Upregulation Of HOXA10 Research Articles

Targeting Oncogenic Enhancers in ASXL1-Mutant Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR<2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

ALKBH5-mediated m6A modification of lncRNA KCNQ1OT1 triggers the development of LSCC via upregulation of HOXA9.

It has been shown that N6‐methyladenosine (m6A) modification is involved in the development of complex human diseases, especially in the development of cancer. Our research investigated the role and mechanism of the m6A modification of lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) in Laryngeal squamous cell carcinoma (LSCC) progression. Microarray analysis was used to quantitatively detect the m6A apparent transcriptional modification level of lncRNA in LSCC tissue. Methylated RNA immunoprecipitation‐qPCR (MeRIP‐qPCR), in situ hybridization (ISH) and quantitative real‐time PCR (qRT‐PCR) were used to examine the m6A modification and expression of KCNQ1OT1. In addition, in vivo and in vitro experiments have tested the effects of KCNQ1OT1 knockdown on the proliferation, invasion and metastasis of LSCC. Mechanically, we found the N6‐methyladenosine (m6A) demethylase ALKBH5 mediates KCNQ1OT1 expression via an m6A‐YTHDF2‐dependent manner and KCNQ1OT1 could directly bind to HOXA9 to further regulate the proliferation, invasion and metastasis of LSCC cells. In general, our research indicates that ALKBH5‐mediated m6A modification of KCNQ1OT1 triggers the development of LSCC via upregulation of HOXA9.

TIFAB accelerates MLL-AF9−Induced acute myeloid leukemia through upregulation of HOXA9

SummaryWe previously showed stabilization of NIK−induced activation of NF-κB non-canonical signaling suppresses MLL-AF9−induced AML. In the current study, we demonstrate that deletion of NF-κB non-canonical RelB prevents the inhibitory effect of NIK stabilization in MLL-AF9 AML. Mechanistically, RelB suppresses its direct target, TIFAB, which is upregulated in human AML and correlates negatively with the survival of AML patients. Forced expression of TIFAB reverses NIK−induced impaired AML development through downregulation of RelB and upregulation of HOXA9. Consistent with upregulation of HOXA9, gene set enrichment analysis shows that forced expression of TIFAB blocks myeloid cell development, upregulates leukemia stem cell signature and induces similar gene expression patterns to those of HOXA9-MEIS1 and HOXA9-NUP98, and upregulates oxidative phosphorylation. Accordingly, forced expression of HOXA9 also largely releases the inhibitory impact of NIK stabilization via downregulation of RelB and upregulation of RelA. Our data suggest that NIK/RelB suppresses MLL-AF9−induced AML mainly through downregulation of TIFAB/HOXA9.

Expression Profile and Prognostic Values of HOXA Family Members in Laryngeal Squamous Cell Cancer.

The homeobox A cluster (HOXA) gene family, comprising 11 members, is involved in a wide spectrum of biological functions in human cancers. However, there is little research on the expression profile and prognostic values of HOXA genes in laryngeal squamous cell cancer (LSCC). Based on updated public resources and integrative bioinformatics analysis, we assessed the expression profile and prognostic values of the HOXA family members. Expression and methylation data on HOXA family members were obtained from The Cancer Genome Atlas (TCGA). The prognostic values of HOXA members and clinical features were identified. A gene set enrichment analysis (GSEA) was conducted to explore the mechanism underlying the involvement of HOXA members in LSCC. The associations between tumor immune infiltrating cells (TIICs) and the HOXA family members were evaluated using the Tumor Immune Estimation Resource (TIMER) database. HOXA2 and HOXA4 were downregulated and HOXA7 and HOXA9–13 were upregulated in LSCC. Upregulation of HOXA10, HOXA11, and HOXA13, along with two clinical characteristics (M stage and gender), were associated with a poor LSCC prognosis based on the results of univariate and multivariate Cox proportional hazards regression analyses. Although there were no significant correlations between TIICs and HOXA members, the GSEA results indicated that HOXA members participate in multiple biological processes underlying tumorigenesis. This study comprehensively analyzed the HOXA members, providing insights for further investigation of the HOXA family members as potential targets in LSCC.

Expression Profile and Prognostic Values of <i>HOXA</i> Family Members in Laryngeal Squamous Cell Cancer

The homeobox A cluster (HOXA) gene family, comprising 11 members, is involved in a wide spectrum of biological functions in human cancers. However, there is little research on the expression profile and prognostic values of HOXA genes in laryngeal squamous cell cancer (LSCC). Based on updated public resources and integrative bioinformatics analysis, we assessed the expression profile and prognostic values of the HOXA family members. Expression and methylation data on HOXA family members were obtained from The Cancer Genome Atlas (TCGA). The prognostic values of HOXA members and clinical features were identified, and a nomogram predicting survival was generated. A gene set enrichment analysis (GSEA) was conducted to explore the mechanism underlying the involvement of HOXA members in LSCC. The associations between tumor immune infiltrating cells (TIICs) and the HOXA family members were evaluated using the Tumor Immune Estimation Resource (TIMER) database. HOXA2 and HOXA4 were downregulated and HOXA7 and HOXA9–13 were upregulated in LSCC. Upregulation of HOXA10, HOXA11 and HOXA13, along with two clinical characteristics (M stage and gender), were associated with a poor LSCC prognosis based on the results of univariate and multivariate Cox proportional hazards regression analyses. In this study, we generated a nomogram to visualize the associations between each prognostic indicator and LSCC prognosis. Although there were no significant correlations between TIICs and HOXA members, the GSEA results indicated that HOXA members participate in multiple biological processes underlying tumorigenesis. This study comprehensively analyzed the HOXA members, providing insights for further investigation of the HOXA family members as potential targets in LSCC. Funding Statement: This research was supported by the Key Discipline Foundation of HwaMei Hospital, University of Chinese Academy of Sciences, China [grant number 2016012] and Ningbo Health Branding Subject Fund [grant number PPXK2018-02]. Declaration of Interests: The authors declared: None of the authors has any commercial or other associations that might pose a conflict of interest. Ethics Approval Statement: Not required.

Abstract 3898: Structure-based optimization of small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia (MLL)

Abstract Chromosomal translocations that affect the Mixed Lineage Leukemia (MLL) gene result in acute myeloid and lymphoid leukemias. Fusion of MLL with one of 60 different partner genes generates MLL fusion proteins which lead to enhanced cell proliferation, up-regulation of Hoxa9 and Meis1 genes and block hematopoietic differentiation, ultimately leading to acute leukemia. Importantly, the N-terminal region of MLL is retained in all MLL fusion proteins and represents the interaction site of menin, a critical oncogenic cofactor for MLL fusion proteins. As a result, inhibition of the menin-MLL interaction should abrogate the development and progression of MLL leukemia. Therefore, disruption of the menin-MLL interaction with small molecule inhibitors might represent a therapeutic strategy for patients harboring MLL-rearrangements. The development of potent small molecule inhibitors of protein-protein interactions with optimized drug-like properties represents a challenging task in the lead optimization process. Here, we employed structure-based design with extensive medicinal chemistry efforts to optimize the thienopyrimidine class of menin-MLL inhibitors represented by the MI-136 compound. Our efforts resulted in development of MI-538, which represents the most potent small molecule inhibitor of the menin-MLL interaction developed to date. MI-538, which binds to menin with a Kd of 6.5 nM demonstrated superior cellular activity in MLL-AF9 transformed leukemic cells with a GI50 = 83 nM. In a mouse xenograft model utilizing MV4:11 human MLL leukemia cells, treatment with MI-538 significantly reduced tumor growth. Overall, systematic exploration of substituents on MI-136 led to the identification of MI-538 with improved activity, selectivity, polarity and pharmacokinetic profile, making it suitable for in vivo studies. Interestingly, we found that simultaneous incorporation of multiple substituents optimized for each site on the cyanoindole ring of MI-136 does not always positively affect the activity or drug-like properties of these compounds. This study demonstrates challenges in optimizing inhibitors of protein-protein interaction for potential therapeutic applications. Citation Format: Jonathan Pollock, Dmitry Borkin, Katarzyna Kempinska, Trupta Purohit, Xiaoqin Li, Bo Wen, Ting Zhao, Hongzhi Miao, Shirish Shukla, Miao He, Duxin Sun, Tomasz Cierpicki, Jolanta Grembecka. Structure-based optimization of small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia (MLL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3898.

Mouse Modeling of GATA2 Related MDS/AML

GATA2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis as well as lymphatic angiogenesis. Human GATA2 deficiency disease is caused by a variety of mutations in GATA2 which lead to the clinical phenotypes variously known as monocytopenia and mycobacterial disease (MonoMac), familial MDS/AML, dendritic cell, myeloid and NK cell lymphopenia (DCML), Emberger syndrome, and classical NK cell deficiency. Patients with GATA2 deficiency have an immunodeficiency with monocytopenia, NK, and B cell leukopenia; are susceptible to mycobacterial, viral and fungal infections as well as MDS and progression to AML. Additionally, patients may suffer from lymphedema and pulmonary alveolar proteinosis. GATA2 mutations have also been associated with aplastic anemia and severe congenital neutropenia.In order to understand the mechanisms underlying the development of MDS/AML in GATA2 deficiency we developed and analyzed a unique mouse model. The previously characterized Gata2+9.5+/- mouse has an intronic regulatory region mutation identical to one found in some patients. However, these mice do not develop MDS/AML spontaneously. We hypothesized that crossing these mice to other MDS/AML prone strains would accelerate the progression of MDS/AML. Specific HOX genes are implicated in this disease process. GATA2-deficient patients often develop monosomy 7 and ASXL1 mutations, both of which lead to the overexpression of HOX genes including HOXA9. The known AML translocation t(2;11)(q31;p15) results in the fusion of NUP98 and HOXD13 (NHD13) and the upregulation of HOX gene expression. Gata2 was identified in a mouse retroviral insertion screen as a potential collaborator in NHD13-mediated leukemogenesis. We hypothesized that hematopoietic specific Hox gene overexpression in a mouse with germline Gata2 haploinsufficiency would lead to the development of MDS/AML. This was done using the established NHD13 transgenic strain crossed to the Gata2+9.5+/- strain. The NHD13 mice express the fusion protein under the control of the hematopoietic specific vav promoter, and have up-regulation of Hoxa5, Hoxa7, Hoxa9, and Hoxa10. Secondly, elevated Flt3 ligand levels have been detected in GATA2-deficient patients as they progress from normal bone marrow morphology to MDS/AML. To model the hyper-activation of the receptor Flt3 by Flt3 ligand, we used a model of constitutively active Flt3, the Flt3-ITD mouse.Gata2+9.5+/- mice were bred to the NHD13 and the Flt3-ITD transgenic strains and compound heterozygotes were analyzed. Gata2+9.5+/-;NHD13 compound heterozygous mice were born normally and at Mendelian ratios. No significant differences in blood cell counts were noted in Gata2+9.5+/-;NHD13 compound heterozygotes until disease progression. These mice developed aggressive AML at 9-14 months of age, similar ages and rates as the NHD13 only mice. Whereas compound Gata2+9.5+/-;Flt3-ITD mice are also born in Mendelian ratios, starting at 10 weeks of age they developed B-cell lymphopenia, similar to that which is seen in human GATA2 deficiency patients displaying B-cell loss early in disease. The Gata 2+9.5+/-; Flt3-ITD mice exhibited increased circulating c-Kit-positive cells in the peripheral blood. Studies to further characterize hematopoiesis in the Gata2+9.5+/-; Flt3-ITD mice, and analyze for the development of MDS/AML are in progress. The Gata 2+9.5+/-; Flt3-ITD mouse model holds promise for the further study of MDS/AML in the context of GATA2 haploinsufficiency. DisclosuresAplan:NIH Office of Technology Transfer: Patents & Royalties.

Traditional Chinese Medicine, the Zishen Yutai Pill, Ameliorates Precocious Endometrial Maturation Induced by Controlled Ovarian Hyperstimulation and Improves Uterine Receptivity via Upregulation of HOXA10.

Controlled ovarian hyperstimulation (COH) is widely used in assisted reproductive technology (ART), but it often leads to precocious maturation of the endometrium such that it impairs embryonic implantation and limits pregnancy rates. Previous studies have shown the traditional Chinese medicine, the Zishen Yutai pill (ZYP), to be effective in treatment of threatened as well as recurrent miscarriages, and it can improve embryonic implantation rates in patients undergoing IVF treatment. In the present study, the ZYP has been found to ameliorate precocious endometrial maturation in a mouse model of different COH. Molecular evaluations, real-time PCR, relative RT-PCR, Western blotting, and immunohistochemistry have indicated that the ZYP increased the expression of HOXA10, an important marker of uterine receptivity. Elevation of HOXA10 led to further upregulation of its target gene, integrin β3, and downregulation of EMX2, two additional markers of uterine receptivity. In this way, the ZYP may mitigate COH-induced precocious maturation of the endometrium and improve uterine receptivity by upregulating HOXA10.

Frequency of a Highly Polymorphic Variant, S36N GFI1, Is Increased in CML Patients

To date no single molecular biomarker, with proven clinical utility, that is predictive and identifies patients with more aggressive chronic myeloid leukaemia (CML) at diagnosis, has been reported. Such a molecular biomarker would enable stratification of patients according to aggressiveness of the malignancy and therefore at greater risk of progressing to blast crisis (BC). If these patients could be identified a more potent TKI could be prescribed at presentation or be considered for stem cell transplant up front. Recently a common polymorphic variant of growth factor independent 1 (GFI1), a zinc finger DNA binding transcriptional repressor, whereby serine is substituted by asparagine at residue 36 (S36N), was reported (Khandanpour et al. Blood. 2010; 115:22462) to be associated with a 1.6 fold increased risk of developing acute myeloid leukemia (AML). In addition, investigators reported a frequency of 11% of the S36N GFI1 allele among AML patients, compared with 3-7% among the European populations. Reports suggest S36N GFI1 variant fails to repress the target genes, including HOXA9, and as a consequence their expression levels are elevated. These observations are consistent with a critical role for GFI1 in myeloid differentiation. In view of these findings we hypothesized that the GFI1 S36N allele might influence CML progression, via up-regulation of HOXA9, from a relatively indolent chronic phase (CP) to invariably fatal BC, characterized by presence of leukaemic stem cells, resistant to tyrosine kinase inhibitors in peripheral blood. Therefore, we assessed if S36N allele frequency was increased in CML and quantified GFI1expression.We retrospectively studied 106 cDNA samples, synthesized using 1 µg RNA extracted from nucleated cellular pellets isolated from peripheral blood or bone marrow aspirate. Total RNA was reverse transcribed using reverse transcriptase primed with random hexamers oligonucleotides. Of the 196 sample 89 were from highly heterogeneous CML patients and the remaining 17 comprised of randomly selected normal control samples from healthy adult volunteer blood donors. The 89 CML patients included 24 (accelerated phase: 2 ; BC:22) with 51 years median age (range 20-75) who had progressed to advanced disease (AD) of these 13 were males. The remaining 65 CML patients (M:43 ; F: 22) were in chronic phase (CP) with median age 74 years (range 7-90). Polymerase chain reaction products, containing GFI1 exons 2, 3 and 4 were subjected to restriction fragment length polymorphism using BfaI restriction enzyme to test for the S36N GFI1 allele. Samples with the S36N allele were confirmed by Sanger sequencing. Total GFI1 (i.e. serine and asparagine alleles), HOXA9 and GUSß (endogenous control gene) mRNA transcripts were quantified by quantitative real time using Taqman assay. Each Taqman assay included a standard curve constructed using 2 plasmids containing HOXA9 or GUSß insert. Serially diluted cDNA derived from MEG01 hematopoietic cell line was used to prepare the standard curve for GFI1quantification.S36N GFI1 allele frequency was significantly increased (p<0.001) among the CML patients (11/89) compared with the normal control group (1/17), but we detected no significant difference between AD and CP samples. However, the total GFI1 mRNA expression was significantly increased in CP samples compared with the control group (p<0.0001). But its expression was significantly decreased in AD compared with those in CP (p=0.0096). Interestingly HOXA9 expression was significantly increased (p=0.018) among individuals expressing the asparagine allele (i.e. heterozygous and homozygous subjects). But we observed no correlation between HOXA9 and GFI1 expression in CML patients.These data suggest an association of S36N GFI1 allele with CML. Furthermore, the study implies the variant allele is associated with increased HOXA9 expression, which may influence disease progression viaMusashi 2. HOXA9 is reported to upregulate Musashi 2, which we and others have shown is increased in CML BC patients. The observed inverse expression of GFI1 levels between CP and AD is consistent with its reported role in myeloid cellular differentiation. These observations imply two mechanisms by which GFI influences CML disease kinetics: decreased GFI1 expression and/or elevated HOXA9 levels due to the presence of the S36N GFI1 allele. Disclosuresle Coutre:Novartis: Honoraria.

Cell fate decisions in malignant hematopoiesis: leukemia phenotype is determined by distinct functional domains of the MN1 oncogene.

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.

Upregulation HOXA10 homeobox gene in endometrial cancer: role in cell cycle regulation

Homeobox genes encode a group of transcription factors that regulate embryonic development, organ differentiation and cell proliferation. Some researchers found that homeobox gene family could regulate the expression of cell cycle-related factors which are involved in tumorigenesis. The aim of the present study was to determine whether HOXA10 gene could regulate the expression of p21 and control the cell cycle in endometrial cancer. Real-time PCR and Western blot were used to analyze HOXA10 and p21 gene expression in tissue samples of normal endometrium and endometrial cancer. Upregulate and downregulate HOXA10 gene in endometrial cancer cells by HOXA10-vector and HOXA10-siRNA, p21 mRNA and protein expression were tested by real-time PCR and Western blot. FCM analyze the cell cycle alteration, and cell count and EdU assay were recruited to detect cell proliferative activity after upregulation of HOXA10. Expression of HOXA10 and p21 gene decreased in endometrial cancer, and the expression level of HOXA10 was correlated with cancer grade. The percentage of G1 phase cells increased, and cell proliferative activity decreased after upregulate HOXA10 expression. The expression of p16, p27, Myc, Cyclin D1, Cyclin E, CDK2, CDK4 and CDK6 did not alter after upregulation HOXA10. P21 gene mRNA and protein increased/decreased after upregulation/downregulation HOXA10 expression in cancer cells. HOXA10 regulates G1 phase arrest in endometrial cancer which may be mediated by p21.

The O-Linked N-Acetylglucosamine Transferase Modifies HOXA9 and Inhibits HOXA9-Driven Immortalization

The homeodomain-containing transcription factor HOXA9 is a core element of the HOXA9 enhanceosome, a critical DNA-protein complex that regulates hematopoietic stem cell self-renewal during hematopoiesis. Several genetic mutations observed in acute myeloid leukemia (AML) patients, including MLL translocations, are associated with aberrant up regulation of HOXA9, thus disrupting the hematopoietic balance towards leukemogenesis. While analyses of HOXA9 and cofactors have uncovered fundamental aspects of the mechanisms through which these proteins mediate their functions, questions remain. For example, what molecular mechanisms contribute to switching HOXA9 enhanceosomes off during myeloid differentiation? Could these mechanisms be targeted for the therapeutic benefit of leukemia patients? Characterization of the molecular interactions in which HOXA9 enhanceosome proteins are involved should shed light on the mechanisms that govern these proteins during both normal hematopoiesis and leukemogenesis. We recently discovered that HOXA9 interacts physically with OGT, the only O-linked N-acetyl glucosamine transferase in humans. We also demonstrated that HOXA9 is O-GlcNAcylated by OGT. Investigation of the functional relevance of this interaction to HOXA9-driven leukemogenesis is currently under way using interaction- and O-GlcNAcylation-deficient alleles of HOXA9 in a colony formation assay. Our preliminary results suggest that OGT inhibits HOXA9's ability to transform primary bone marrow cells, thus suggesting OGT is a potential tumor suppressor of HOXA9-driven leukemogenesis. Current efforts focus on further dissecting the molecular interplay occurring between HOXA9 and OGT on chromatin, its impact on the regulation of HOXA9 targets and its role in HOXA9-driven leukemogenesis. Work is also under way to identify factors involved in the OGT-mediated regulation of HOXA9 enhanceosomes. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Disorder May Cause Downregulation of HOXA10 in the Eutopic Endometrium of Fertile Women With Endometriosis

HOXA10 is an important gene for endometrial receptivity and plays a regulatory role in the adult female reproductive tract. It is regulated by epigenetic modulation in the CpG clusters of promoter in some cases. The aim of this study was to investigate HOXA10 expression and the epigenetic regulation in the eutopic endometrium of fertile women with endometriosis by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot. The effect of 5-azacytidine (5-ac), a demethylation agent on HOXA10 expression was determined on endometrium stromal cells (ESCs) from these women with endometriosis. Results revealed that in normal endometrium (NE), HOXA10 messenger RNA (mRNA) and protein expression at the secretory phase were significantly higher than that at the proliferative phase. The HOXA10 mRNA and protein expression in the eutopic endometrium of endometriosis were significantly lower than in NE. The HOXA10 mRNA and protein levels in cultured stromal cells from endometriosis in vitro were significantly increased in a 5-ac treatment group compared with a nontreatment group. Our results indicated that the level of HOXA10 decrease in the eutopic endometrium of patients with endometriosis. Upregulation of HOXA10 in ESCs after treatment with 5-ac suggests that HOXA10 expression is controlled by methylation of the promoter. An epigenetic aberration is likely the main cause of endometriosis.

Mechanisms of Transcriptional Regulation and Transformation by HOXA9

Abstract SCI-30HOXA9 plays important roles in both development and hematopoiesis and is overexpressed in more than 50 percent of acute myeloid leukemias (AML). Nearly all cases of AML with mixed lineage leukemia (MLL) translocations show increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. In most cases, upregulation of HOXA9 is accompanied by upregulation of its homeodomain-containing cofactor MEIS1, which directly interacts with HOXA9. While HOXA9 alone is sufficient for transformation of hematopoietic stem cells in culture, the addition of MEIS1 increases the transformation efficiency and results in rapidly fatal leukemias in transplanted animals. Despite the crucial role that HOXA9 plays in development, hematopoiesis, and leukemia, its transcriptional targets and mechanisms of action are poorly understood. We have used ChIP-seq to identify Hoxa9 and Meis1 binding sites on a genome-wide level in myeloblastic cells, profiled their associated epigenetic modifications, identified the target genes regulated by HOXA9 and identified HOXA9 interacting proteins. HOXA9 and MEIS1 cobind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/P300 binding. These include many proleukemogenic gene loci, such as Erg, Flt3, Myb, Lmo2, and Sox4. In addition, HOXA9 binding sites overlap a subset of enhancers previously implicated in myeloid differentiation and inflammation. HOXA9 binding at enhancers stabilizes association of MEIS1 and lineage-restricted transcription factors, including C/EBPα, PU.1, and STAT5A/B thereby promoting CBP/p300 recruitment, histone acetylation, and transcriptional activation. Current efforts are focused on using both biochemical and genetic approaches to assess the role of HOXA9 “enhanceosome” components C/EBPα, PU.1, and STAT5A/B in transcriptional regulation and leukemogenesis. Studies to date suggest that C/EBPα and PU.1 binding can occur in the absence of HOXA9/MEIS1, supporting a model in which these proteins act as pioneer transcription factors for establishment of poised, but not activated, HOXA9-regulated enhancers. Work is under way to assess the impact of high-level HOXA9 and MEIS1 on enhanceosome assembly and the role of recruitment of transcriptional coactivators involved in target gene up- or downregulation, including histone acetyltransferases and chromatin remodeling complexes. Collectively, our findings suggest that HOXA9-regulated enhancers are a fundamental mechanism of HOX-mediated transcription in normal development that is deregulated in leukemia. Disclosures:No relevant conflicts of interest to declare.

Upregulation of HOXA10 in gastric cancer with the intestinal mucin phenotype: reduction during tumor progression and favorable prognosis

Gastric cancer (GC) is one of the most common malignancies worldwide. Better knowledge of the changes in gene expression that occur during gastric carcinogenesis may lead to improvements in diagnosis, treatment and prevention. In this study, we screened for genes upregulated in GC by comparing gene expression profiles from microarray and serial analysis of gene expression and identified the HOXA10 gene. The aim of the present study was to investigate the significance of HOXA10 in GC. Immunohistochemical analysis demonstrated that 221 (30%) of 749 GC cases were positive for HOXA10, whereas HOXA10 was scarcely expressed in non-neoplastic gastric mucosa except in the case of intestinal metaplasia. Next, we analyzed the relationship between HOXA10 expression and clinicopathological characteristics. HOXA10 expression showed a significant inverse correlation with the depth of invasion and was observed more frequently in the differentiated type of GC than in the undifferentiated type of GC. HOXA10 expression was associated with GC with the intestinal mucin phenotype and correlated with CDX2 expression. Furthermore, the prognosis of patients with positive HOXA10 expression was significantly better than in the negative expression cases. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and wound healing assay revealed that knockdown of HOXA10 in GC cells by short interfering RNA transfection significantly increased viability and motility relative to the negative control, indicating that HOXA10 expression inhibits cell growth and motility. These results suggest that expression of HOXA10 may be a key regulator for GC with the intestinal mucin phenotype.

Implication of HOXA9, MEIS1, AML1 and IRF4 In Childhood Acute Leukemia (AL) In Greek Patients

AbstractAbstract 4643 Introduction:Childhood AL is characterized by abnormal chromosomal translocations and gene fusions. The most common cytogenetic aberrations are: TEL/AML1 (25%), E2A/PBX1 (4.8%), BCR/ABL (1.6%), MLL rearrangements (1.6%) and AML1/ETO (exclusively found in acute myeloid leukemia (AML)). Their high prognostic significance determines the intensification (eg. for MLL rearrangements) or de-intensification (eg. for TEL/AML1) of therapy, even though the mechanisms through which these fusions affect the prognostic outcome are largely unknown. However, the majority (~70%) of childhood patients is negative for these translocations and, even though most present with numeric cytogenetic abnormalities (eg. aneuploidies) at diagnosis, they are treated with the same therapeutic protocols as those with known translocations. Only BCR/ABL+ AL patients receive individualized treatment (addition of imatinib mesylate) that offers a better prognostic outcome, thus representing an example of how childhood leukemia can benefit from individualized therapy. The study of the genes implicated in leukemic lesions can give insight into the pathogenesis of leukemia and hence provide new diagnostic markers and therapeutic targets. It is therefore fundamental to make scientific hypotheses based on the behaviour of such genes and then perform experiments to validate or reject them. In the present study we investigate the expression of 4 such genes: HOXA9 and MEIS1, implicated in both normal hematopoiesis and MLL+ leukemias; AML1, an important regulator of B-cell differentiation and the most frequent target of chromosomal translocations; and IRF4, a transcription factor of lymphocyte differentiation, also implicated in leukemias and lymphomas. The aim of this study is to examine whether there is a correlation between the expression of the above genes and the leukemic cytogenetic profile. Materials and Methods:RNA was extracted with Trizol (Invitrogen Inc.) from bone marrow samples of 43 newly diagnosed children with leukemia (39 acute lymphoblastic leukemia (ALL) and 4 AML), 20 healthy children (controls) and from 4 cell lines, each representing a different type of childhood leukemia: CCRF-CEM (T-cell ALL), CCRF-SB (B-cell ALL), Reh (non-T, non-B ALL) and THP-1 (acute monocytic leukemia). Gene expression was investigated with Real-Time Reverse Transcription PCR (qRT-PCR), using the Plexor one-step qRT-PCR kit (Promega Inc.). Fluorescence in situ hybridization (FISH) was performed with dual-color probes (Abbott Molecular, Inc.). Results:Cytogenetic data were available for 41 patients (pts): TEL/AML1+: 5 pts (ALL) (12.2%), MLL+: 4 pts (3 ALL and 1 AML) (9.7%), BCR/ABL+: 3 pts (ALL) (7.3%), E2A/PBX+: 2 pts (ALL) (4.8%), AML1/ETO+: 1 pt (AML); the remaining 26 patients (63%) were negative for these fusions but 6 (14.6%) had extra copies of the AML1 gene. Gene expression was as follows: HOXA9: up-regulated in 20 patients (46.51%) and the CCRF-SB, Reh and THP-1 cell lines; MEIS1: up-regulated in 19 patients (44.19%); AML1: up-regulated in 21 patients (48.84%) and all 4 cell lines; IRF4: up-regulated in 25 patients (58.14%) and down-regulated in the CCRF-SB cell line. Simultaneous co-expression of HOXA9 and MEIS1 was present in 15 patients (34.88%). Of the 6 patients having extra copies of AML1, only one showed over-expression of the gene. Conclusions:In our group of patients the frequency of the TEL/AML1 fusion appears to be less, whereas the frequency of the MLL rearrangement appears to be higher, than that reported for western European countries. We find all of the 4 genes studied significantly up-regulated in certain groups of patients, as compared to controls, regardless of leukemic subtype. Up-regulation of HOXA9 and MEIS1 did not appear to be an exclusive characteristic of the MLL+ group. In the case of the AML1 gene, we find that gene amplification does not correlate with over-expression of the gene. In addition, IRF4, a known tumor suppressor gene which would be expected to be down-regulated in newly diagnosed leukemias, seems to be the one most frequently up-regulated (58.14% of patients). Our findings lead us to suggest that there might be common patterns of aberrant gene expression among childhood leukemia patients regardless of cytogenetic subtype. Such patterns could be further investigated in an attempt to gain insight into the etiology of leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

NUP98-HOXA9 Reprograms Embryonic Hematopoiesis, Suppresses Cellular Apoptosis, and Causes Malignant Tissue Infiltrates in Transgenic Zebrafish.

Abstract 3961Poster Board III-897The NUP98-HOXA9 fusion oncogene results from a t(7;11)(p15;p15) chromosomal translocation and is associated with inferior prognosis in de novo and treatment-related acute myeloid leukemia (AML), as well as blast crisis in chronic myeloid leukemia (CML). HOXA9 belongs to the highly-conserved HOX gene family of developmental transcription factors and is critical for vertebrate hematopoiesis. Upregulation of HOXA9 is a frequent phenomenon in human AML, implicating a central role in myeloid disease. Thus, elucidating the activity of oncogenic NUP98-HOXA9 may reveal universal mechanisms of leukemogenesis and lead to design of targeted therapies. The zebrafish is a robust model for studying vertebrate hematopoiesis and leukemogenesis, by virtue of its ex utero development, and conserved genetics and cell biology. We have engineered a Cre-lox inducible transgenic zebrafish harboring the human NUP98-HOXA9 fusion oncogene downstream of the zebrafish pu.1 promoter. NUP98-HOXA9 perturbed zebrafish embryonic hematopoiesis, leading to upregulated expression of myeloid-specific genes, pu.1, lysC, and l-plastin, and downregulation of the erythroid-specific gene, gata1. These changes in gene transcription affect both ‘primitive' and ‘definitive' waves of zebrafish hematopoiesis (including the recently-described erythro-myeloid progenitors [EMPs]), suggesting that NUP98-HOXA9 reprograms blood cell precursors to a predominantly myeloid fate. Preferential upregulation of pu.1 during ‘primitive' hematopoiesis further suggests an impaired potential for terminal myeloid differentiation. These effects appear developmentally mediated, as cell proliferation was not increased as measured by phosphorylated histone-H3 (pH3) staining. However, following ionizing irradiation (IR), NUP98-HOXA9 expression preserved cell proliferation and suppressed apoptosis in the presence of DNA damage. Irradiated transgenic embryos displayed wild-type levels of pH3, suggesting inhibition of cell cycle checkpoints. Irradiated embryos also displayed reduced levels of the conserved apoptotic marker, activated caspase-3, as well as downregulation of the puma pro-apoptotic gene, and upregulation of bcl2 and bcl-xL anti-apoptotic genes. These data postulate suppression of caspase-3-dependent apoptosis as a mechanism for NUP98-HOXA9 -mediated oncogenesis in vivo. Incomplete phenotypic ‘rescue' with bcl-xL morpholino suggests insufficiency of single-target therapy, with a need for multi-target interruptions to restore radiosensitivity. Between 20-24 months post fertilization, a proportion of these NUP98-HOXA9 transgenic zebrafish (n=9/19; 47%) presented with gross abdominal masses and/or abnormal swimming behavior. Thus far, histological sectioning has demonstrated enlarged kidneys and livers, with evidence of infiltrative, pleiomorphic, mitotically-active cells with myeloid morphology. This pathology is reminiscent of the myeloproliferative disease (MPD) observed in NUP98-HOXA9 transgenic mice. Taken together, these data provide insights into the mechanisms underlying NUP98-HOXA9-mediated leukemogenesis and provide an unprecedented opportunity for in vivo chemical modifier screens to identify promising therapeutic agents that restore a normal phenotype. Disclosures:No relevant conflicts of interest to declare.

Establishment and Characterization of A New Leukemia Mouse Model Induced by MLL/AF4 Fusion Protein.

Abstract 3573Poster Board III-510 BackgroundMLL/AF4 fusion is the most common abnormality in infant leukemia and is associated with a poor prognosis. Although further study and treatment should be groped for acute lymphoblastic leukemia (ALL) with MLL/AF4, the lack of a proper animal model has impeded understanding of the molecular mechanism of leukemia associated with the MLL/AF4 fusion gene. Previous mouse models suggest that the tumorigenesis capacity of MLL/AF4 alone is insufficient for development mixed-lineage leukemia. MLL/AF4 is capable of inducing lymphoid malignancy with long latency, which is very different from aggressive pro-B ALL in humans. Recently, we have reported that enhancement of S100A6 (calcyclin) expression played an important role in MLL/AF4-leukemogenesis using murine 32Dc cell line. In this study, we tried to make improved MLL/AF4 transgenic (Tg) mice which reflect human leukemogenicity and analyzed molecular mechanism of MLL/AF4 fusion gene. MethodsTo get high and sustained expression of MLL/AF4 in hematological lineage, we used MSCV promoter to express MLL/AF4 fusion gene. After MLL/AF4 expression plasmids were injected into fertilized eggs of mice (C57BL/6), the manipulated embryos were transferred into the oviducts of pseudopregnant females to make MLL/AF4 Tg mice. Finally we characterized MLL/AF4 Tg mice and analyzed molecular mechanism of MLL/AF4-leukemogenesis. ResultsOur new established MLL/AF4 Tg mice had lymphoma earlier than previous MLL/AF4 knock-in mice (median 12M vs 17M). Infiltration of lymphoid cells was located in liver, lung, kidney, and spleen and they consist of pro B-cell (CD45R+CD43+CD19-) lineage. Leukemic change developed two months (median 14M) after appearance of lymphoma. More than 30% of abnormal lymphocytes and severe anemia (RBC: 276×104 ml, Hb: 4.9 g/ml) were found in peripheral blood in MLL/AF4 Tg mice. Western blot analysis showed that up-regulation of HoxA9 and S100A6 was detected in MLL/AF4 Tg mouse compared to wild type C57BL/6. ConclusionWe succeeded in establishment of improved more aggressive MLL/AF4 Tg mice which reflect MLL/AF4 leukemogenicity of humans. Appearance of lymphoma in our MLL/AF4 Tg mice needed short er latency than those of the previous knock-in mice. Enhancement of HoxA9 and S100A6 expression was involved in MLL/AF4-associated leukemogenesis. We believe that this new MLL/AF4 Tg mouse is useful for study of the molecular mechanisms of MLL/AF4 leukemogenesis and development of new therapy for mixed-lineage leukemia. Disclosures:No relevant conflicts of interest to declare.

A Novel Approach for Analyzing Gene Expression Profiles Defines Two Distinct Subgroups of t(4;11) Positive Infant Acute Lymphoblastic Leukemia Patients.

Gene expression profiles (GEPs) are becoming increasingly more important for diagnostic procedures, allowing clinical predictions including treatment response and outcome for various types of cancer. However, established gene signatures not always appear helpful in understanding underlying disease mechanisms. Therefore, we explored an alternative approach for analyzing GEPs of a group of t(4;11) positive infant acute lymphoblastic leukemia patients (n=15), a highly aggressive type of MLL rearranged leukemia. We developed a method that uses a relational database program in combination with a normalization approach and specific discriminators. For normalization, every GEP of a given t(4;11) positive ALL sample was compared with 3 GEPs of normal bone marrow aspirates derived from healthy donors. Using the GeneChip Analysis Suite 5.0 program for single comparison analysis, the resulting gene lists were then compared with each other and only consistently identified genes (present on all three gene lists) were used for further analysis. This noise reduction decreased the amount of potentially deregulated target genes to about 30–40%. The remaining gene lists represented highly significant target genes that were then incorporated into a relational database program using specific discriminators. These discriminators were:1.upregulation of HOXA9 and HOXA10,2.presence/absence of the AF4-MLL fusion transcripts in addition to the MLL-AF4 fusion, and3.the localization of the genomic breakpoint within the MLL gene.This pilot study led to promising results, surprisingly classifying individual t(4;11) positive ALL patients into two distinct subgroups. Both subgroups share about 80 target genes, but also display particular sets of subgroup specific target genes. Importantly, these identified target genes can directly be linked to biological properties of t(4;11) positive leukemia cells, and therefore, allow important novel insights into this aggressive type of leukemia in infants.

Gene Expression Profiling of Acute Myeloid Leukemia with Translocation t(8;16)(p11;p13) and MYST3-CREBBP Rearrangement Reveals a Distinctive Signature with a Specific Pattern of HOX Gene Expression

Acute myeloid leukemia (AML) with translocation t(8;16)(p11;p13) is an infrequent leukemia subtype with characteristic clinicobiological features. This translocation leads to fusion of MYST3 (MOZ) and CREBBP (CBP) genes, probably resulting in a disturbed transcriptional program of a myelomonocytic precursor. Nonetheless, its gene expression profile is unknown. We have analyzed the gene expression profile of 23 AML patients, including three with molecularly confirmed MYST3-CREBBP fusion gene, using oligonucleotide U133A arrays (Affymetrix). MYST3-CREBBP cases clustered together and clearly differentiated from samples with PML-RARalpha, RUNX1-RUNX1T1, and CBFbeta-MYH11 rearrangements. The relative expression of 46 genes, selected according to their differential expression in the high-density array study, was analyzed by low-density arrays in an additional series of 40 patients, which included 7 MYST3-CREBBP AML cases. Thus, genes such as prolactin (PRL) and proto-oncogene RET were confirmed to be specifically overexpressed in MYST3-CREBBP samples whereas genes such as CCND2, STAT5A, and STAT5B were differentially underexpressed in this AML category. Interestingly, MYST3-CREBBP AML exhibited a characteristic pattern of HOX expression, with up-regulation of HOXA9, HOXA10, and cofactor MEIS1 and marked down-regulation of other homeobox genes. This profile, with overexpression of FLT3, HOXA9, MEIS1, AKR7A2, CHD3, and APBA2, partially resembles that of AML with MLL rearrangement. In summary, this study shows the distinctive gene expression profile of MYST3-CREBBP AML, with overexpression of RET and PRL and a specific pattern of HOX gene expression.