusion gene. Methods: The role of EVI1 expression was studied in iMLL-AF9 transgenic mice carrying an Evi1-IRES GFP reporter in vitro using flow cytometry, colony formation and RT-qPCR assays, ex vivo with high-resolution bone marrow (BM) imaging, and in vivo, by transplantation of enriched naïve Evi1+ iMLL-AF9 hematopoietic stem and progenitor cells (HSPC) into irradiated recipients on DOX. Haematopoiesis of symptomatic mice was analysed by flow cytometry and histology. For mechanistic studies, single cell and bulk RNA sequencing was performed on enriched HSPC or BM samples from diseased mice. Results: Analysis of BM cells from Evi1-IRES-GFP reporter mice revealed that not only the mostly quiescent LT-HSC but also fractions of the more proliferating multipotent progenitors (MPP1-3) express abundant Evi1 (“Evi1high”). Induction of the iMLL-AF9 fusion did not result in significant changes in numbers of Evi1+ cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments. However, in colony assays, Evi1high iMLL-AF9 cells retained a more immature phenotype and produced more colonies with an invasive morphology than Evi1low cells (n=11, p<0.05). While Evi1 expression did not influence disease induction upon transplantation of LT-HSC, recipients of Evi1+ MPP1 cells developed AML earlier than Evi1- MPP1 (n=11, 79 vs. 269d, p<0.05). Disease induced by Evi1+ cells presented with more extensive leukemic organ infiltration than Evi1- AML. Evi1 expression also correlated with in vitro Ara-C resistance. We also examined whether some exogenous factors may increase AML susceptibility by expanding the Evi1+ HSPC. Although a single injection of recombinant mouse thrombopoietin (TPO) only increased the number of LT-HSC and not of MPP1, the Evi1high cell fraction was enlarged in both compartments (LT-HSC: 23 vs. 50%; MPP1: 22 vs. 47%; n=29, p<0.0001) supported by high-resolution imaging. Interestingly, increased TPO induced HSPC cycling was confined to the Evi1high cell population (n=3, p<0.05). Transplantation of TPO-treated iMLL-AF9 LT-HSC or MPP1 resulted in a significantly faster induction of Evi1+ AML than controls (n=19, MPP1: 35 vs. 79d, p<0.001; LT-HSC: 41 vs. 90d, p<0.001). To better understand mechanisms of aggravated AML after TPO mediated expansion of Evi1+ HSCP we performed multiplexed single cell RNA sequencing of highly enriched HSPC cells in iMLL-AF9 and control mice. While we observed no changes of cellular cluster organisation 2 days after TPO injection, we found some differentially expressed genes in TPO-stimulated cycling iMLL-AF9 HSPC, including potential stemness regulators. Summary/Conclusion: Our results suggest that expansion of Evi1-expressing HSPC by exogenous factors can result in a more aggressive MLL-AF9-driven AML. Ongoing data exploration and validation may characterize aberrantly expressed genes in TPO-stimulated Evi1+ iMLL-AF9-expressing HSPC as potential therapeutic targets to impair stemness of AML cells.

EVI1 Promotes the Proliferation and Invasive Properties of Human Head and Neck Squamous Cell Carcinoma Cells.

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor prognosis. So far, the EGFR inhibitor cetuximab is the only approved targeted therapy. A deeper understanding of the molecular and genetic basis of HNSCC is needed to identify additional targets for rationally designed, personalized therapeutics. The transcription factor EVI1, the major product of the MECOM locus, is an oncoprotein with roles in both hematological and solid tumors. In HNSCC, high EVI1 expression was associated with an increased propensity to form lymph node metastases, but its effects in this tumor entity have not yet been determined experimentally. We therefore overexpressed or knocked down EVI1 in several HNSCC cell lines and determined the impact of these manipulations on parameters relevant to tumor growth and invasiveness, and on gene expression patterns. Our results revealed that EVI1 promoted the proliferation and migration of HNSCC cells. Furthermore, it augmented tumor spheroid formation and the ability of tumor spheroids to displace an endothelial cell layer. Finally, EVI1 altered the expression of numerous genes in HNSCC cells, which were enriched for Gene Ontology terms related to its cellular functions. In summary, EVI1 represents a novel oncogene in HNSCC that contributes to cellular proliferation and invasiveness.

Establishment of a High-risk MDS/AML Cell Line YCU-AML1 and its Xenograft Model Harboring t(3;3) and Monosomy 7.

Acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) with both inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and monosomy 7 defines an extremely aggressive myeloid cancer whose molecular pathogenesis and optimal therapeutic strategy still remain unclear. We established a new MDS/AML cell line, YCU-AML1, and its patient-derived xenograft (PDX) model from a high-risk MDS patient who later transformed into AML harboring both t(3;3)(q21;q26.2) and monosomy 7. YCU-AML1 cells propagated in co-culture system with stromal cells in granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent manner. CD34+ bone marrow cells derived from our PDX model showed high EVI1 and low GATA2 expression. Moreover, mutational profile of our MDS/AML model was consistent with recently published mutational spectrum of myeloid malignancies with inv(3)/t(3;3). These data suggest that YCU-AML1 cells and its MDS/AML model strongly mimics a high-risk human myeloid cancer with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and monosomy 7 in terms of both clinical phenotype and molecular basis. We believe our model can be used as a feasible tool to further explore molecular pathogenesis and novel treatment strategy of high-risk MDS/AML with t(3;3)(q21;q26.2) and monosomy 7.

The role of EVI1 gene quantification in AML patients with 11q23/MLL rearrangement after allogeneic hematopoietic stem cell transplantation.

It remains unclear about the role of the EVI1 gene in AML patients with 11q23/MLL rearrangement (MLL-r AML) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We analyzed the clinical value of EVI1 gene quantification in 96 MLL-r AML patients. High EVI1 expression was found in 73% (70/96) of MLL-r AML patients, and EVI1-high MLL-r AML patients were characterized by high WBC counts (P = 0.046) and low platelet counts (P < 0.001) and commonly had t(6;11) (P = 0.032). In addition, a significant difference was observed in the SETD2 gene mutation between the EVI1 high and low groups (0% vs. 50%, P < 0.001). EVI1-high MLL-r AML patients had worse 2-year OS (49.8% vs. 79.7%, P = 0.01) and 2-year PFS (40.2% vs. 68.1%, P = 0.014) than EVI1-low patients. In 57 MLL-r AML patients undergoing allo-HSCT, poorer 2-year PFS (48.6% vs. 72.4%, P = 0.039) and higher CIR (33.2% vs. 11.1%, P = 0.035) were observed in the EVI1-high patients. Multivariate analysis revealed that pre-EVI1+ was the sole independent factor of high CIR (P = 0.035, HR = 4.97, 95% CI: 1.12-22.04). EVI1+ at 100 days post allo-HSCT was associated with a significantly higher 2-year CIR (P = 0.017). The quantification of the EVI1 gene could be used as an additional marker for early predicting relapse in allo-HSCT MLL-r AML patients.

Coexistence and Prognostic Significance of EVI1 Expression and Driver Mutations in KMT2A-Rearranged Acute Myeloid Leukemia

Background: KMT2A(MLL)-rearrangements are among the most frequent chromosomal abnormalities in acute myeloid leukemia (AML) and high EVI1 expression is known as an adverse prognostic factor in this subgroup. A recent study showed driver mutations are associated with poor prognosis in pediatric KMT2A-rearranged AML, however, the relationship between EVI1 expression and driver mutations is unclear. In this study, we examined coexistence and prognostic significance of these genetic abnormalities in pediatric and adult KMT2A-rearranged AML. Patients and Methods: Forty-four pediatric KMT2A-rearranged AML samples collected in the AML-05 study, conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG), were analyzed for 338 genes by targeted sequencing. In addition, 85 adult KMT2A-rearranged AML samples in MLL Munich Leukemia Laboratory were analyzed for 16 genes (ASXL1, BRAF, CBL, CEBPA, FLT3-ITD, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1, TP53 and WT1) by amplicon deep-sequencing, direct Sanger sequencing, or melting curve analysis. In both studies, EVI1 expression was measured using quantitative RT-PCR. Results and Discussion: In pediatric KMT2A-rearranged AML (n=44), among 28 patients with low EVI1 expression, 13 patients (EVI1-WT, 46.4%) had no driver mutations and 15 patients (EVI1-MT, 53.6%) had one or more driver mutations. Among 16 patients with high EVI1 expression, 2 patients (EVI1+WT, 12.5%) had no driver mutations and 14 patients (EVI1+MT, 87.5%) had one or more driver mutations. Mutations in activated signaling pathway genes (FLT3, NRAS, KRAS, PTPN11, CBL, and BRAF) were detected in most patients with EVI1-MT (12/15, 80.0%) and EVI1+MT (13/14, 93.3%). The frequency of mutations in epigenetic regulator genes (SETD2, ASXL1, ASXL2, BCOR, KDM6A, and CREBBP) was significantly higher in EVI1-MT patients (7/15, 46.7%), compared with EVI1+MT patients (1/14, 7.1%) (P=0.04). By contrast, the frequency of mutations in cohesion complex genes (STAG2 and SMC3) was significantly lower in patients with EVI1-MT patients (0/15, 0.0%), compared with EVI1+MT patients (4/14, 28.6%) (P=0.04). The frequency of mutations in transcription factor genes (WT1, MECOM, SPI1, GATA2, and RUNX1) was also lower in EVI1-MT patients (2/15, 13.3%), compared with EVI1+MT patients (5/14, 35.7%) (P=0.21). In adult patients, the frequency of ASXL1 mutations was higher in EVI1-MT patients (3/18, 18.8%), compared with EVI1+MT patients (1/29, 3.4%) (P=0.15) and those of WT1 and RUNX1 mutations were lower in EVI1-MT patients (0/18, 0.0%), compared with EVI1+MT patients (4/29, 13.8%) (P=0.28), which were compatible with the tendency in pediatric KMT2A-rearranged AML. Next, we examined the prognostic significance of EVI1 expression and driver mutations. Compared with EVI1-WT patients, EVI1-MT patients had lower event-free survival (EFS) (3-years EFS: 84.6% vs. 65.2%, P=0.24). EFS of EVI1+MT patients (3-years EFS: 30.8%) was significantly lower than that of EVI1-WT patients (P=0.001) and EVI1-MT patients (P=0.04). EFS of EVI1+WT patients (3-years EFS: 0.0%) was also lower than that of EVI1-WT patients (P=0.003) and EVI1-MT patients (P=0.09). There was no significant difference in EFS between EVI1+WT and EVI1+MT patients (P=0.88). The results of overall survival (OS) were similar except for EVI1+WT patients (n=2) (3-years OS: EVI1-WT 92.3%, EVI1-MT 70.6%, EVI1+WT 100.0%, EVI1+MT 46.6%). Multivariate analysis including EVI1 expression, driver mutations, age, white blood cell count, and KMT2A-MLLT4 fusion showed EVI1+ is an independent prognostic factor for EFS (hazard ratio (HR): 3.02, 95% confidence interval (CI): 1.08-9.48, P=0.04). There was no prognostic significance in driver mutations (HR:1.24, 95%CI: 0.39-4.74, P=0.73). As a whole, adult patients' survival data were lower, however, the tendency was similar to that of pediatric data (3-years EFS: EVI1-WT 41.7%, EVI1-MT 24.5%, EVI1+WT 9.1%, EVI1+MT 13.5%; 3-years OS: EVI1-WT 55.6%, EVI1-MT 30.7%, EVI1+WT 18.2%, EVI1+MT 36.2%). These data showed that there is an association between EVI1 expression and the pathway of driver mutations, suggesting these abnormalities may have some cooperative mechanisms in leukemogenesis. Compared with driver mutations, high EVI1 expression may have a stronger impact on poor prognosis in KMT2A-rearranged AML, however, the results should be confirmed in the larger cohort. Disclosures Ogawa: RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents &amp; Royalties. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract 2611: Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression

Abstract EVI1 (ecotropic viral integration site 1) is a transcription factor essential for the development and progression of acute myeloid leukemia (AML). Since high EVI1 expression, seen in approximately 10% of AML patients (EVI1high AML), is associated with poor clinical outcome, development of a novel therapeutic strategy is required. In this study, we investigated key molecules that are induced by aberrant EVI1 expression and modulate the metabolomics of leukemia cells. We conducted whole transcriptome analysis by RNA-sequencing in murine leukemia cells induced by retroviral transduction with EVI1 followed by transplantation into irradiated mice. We identified 45 genes with more than 10-fold expression in EVI1-transduced bone marrow cells compared to those with the mock vector at both pre-leukemic phase and leukemia phase. Intriguingly, the fructose bisphosphatase 1 gene (Fbp1), which encodes a key enzyme of gluconeogenesis was highly upregulated in EVI1-induced AML cells. We confirmed that FBP1 mRNA expression is quickly upregulated by ectopic expression of EVI1 in murine LSK (Linneg, c-Kitpos, Sca-1pos) cells. Moreover, we observed an enrichment of EVI1 in the promoter and enhancer region of Fbp1 by chromatin immunoprecipitation followed by qPCR analysis in murine hematopoietic cells, suggesting that Fbp1 expression is directly regulated by EVI1. Although high FBP1 expression suppresses glycolytic metabolism and negatively affects cellular proliferation in various types of solid tumor, its role in AML has not been investigated. In contrast to the results previously reported in other types of cancers, pharmacologic inhibition or short hairpin RNA (shRNA)-mediated knockdown of FBP1 significantly decreased colony-forming cell (CFC) capacity in EVI1-transduced murine LSK cells and significantly delayed progression of leukemia in the mice secondarily transplanted with EVI1-induced AML cells in vivo. These results suggest that aberrant expression of FBP1 is important for progression of EVI1high AML. While high FBP1 enzymatic activity negatively affects the glycolytic pathway, it in turn enhances the pentose phosphate pathway (PPP) through elevating the fructose-6-phosphate level. Activated PPP results in enhanced nucleotide synthesis essential for rapidly dividing cells. Importantly, individual knockdown of main PPP enzymes (G6pd, Pgd, Rpia) by shRNA transduction significantly decreased the CFC capacity of EVI1 transduced murine LSK cells, further confirming an important role of PPP in driving proliferation of EVI1high leukemia cells. Collectively, these results indicate that the activated PPP through transcriptional upregulation of FBP1 is crucial for progression of EVI1-driven leukemia. Since inhibition of FBP-1 did not compromise normal hematopoiesis, targeting the enzyme can be a promising therapeutic approach for the EVI1high AML. Citation Format: Hideaki Mizuno, Yuki Kagoya, Akira Chiba, Junji Koya, Yosuke Masamoto, Mineo Kurokawa. Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2611.

EVI1 in Acute Myeloid Leukemia Triggers Metabolic Reprograming Associated with Leukemogenesis and Increases Sensitivity to L-Aspalaginase

Leukemia cells survive and proliferate under conditions of metabolic stress by acquiring mutations that increase energy metabolism. Here, we aimed to identify a specific metabolic inhibitor and examine transcription factor-enhanced changes in energy metabolism by refractory leukemia cells. Overexpression of Ecotropic Virus Integration site 1 protein homolog (EVI1) in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. We focused on metabolic reprograming of MLL leukemia cells expressing EVI1, since the metabolic relationship between MLL and EVI1 is unclear. We used an extracellular flux analyze to examine metabolic changes during leukemia development in a mouse model of MLL-r AML expressing high levels of EVI1 (EVI1+).To examine whether EVI1 regulates energy metabolism in MLL-rearranged leukemia cells, we used transgenic mice expressing EVI1 (TG) in LSK and GMP cells model in which AML is driven by the MLL-AF9 oncogene. We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a flux analyzer. TG MLL-AF9 mice showed a significantly higher basal and capacity of OCR than WT MLL-AF9 mice ex vivo. EVI1+ cells showed accelerated oxidative phosphorylation (OXPHOS) prior to activation of glycolysis, and higher dependency on glutamine as an energy source. To identify the metabolic pathways regulated by EVI1, we performed capillary electrophoresis time-of-fight mass spectrometry-based metabolome profiling of WT and TG MLL-AF9 leukemia cells. We found significant differences between the cells in terms of the amounts of metabolites derived from the glycolytic and TCA cycles. Fructose 1,6-bisphosphate and lactate were up-regulated in TG MLL-AF9 cells, implying activation of glycolysis. Moreover, the amounts of fumarate and malate (metabolites of the TCA cycle) were significantly higher in TG MLL-AF9 cells. EVI1 played a role in glycolysis as well as driving expression of genes engaged in the tricarboxylic acid cycle. Next, we tested whether pharmacological inhibition of glycolysis and glutaminolysis suppresses MLL-AF9. L-asparaginase (ASP) [which catalyzes hydrolysis of asparagine (Asn) and glutamine (Gln) to asparatic acid or glutamic acid, respectively] markedly suppressed proliferation of TG MLL-AF9 cells, EVI1highAML cell lines. To examine the therapeutic potential of ASP in vivo, we treated secondary recipients of TG MLL-AF9 AML cells with ASP or control (vehicle), beginning 5 days post-transplantation. Mice then received intraperitoneal injections (five times per week) of distilled water or ASP (1000 U/kg). ASP led to a significant reduction in the number of GFP+ AML cells in the peripheral blood and increased the survival of recipient mice. Next, we examined an AML xenograft model. Two groups of NOG mice were injected subcutaneously with UCSD/AML1 cells and then treated with ASP or control. ASP -treated mice showed a significant reduction in the growth of AML tumors. Overall, these findings indicate that ASP -mediated inhibition of OXPHOS is a potential treatment for AML.We clarified that increased glutamine dependency by MLL-r AML cells showing high EVI1 expression makes them sensitive to ASP. We found that the energy advantage of AML cells is acquired via transcription factor-mediated activation of mitochondrial metabolism, leading to a poor prognosis. Furthermore, we show that new therapeutic options can be identified by examining the energy-based metabolic characteristics of leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

Expression of EVI1 in Bone Marrow of Patients with AML and ALL and Its Correlation with Leukemia Subtyping, Clinical Features and Prognosis of Patients

To explore the expression of EVI1 gene in AML and the ALL patients' bone marrow cells, and its correlation with the leukemic typing, clinical characteristics and prognosis. Three hundred and thirty-three cases of leukemia in our hospital from 2012 to 2016 were enrolled in this study. Among them there were 263 cases of AML, 70 cases of ALL, and 13 volunteers were selected as normal controls. The bone marrow samples of the patients and volunteers were aspirated. Firstly, the blood mononuclear cells(PBMNC) were separated from the samples through the Ficoll-Hypaque density gradient centrifugation. Secondly, the total DNA was extracted, and the expression of EVI1 mRNA were assayed by real-ime quantitative PCR, which help to find out the EVI1 mRNA expression difference between AML and ALL. Finally, the correlation of EVI1 expression with the acute leukemia typing, clinical characteristics and prognosis were analyzed by a series of statistical method. Based on the expression levels of the EVI1 mRNA determined by real-time quantitative PCR, the expression levels in AML and ALL were both higher, but without significant difference. The rates of both EVI1 mRNA overexpression and low expression of the EVI1 mRNA in AML patients were higher than those in AML group, however, the rate of normal expression in ALL group was statistically significantly lower than that in the AML. The level of Plt in patients with AML-M1 and T-ALL positively correlated with the level of EVI1 expression（r=0.393,P<0.05；ρ=0.442，P<0.05）while the level of blasts(%) in patients with AML-M3 negatively correlated with the level of EVI1 expression. The expression of EVI1 correlated with the age of patients（χ2 =6.684,P<0.05）. The prognosis of AML patients with high expression of EVI1 mRNA was poor（Log-Rank test，P<0.05）and AML-M3 patients with high expression of EVI1 had a significantly poorer prognosis than that of patients without high expression（Log-Rank test，P<0.01）. In expression level of EVI1 no difference has been found in AML and ALL, but the distribution of EVI1 expression shows obvious difference and the difference correlats with the age of patients, at the same time, the some clinical features and subtype of acute leukemia correlate with the expression level of EVI1.

Suppression of GPR56 expression by pyrrole-imidazole polyamide represents a novel therapeutic drug for AML with high EVI1 expression

G protein-coupled receptor 56 (GPR56) is highly expressed in acute myeloid leukemia (AML) cells with high EVI1 expression (EVI1high AML). Because GPR56 is a transcriptional target of EVI1 and silencing of GPR56 expression induces apoptosis, we developed a novel drug to suppress GPR56 expression in EVI1high AML cells. For this purpose, we generated pyrrole-imidazole (PI) polyamides specific to GPR56 (PIP/56-1 or PIP/56-2) as nuclease-resistant novel compounds that interfere with the binding of EVI1 to the GPR56 promoter in a sequence-specific manner. Treatment of EVI1high AML cell lines (UCSD/AML1 and Kasumi-3) with PIP/56-1 or PIP/56-2 effectively suppressed GPR56 expression by inhibiting binding of EVI1 to its promoter, leading to suppression of cell growth with increased rates of apoptosis. Moreover, intravenous administration of PIP/56-1 into immunodeficient Balb/c-RJ mice subcutaneously transplanted with UCSD/AML1 cells significantly inhibited tumor growth and extended survival. Furthermore, organ infiltration by leukemia cells in immunodeficient Balb/c-RJ mice, which were intravenously transplanted using UCSD/AML1 cells, was successfully inhibited by PIP/56-1 treatment with no apparent effects on murine hematopoietic cells. In addition, PIP treatment did not inhibit colony formation of human CD34+ progenitor cells. Thus, PI polyamide targeting of GPR56 using our compound is promising, useful, and safe for the treatment of EVI1high AML.

Erratum: EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

Retrospective Evaluation of Correlations Between Genetic Backgrounds and Stem Cell Transplantation for De Novo Pediatric Acute Myeloid Leukemia: A Study from the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Clinical Trial

IntroductionPediatric acute myeloid leukemia (AML) comprises less than 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Approximately 40% of patients still have a relapse after first-line therapies, and the expected long-term survival rate decreases following relapse. Stem cell transplantation (SCT) was a conclusive strategy for de novo AML patients with a high risk and relapsed or refractory patients with standard and intermediate risk in a recent clinical trial. AML is a molecularly and clinically heterogeneous disease caused by various genetic alterations. Thus, it is difficult to accurately evaluate risk stratification even if known representative molecular markers, including KIT, FLT3-ITD, t(8;21)/RUNX1-RUNX1T1, and KMT2A (also known as MLL)-rearrangements, are used.MethodsWe investigated differences in the genetic background between SCT and non-SCT groups in participants of the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. Among 369 patients with de novo AML, 175 patients received SCT. A standardized form was used to record clinical variables, including patient demographic information. The clinical data of patients in each risk group were followed for 3 years after the date of final registration. JPLSG performed a central review of morphologic classification and karyotyping based on the World Health Organization Classification, French-American-British classification, and cytogenetic analysis using conventional G-banding. Molecular characterization included mutational analyses of KIT (exons 8 and 17), N- and K-RAS (exons 1 and 2), NPM1 (exon 12), CEBPA (exon 12), FLT3-ITD, NUP98-NSD1,and CBFA2T3-GLIS2 gene rearrangement, as well as KMT2A- partial tandem duplication (MLPA methods). We also evaluated the gene expression of MECOM (also known as EVI1) and PRDM16 (also known as MEL1) because their high expressions are known poor prognostic markers. Overall survival (OS) was defined as the time from AML diagnosis to death or censorship at the last follow-up. Event-free survival (EFS) was defined as the time from AML diagnosis to treatment failure, relapse, death, or last follow-up.ResultsThe 3-year OS among SCT patients (n = 175) was approximately 50%. It was significantly worse than that of non-SCT patients (n = 194, 90%; P < 0.001). Among 137 CBF-AML patients, 44 patients (32%) received SCT and their 3-year OS was 80%. On the other hand, among 232 non-CBF-AML patients, 131 patients (57%) received SCT, and their 3-year OS was only 35%. This result indicated that SCT is beneficial for relapsed CBF-AML, whereas most non-CBF patients who received SCT did not obtain the clinical benefit. In terms of the molecular characteristics, among 58 patients with high EVI1 expression, 39 patients (67%) received SCT, and their 3-year OS was approximately 35%. EVI1 expression was especially useful when using with MLL rearrangement because prognosis of patients with both MLL rearrangement andhigh EVI1 expression were extremely poor. On the other hand, among 84 patients with high PRDM16 expression, 63 patients (75%) received SCT, and their 3-year OS was approximately 20%. Although all AML patients with FLT3-ITD were assigned to receive SCT in the AML-05 trial, FLT3-ITD(+) patients withlow PRDM16 expression had a better outcome than FLT3-ITD(+) patients with high PRDM16 expression (3-year OS: 70% vs. 21%; P < 0.001). This result indicated that FLT3-ITD itself might not necessarily be associated with poor prognosis. PRDM16 gene expression is a useful marker to select patients needing SCT, particularly for patients with FLT3-ITD.ConclusionSCT was beneficial for patients with CBF-AML, whereas SCT was insufficient to rescue patients with non-CBF AML who relapsed, particularly patients with both FLT3-ITD(+) and high PRDM16 expression. New strategies, such as gemtuzumab ozogamicin or haploidentical SCT, are urged to rescue high risk/refractory patients. DisclosuresNo relevant conflicts of interest to declare.

Targeting the Creatine Kinase Pathway in EVI1-Positive Acute Myeloid Leukemia

Abnormal expression of the transcription factor EVI1 through chromosome 3q26 rearrangements has been implicated in the development of one of the most therapeutically challenging high-risk subtypes of acute myeloid leukemia (AML). Here we integrated genomic and metabolic screening of hematopoietic stem cells to reveal that EVI1 overexpression altered cellular metabolism. A pooled shRNA screen targeting metabolic enzymes identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a druggable dependency in EVI1-positive AML.Of 18 screened AML cell lines harboring various genetic alterations, only the four EVI1-expressing lines exhibited markedly elevated CKMT1 protein expression and activity. Treatment of this cell line panel with either CKMT1-targeting shRNAs or cyclocreatine, an analog of the CKMT1 substrate creatine and inhibitor of the creatine biosynthesis pathway, showed that elevated CKMT1 protein expression correlated with sensitivity to CKMT1 pathway inhibition. Consistent with these data, flow cytometry analysis of a panel of 68 unselected primary AML patient specimens revealed that the four leukemias with the highest levels of EVI1 expression also had elevated CKMT1 protein levels and enhanced sensitivity to cyclocreatine treatment.We next established that enforced EVI1 expression increased CKMT1 protein and mRNA levels and that three independent shRNA molecules targeting EVI1 drastically reduced CKMT1 expression in two EVI1-positive AML cell lines. A luciferase-based reporter system established that RUNX1 represses CKMT1 expression through direct binding to its promoter. ChIP-qPCR approaches were then applied to dissect the sequential events involved in EVI1-induced CKMT1 upregulation and the possible role of RUNX1 as an intermediate. In both primary AML samples and cell lines, we determined that EVI1 represses RUNX1 expression by directly binding to its promoter. This, in turn, eliminates repressive RUNX1 binding at the CKMT1 promoter and thereby promotes CKMT1 expression. Based on these data, we explored the relationship between EVI1 and RUNX1 expression with CKMT1 mRNA levels in two AML transcriptional datasets (GSE14468 and GSE10358). We divided these cohorts into four subgroups with high versus low expression of EVI1 and RUNX1. Consistent with our mechanistic analysis, primary AML samples within the EVI1high/RUNX1low subgroup were significantly more likely to express high levels of CKMT1 than AML samples in the other three subgroups.CKMT1 promotes the metabolism of arginine to creatinine. To determine the effect of CKMT1 suppression on this pathway, we measured the metabolic flux of stable-isotope labeled L-arginine 13C6 through creatine synthesis using mass spectrometry. CKMT1-directed shRNAs or cyclocreatine selectively decreased intracellular phospho-creatine and blocked production of ATP by mitochondria. Salvage of the creatine pathway by exogenous phospho-creatine restored normal mitochondrial function, prevented the loss of viability of human EVI1-positive AML cells induced by cyclocreatine or CKMT1-directed shRNAs, and maintained the serial replating activity of Evi1-transformed bone marrow cells.Primary human EVI1-positive AML is frequently associated with somatic NRAS mutations. Thus, to investigate whether EVI1 over-expression sensitizes primary AMLs to CKMT1 inhibition in vivo, we transplanted primary NrasG12D mutant AMLs with and without elevated Evi1 expression into congenic recipient mice. In this system, Ckmt1 knockdown did not significantly alter the outgrowth of control Nras mutant AML cells compared to a shControl (63% versus 71%). By contrast, NrasG12D AML cells characterized by elevated Evi1 expression were profoundly depleted by Ckmt1 suppression to 2% versus 58% in shControl recipients. Consistent with these results, pharmacologic or genetic inhibition of the CKMT1-dependent pathway blocked disease progression and prolonged the survival of mice injected with human EVI1-positive cells but not with EVI1-negative cells, without noticeable cytotoxic effect on normal murine cells.In conclusion, we have integrated "omic" approaches to identify CKMT1 as a druggable liability in EVI-positive AML. This study supports a potential therapeutic avenue for targeting the creatine kinase pathway in EVI1-positive AML, which remains one of the worst outcome subtypes of AML. DisclosuresDeAngelo:Incyte: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Pfizer: Consultancy; Ariad: Consultancy. Stone:Pfizer: Consultancy; Agios: Consultancy; Jansen: Consultancy; Celator: Consultancy; Merck: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Novartis: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Sunesis Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy.

EVI1 expression, clinical and cytogenetical characteristics in 447 patients with acute myeloid leukemia

目的分析447例急性髓系白血病（AML）患者EVI1基因的表达及其临床和细胞遗传学特征。方法检测2007年1月至2015年4月收治的447例初诊AML患者的EVI1基因表达水平，分析其临床和细胞遗传学资料以及部分患者的分子学突变资料，总结EVI1基因高表达患者的特征。结果EVI1基因高表达者占17.9%，低表达者占82.1%。两组患者的年龄、性别、外周血血红蛋白水平、白细胞计数、血小板计数差异均无统计学意义；EVI1基因高表达组M0、M5和M6患者比例较低表达组显著增高（P值分别为0.027、0.004和0.011）。在细胞遗传学特征方面，EVI1基因高表达组11p15重排、11q23/MLL重排、3q26重排、−7/7q−以及t (9;11）患者比例较低表达组显著增高（P值分别为<0.001、< 0.001、<0.001、<0.001、0.014）；而正常核型、inv (16）、t (8;21）在EVI1基因低表达组占优势（P值分别为0.001、0.009、0.002）。EVI1基因高表达更多见于细胞遗传学高危组。NPM1突变更倾向分布于EVI1基因低表达组（P<0.001）。EVI1基因高表达组缓解率明显低于低表达组（P<0.001）。异基因造血干细胞移植（allo-HSCT）显著改善EVI1基因高表达者的无白血病生存。结论EVI1基因高表达多见于M5；核型分布中11p15重排、11q23/MLL重排、3q26重排、−7/7q−以及t (9;11）占优势；缓解率低，allo-HSCT能够改善预后。

Predictive value of high EVI1 expression in AML patients undergoing myeloablative allogeneic hematopoietic stem cell transplantation in first CR.

The EVI1 gene is a transcriptional regulator of hematopoietic stem cell self renewal and its overexpression is associated with adverse prognosis in de novo AML. Whether the overexpression of EVI1 also predicts poor outcome of AML patients undergoing myeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first CR (CR1) is still unclear. Thirty-two (21.2%) out of 151 patients were categorized as high EVI1 expression (EVI1+), and 119 (78.8%) patients were categorized as low EVI1 expression (EVI1-). The frequency of EVI1+ was much higher in the adverse-risk group than the intermediate-risk group (53% vs 19%, P=0.005). EVI1+ patients were significantly likely to harbor with translocations involving the MLL gene on 11q23 (8/9). Significantly poor results were observed in the EVI1+ cohort in terms of leukemia-free survival (LFS) (in 24 months 52.6 vs 71.0%, P=0.027), overall survival (OS) (in 24 months 52.8 vs 72.4%, P=0.012), and cumulative incidence of relapse (in 24 months 39.5 vs 22.5%, P=0.013). Multivariable analysis revealed that low EVI1 expression as an independent prognostic factor favoring LFS (hazards ratio=0.47, 95% confidence interval 0.26-0.86, P=0.01) but not OS. Our results indicate high EVI1 expression might predict high risk of relapse in AML patients undergoing myeloablative allo-HSCT in CR1.

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.