NUP98-HOXA9 Research Articles

AML-158 Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation

Rearrangements of the nucleoporin 98 gene (NUP98) define a high-risk subset of childhood acute myeloid leukemia (AML). The resulting fusion oncoproteins (FOs) involve the N-terminal, intrinsically disordered region of NUP98, and the C-terminal portion of one of more than 30 identified fusion partners. Approximately one third of fusion partners have DNA-binding homeodomains, and the remaining partners have other domains involved in gene regulation. NUP98 FOs have long been known to localize in nuclear puncta. Here, we investigated whether these puncta form by liquid-liquid phase separation (LLPS) and how they might contribute to cell transformation. We first focused on the NUP98::HOXA9 (NHA9) FO, in which the HOXA9 fusion partner includes a DNA-binding homeodomain. We expressed GFP-tagged NHA9 in HEK293T cells and characterized the resulting FO-associated nuclear puncta. We also investigated lentiviral FO expression in mouse hematopoietic stem and progenitor cells (HSPCs), studying localization using confocal imaging, self-renewal using colony forming unit assays, and gene expression using RNA sequencing. We next mutated the phenylalanine glycine (FG) repeats of NUP98 to disrupt interactions with other FOs and interacting proteins or mutated the homeodomain of HOXA9 to disrupt DNA binding. Finally, we investigated the applicability of our findings to other NUP98 FOs with and without homeodomains. NHA9 localizes in nuclear puncta in HEK293T and HSPCs, and mutation of FG repeats or the HOXA9 homeodomain abrogates puncta formation. Furthermore, expression of NHA9 confers self-renewal, but cell transformation does not occur in FO mutants that fail to form puncta. Upregulation of NUP98 FO target genes, including the HOXA cluster, occurred with expression of NHA9 and transforming, puncta-positive mutants but not in mutant cells that did not form puncta or transform HSPCs. Expression of additional NUP98 FOs (including NUP98::KDM5A, NUP98::LNP1, and NUP98::PRRX1) led to both puncta formation and cell transformation. Finally, cells from a NUP98::KDM5A patient derived xenograft confirmed puncta formation with endogenous FO expression. Taken together, these results demonstrate that NUP98 FOs undergo LLPS, show that phase separation is critical for cell transformation and gene expression changes, and provide rationale to further explore the exploitation of NUP98 FO-associated puncta for therapeutical benefit.

The role of phase separation in oncogenesis by fusion oncoproteins

NUP98 fusion oncoproteins (FOs) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid‐liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98‐HOXA9 (NHA9), retains a DNA‐binding homeodomain, whereas others harbor other types of DNA‐ or chromatin‐binding domains. NUP98 FOs have long been known to form puncta, but long‐standing questions are how nuclear puncta form, and how they drive leukemogenesis. We will discuss our studies of NHA9 condensates, showing that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three other leukemia‐associated NUP98 FOs form nuclear puncta and transform hematopoietic cells. To extend our findings, we tested ~150 additional fusion oncoproteins associate with a wide range of human cancers for puncta formation in cells. We will discuss our observation that >50% of these formed cellular condensates, with this behavior for some linked with aberrant gene expression and cell transformation. Our findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs and likely contributes to oncogenesis driven by many other fusion oncoproteins.

The role of phase separation by NUP98 fusion oncoproteins in leukemia

NUP98 fusion oncoproteins (FOs) are hallmarks of high-risk acute leukemia in children. NUP98 FOs universally contain the intrinsically disordered N-terminal region of NUP98, which harbors multiple “sticky” phenylalanine-glycine (FG) motifs. NUP98 fusion partners include a diverse set of genes that predominantly encode transcriptional or epigenetic regulators bearing folded DNA or chromatin-binding domains. Here, we focused on NUP98-HOXA9 (NHX9), an FO linking the N-terminal FG repeat domains of NUP98 (NUP98-FG) and the DNA-binding homeodomain (HD) of HOXA9.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

The Retinoblastoma Tumor Suppressor Is Required for the NUP98-HOXA9-Induced Aberrant Nuclear Envelope Phenotype.

Chromosomal translocations involving the nucleoporin NUP98 gene are recurrently identified in leukemia; yet, the cellular defects accompanying NUP98 fusion proteins are poorly characterized. NUP98 fusions cause changes in nuclear and nuclear envelope (NE) organization, in particular, in the nuclear lamina and the lamina associated polypeptide 2α (LAP2α), a regulator of the tumor suppressor retinoblastoma protein (RB). We demonstrate that, for NUP98-HOXA9 (NHA9), the best-studied NUP98 fusion protein, its effect(s) on nuclear architecture largely depend(s) on RB. Morphological alterations caused by the expression of NHA9 are largely diminished in the absence of RB, both in human cells expressing the human papillomavirus 16 E7 protein and in mouse embryonic fibroblasts lacking RB. We further show that NHA9 expression associates with distinct histone modification. Moreover, the pattern of trimethylation of histone H3 lysine-27 is affected by NHA9, again in an RB-dependent manner. Our results pinpoint to an unexpected interplay between NUP98 fusion proteins and RB, which may contribute to leukemogenesis.

A genetic screen in Drosophila uncovers the multifaceted properties of the NUP98-HOXA9 oncogene

Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 to the homeodomain of the transcription factor HOXA9. The mechanisms enabling NA9-induced leukemia are poorly understood. Here, we conducted a genetic screen in Drosophila for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, we discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, our work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets.

NUP98-HOXA9-Initiates Molecular and Biologic Features of Disease Progression in a Humanized Model of CML

Acute myeloid leukemias (AMLs) are heterogenous diseases often resulting from the acquisition of multiple genetic alterations that deregulate hematopoietic precursor proliferation and block normal differentiation. Chronic myeloid leukemia offers a unique opportunity to identify molecular mechanisms that interfere with normal differentiation in the context of a highly proliferative hematopoietic stem cell clone that produces massive number of functional differentiated myeloid cells due to the presence of a BCR-ABL1 fusion gene. Since the NUP98-HOXA9 (NA9) fusion gene has been identified in some CML blast crisis patients or de novo AML, we asked whether a humanized model of CML progression to AML would result from its lentiviral-mediated introduction into primary CML CD34+ cells from 3 chronic phase CML patients in which >97%, >96%, and 44% of the CD34+ longterm culture-initiating cells were Ph+/BCR-ABL1+. In vitro experiments showed the NA9-transduced cells produced a hugely increased number of granulo-monocytic progenitors in long-term cultures (up to 1000-fold, p=0.03; t-ratio test) and enhanced the serial replating activity of directly clonogenic cells as compared to matched samples of cells transduced with a control vector. In vivo experiments showed that 90% of sublethally irradiated NOD-SCID IL2Rgc-null mice expressing human IL3, GM-CSF and SCF constitutively that were transplanted with these same NA9-transduced cells developed evidence of a progressed myeloproliferative neoplasia. This included tissue infiltrates of eosinophils, basophils and mastocytes, exclusive myeloid differentiation and signs of an imminently fatal leukemia between 8 and 27 weeks post-transplant in 50% of cases, although an excess blast population was not seen in these. RNA-sequencing of cells analyzed just 48 hours after transduction revealed a signature of 53 genes that were more highly expressed in CD34+ CML cells carrying the NA9 fusion gene in comparison to control CML cells. This signature included many genes expressed by hematopoietic stem cells (HSCs), such as 5' HOXA genes, 3' HOXB genes, PBX3, MEIS1,ARID5B, AHR, REL, BMP6, GDF10, SFRP5, PPBP,PLA2G4A suggesting that NA9 induces the expression of a partial HSC program in later CML progenitor types that make up the bulk of the CD34+ CML population. Consistent with this hypothesis, the NA9 signature separated the HSCs, CMPs and GMPs of chronic CML patients as well as those of normal subjects (GSE47927; p=1x10-6, p=2x10-4 respectively). Most genes in the NA9 signature were also found to be over-expressed in CD34+ cells of patients in the accelerated phase and in the blast phase of CML, including ARID5B, AHR, STARD9, TOX, or FOXP1 (p=9.10-8; ANOVA) (GSE4170). The NA9 signature was also significantly enriched in transcripts of genes that are over-expressed in the blasts of AML patients carrying NPM1 mutations or MLL fusions and was predictive of overall survival in the AML cohort of the Cancer Genome Atlas (p=0.02; log-rank test, n=200). This stem cell signature was also associated with an increase in the number of H3K27ac marks (on average 7805 ± 960 peaks for NA9 vs 5888 ± 2739 peaks for control) at 48 hours post-transduction. De novo H3K27ac peaks in NA9+ CD34+ cells were located in proximal and distal enhancers of 993 genes (GREAT parameters TSS ± 2kb from TSS and 100kb max extension). These were significantly enriched genes in the GM-CSF signaling pathway (MySigDB, binomial p-value=2.19x10-8) and that are upregulated in granulocytes and monocytes following LPS exposure. H3K27ac peaks also matched with GATA1, RELA, MEF2A and IKZF1 transcription factor binding sites previously mapped by ChIPSeq experiments (adjusted p-value <0.05). Finally, we identified super-enhancers in 12 genes among which were PBX3, ANGPT1, MBNL1 and PRKACB. Overexpression of the NA9 fusion gene in chronic phase CD34+ CML cells thus appears to reprogram the expression of HSC genes as well as those associated with GM-CSF pathway activation and inflammatory responses via H3K27 acetylation of associated loci leading to a picture of advanced accelerated phase/disease progression but not the complete differentiation arrest seen in terminal blast crisis or frank AML. These findings highlight the multiplicity of biologically important molecular alterations that can result from a single epigenetic perturbation but, nevertheless, are insufficient to create an overt AML phenotype. Disclosures Turhan: novartis: Honoraria, Research Funding; Incyte: Consultancy, Honoraria.

The Bone Marrow Niche Distinguishes Young and Old Leukemia

Age is an independent risk factor in the outcome of acute myeloid leukemia (AML). Treatment of pediatric AML is often extrapolated from adult trials assuming disease homogeneity. However, age dependent clinical and molecular differences exist in AML and normal hematopoietic stem cells (HSCs) differ with age. We hypothesized that cellular age influences leukemogenesis. Using the NUP98HOXA9 (NH9) murine leukemia model, we addressed how intrinsic cellular age and the interplay with the bone marrow (BM) microenvironment impacts leukemic transformation. Lin-sca-1+cKit+ (LSK) cells isolated from fetal liver (FL) and BM at 3 week (w), 10w and 52w C57BL/6 mice were transduced with NH9 and in vitro transformation was assessed by colony forming cell (CFC) assays. NH9 transduced young (FL and 3w) and adult (10w and 52w) LSKs serially replated with a similar myeloid immunophenotype showing that LSKs from all ages transformed comparably in vitro in the absence of a BM niche. To investigate age related transformation in a BM microenvironment, NH9 transduced LSKs from all ages (at CFC2) were transplanted into 6w old C57BL/6 recipients. NH9 transduced LSKs from adult BM gave rise to leukemia with a shorter latency compared to young LSKs, with a reduction in median survival of almost 100 days. While adult LSKs resulted in a fully penetrant leukemia, only 50-75% of mice transplanted with young LSKs developed leukemia. A proportion of recipients transplanted with young LSKs developed acute lymphoblastic leukemia expressing lymphoid markers CD19 or B220 with an absence of myeloid markers. All mice transplanted with adult LSKs developed AML determined by morphology and flow cytometry, and immunohistochemistry of the leukemic blasts showed myeloid marker MPO expression. However, in AMLs generated from young LSKs, the blasts showed dual expression of MPO and the lymphoid marker CD3. Therefore, young LSKs retain expression of multi-lineage markers exhibiting lymphoid lineage potential. Together our data show that the presence of an adult microenvironment significantly alters the latency, penetrance and phenotype of the resultant leukemia from different aged stem cells. These effects may be cell intrinsic, or the BM microenvironment may be providing external cues, or may be as a result of the interaction between the leukemic cell and the BM niche cells. To address whether there may be cell intrinsic cues, targeted transcriptional profiling was assessed in NH9 transduced LSKs in vitro and in AMLs generated in vivo. Young LSKs transformed in vitro showed higher expression of genes associated with multiple hematopoietic lineages compared to adult LSKs, supporting the finding that young LSKs retain multi-lineage potential. In contrast, adult LSKs transformed in vitro showed higher expression of genes associated with a stem cell signature and upregulation of the bone morphogenetic protein (BMP) pathway. The BMP receptors BMPR1a, BMPR2, and the responsive SMAD1 were upregulated, and the antagonist Noggin was downregulated, suggesting that in adult transformed cells in vitro, the BMP pathway is primed for activation. The BMP pathway transcriptional targets were also perturbed, including upregulation of ID3 and downregulation of Creb3l1, which have been shown to have pro-oncogenic and tumor suppressive activities respectively. Significantly, upregulation of the BMP pathway was found in adult leukemic cells in the presence of a BM niche in vivo. Global transcriptional profiling by RNA-sequencing performed on in vivo generated AMLs from all 4 ages confirmed upregulation of the BMP pathway in adult AMLs. In further support of the role of the BM microenvironment in adult AML, pathways involved in cell adhesion and localization of the stem cell in the BM niche were enriched in adult AMLs. These data suggest a cell intrinsic role for the BMP pathway that may be exploited by transformed/leukemic cells in the presence of a BM niche. Our data may provide a mechanistic explanation for the enhanced myeloid leukemic potential of adult LSKs or, alternatively, the decreased susceptibility of young LSKs toward AML. DisclosuresWheadon:GlaxoSmithKline: Research Funding.

NUP98 Fusion Proteins Interact with the NSL and MLL1 Complexes to Drive Leukemogenesis

The nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in multiple hematopoietic malignancies. Here, we demonstrate that NUP98 fusion proteins, including NUP98-HOXA9 (NHA9), NUP98-HOXD13 (NHD13), NUP98-NSD1, NUP98-PHF23, and NUP98-TOP1 physically interact with mixed lineage leukemia 1 (MLL1) and the non-specific lethal (NSL) histone-modifying complexes. Chromatin immunoprecipitation sequencing illustrates that NHA9 and MLL1 co-localize on chromatin and are found associated with Hox gene promoter regions. Furthermore, MLL1 is required for the proliferation of NHA9 cells invitro and invivo. Inactivation of MLL1 leads to decreased expression of genes bound by NHA9 and MLL1 and reverses a gene expression signature found in NUP98-rearranged human leukemias. Our data reveal a molecular dependency on MLL1 function in NUP98-fusion-driven leukemogenesis.

Human NUP98-HOXA9 promotes hyperplastic growth of hematopoietic tissues in Drosophila

Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, we sought to functionally dissect NA9 activity using Drosophila. For this, we generated transgenic NA9 fly lines and expressed the oncoprotein during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, our work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings.

Epigenetic therapy restores normal hematopoiesis in a zebrafish model of NUP98-HOXA9-induced myeloid disease.

Acute myeloid leukemia (AML) occurs when multiple genetic aberrations alter white blood cell development, leading to hyperproliferation and arrest of cell differentiation. Pertinent animal models link in vitro studies with the use of new agents in clinical trials. We generated a transgenic zebrafish expressing human NUP98-HOXA9 (NHA9), a fusion oncogene found in high-risk AML. Embryos developed a preleukemic state with anemia and myeloid cell expansion, and adult fish developed a myeloproliferative neoplasm (MPN). We leveraged this model to show that NHA9 increases the number of hematopoietic stem cells, and that oncogenic function of NHA9 depends on downstream activation of meis1, the PTGS/COX pathway and genome hypermethylation through the DNA methyltransferase, dnmt1. We restored normal hematopoiesis in NHA9 embryos with knockdown of meis1 or dnmt1, as well as pharmacologic treatment with DNA (cytosine-5)-methyltransferase (DNMT) inhibitors or cyclo-oxygenase (COX) inhibitors. DNMT inhibitors reduced genome methylation to near normal levels. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of a histone deacetylase inhibitor plus either a DNMT inhibitor or COX inhibitor to block the effects of NHA9 on zebrafish blood development. Our work proposes novel drug targets in NHA9-induced myeloid disease, and suggests rational therapies by combining minimal doses of known bioactive compounds.

Myeloid Cells Having NUP98-HOX Fusion Genes with Simultaneous FLT3, NRAS, or WT1 Mutations Confer Growth Advantage, Inhibit Differeniation/Apoptosis, and Augment Self-Renewal

Introduction: Clinical features and genetic backgrounds of acute myeloid Leukemia (AML) are heterogenous and the prognosis is poor. Cooperating alterations of several genes including oncogenes or tumor suppressor genes lead to development of Leukemia. AML leukemogenesis is thought to require at least two different types of genetic change: class I mutations, which confer a proliferative or survival advantage; and class II mutations, which inhibit myeloid differentiation and apoptotic activity. In hematological malignancies with 11p15 translocations, the NUP98 gene is reportedly fused to various partner genes, often including homeobox genes, such as HOXA9, A11, A13, C11, C13, D11, D13 and PMX. We previously revealed that high frequencies of FLT3 internal tandem duplication (FLT3-ITD), NRAS mutations (NRASMT), and WT1 mutations (WT1MT) in myeloid malignancies with NUP98-HOX fusions were found and their concurrent mutations with NUP98-HOX fusions were associated with clinical prognosis of the patients. However, biological outcome of the combination of NUP98-HOX fusions with FLT3-ITD, NRASMT or WT1MT remains to be investigated. To elucidate the mechanism resposible for the leukemogenesis, we examined the function of NUP98-HOX fusions and the concomitant somatic mutations in vitro. Methods: Full length of NUP98-HOXA9 (NHA9), FLT3-ITD (51 bp-duplication), NRASMT (G13V), and WT1MT (Arg250trp) was cloned into a plasmid that has a mammalian replication initiation region and a nuclear matrix attachment region, which possesses great ability to persist gene amplification for long periods. Each plasmid or the combination of the two plasmids (NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT) were transfected into IL3-dependent mouse myeloid 32D cell line. Cell proliferation was determined in medium without IL3. Cellular differentiation was examined in the medium containing G-CSF using May-Giemsa staining and fluorescence-activated cell sorting (FACS). Apoptosis was quantitated using FACS. Colony forming assay was performed in methyl cellulose medium with stem cell factor and IL3. Moreover, autophagy induced by tamoxifen was checked. Results: Compared to the empty vector, IL3-independent cellular growth was significantly increased in the cells transfected with NHA9 and FLT3-ITD alone, but not in NRASMT and WT1MT alone. In contrast, co-transfection of NRASMT or WT1MT with NHA9 significantly increased the IL3 independent proliferation of 32D cells. Similarly, cells transfected with NHA9/FLT3-ITD showed the enhanced IL3-independent proliferation. CD11b and Gr1, of which represent myeloid differentiation marker, decreased in all the transfected cells except for NRASMT. While transfection with FLT3-ITD, NRASMT and WT1MT increased the morphological nuclear segmentation of the cells, NHA9/FLT3-ITD, NHA9/NRASMT, and NHA9/WT1MT abrogated the nuclear segmentation. Both Annexin V positive/propidium iodide negative cells and active caspase 3-positive cells declined in all transfected cells. Transfection of NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT tended to increase the number of colony forming unit compared to the single gene-transfected cells. After treatment with tamoxifen, monodansylcadaverine, which was used to reveal autophagic vacuoles, was incorporated into empty vector-transfected cells, not into all transfected cells. Conclusions: NHA9 acquired proliferation capacity, blockage of differentiation, and anti-apoptosis activity, FLT3-ITD did proliferation capacity and anti-apoptosis activity, NRASMT and WT1MT did anti-apoptosis activity. By contrast, NHA9 with FLT3-ITD, NRASMT, or WT1MT conferred growth advantage, inhibited differentiation and apoptosis, augmented self-renewality. In vivo study demonstrated that NHA9 or each genetic mutation (FLT3-ITD and NRASMT) alone was insufficient to achieve myeloid leukemia. These suggested that leukemogenesis of NUP98-HOX fusion genes with simultaneous gene mutations might be induced by not only harmonization between class I and class II mutations but also self-renewality and anti-autophagic cell death. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Determination of the Efficacy of Epigenetic Therapy in High Risk Myeloid Disease Using the Zebrafish Model

Epigenetic therapy implies the use of drugs that target regulators of gene expression, such as DNA and histone methylation or histone acetylation, without altering the DNA coding sequence. Epigenetic mechanisms have been found to be perturbed in myeloid diseases, and in fact, the DNA demethylating agents, 5-azacytadine and decitabine are FDA approved drugs for myelodysplastic syndrome. However, use of epigenetic therapies in other types of myeloid disease including acute myeloid leukemia (AML) has been met with variable success, suggesting efficacy might be improved by preselecting subtypes of disease in which there is a high degree of epigenetic dysregulation. AML has recently been found to be associated with a host of epigenetic abnormalities including mutations in DNA methyltransferase 3A (DNMT3A), ten-eleven translocation-2 (TET2) and enhancer of zeste homolog 2 (EZH2).We generated a transgenic zebrafish model of high risk myeloid disease expressing the human NUP98-HOXA9 (NHA9) fusion oncogene, a genetic lesion for which epigenetic dysregulation has not previously been identified. Transgenic embryos exhibit an increase in immature myeloid cells at the expense of erythroid cells and adult fish develop a myeloproliferative neoplasm (MPN). Leveraging this model in a microarray screen, we identified 3-fold elevated levels of the epigenetic regulator, dnmt1, the major maintenance methyltransferase, for the first time in high risk AML. Decitabine specifically inhibits DNMT1 and treatment of NHA9 transgenic embryos with 75µM decitabine restored normal hematopoiesis, as evidenced by normal numbers of leukocytes and red cells. Moreover, using hematopoietic stem cell (HSC) reporter lines and whole mount in situ hybridization, we identified a 2-3 fold increase in this population in NHA9 embryos, suggesting the HSC as the cell of origin in this disease. Interestingly, 75µM decitabine therapy also restored normal HSC numbers. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of DNMT1 inhibitors, decitabine (10-25µM) or zebularine (100µM), plus histone deacetylase inhibitors, valproic acid (25-100µM) or trichostatin A (250nM), to inhibit the effects of NHA9 on hematopoiesis.To determine if NHA9 expression directly results in changes to DNA methylation, we performed MeDIP-Seq on genomic DNA from pools of untreated and decitabine-treated NHA9 or control embryos. Untreated NHA9 embryos displayed significantly higher methylation levels at the regions of gene promoters compared to control embryos, which were restored to control levels following treatment with 75µM decitabine. Similarly, combination therapy with 10µM decitabine and 25µM valproic acid significantly reduced methylation to near control levels following a 5 hour treatment, while prolonged exposures to these same doses resulted in profound global hypomethylation.These findings elucidate underlying mechanisms in the pathogenesis of NHA9-induced myeloid disease and propose novel actionable epigenetic drug targets. Furthermore, we highlight the opportunities inherent in the zebrafish model as a unique in vivo platform for the preclinical screening of epigenetic based combination therapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 472: Interactions of the fusion protein Nup98-Hoxa9 with Pbx3, p300 and HDAC1: widening the targeted therapy window in acute myeloid leukemia (AML)

Abstract The chromosomal translocation t(7;11)(p15,p15), that results in the oncogenic fusion protein Nup98-Hoxa9 (NH), appears in 1% of patients with AML and is associated with very poor prognosis and short overall survival. Despite the large severity of the leukemia induced by this fusion protein, the oncogenic events triggered by NH are poorly understood, although a potential role as an aberrant transcription factor has been proposed. We have generated a human Hematopoietic Progenitors (hHP) cellular model expressing NH constitutively to identify the molecular mechanisms supporting the malignancy of this fusion protein, facilitating the search for therapeutic targets. We identified the DNA binding sites of NH by performing ChIP-seq experiments, which were validated by qRT-PCR analysis on ChIP selected DNA and Luciferase assays. Expression profiling was performed in hHP-NH and co-Immunoprecipitations (Co-IPs) were done to demonstrate the interaction of NH with different transcriptional regulators. Specific drug sensitivity of the hHP-NH model was assessed in cell proliferation assays. Our work provides the first description of the DNA binding sites of NH, most of which are regulatory regions of genes involved in the development of AML. In particular, we demonstrate that NH induces the overexpression of MEIS1, HOXA9 and PBX3, transcription factors forming an activator complex that is a key element in the leukemic onset driven by other chromosome rearrangements. Interestingly, we show that NH directly interacts with this complex through Pbx3. To evaluate the biological relevance of the interaction of the MEIS1-HOXA9-PBX3 complex with NH, we have analyzed the sensitivity of hHP-NH to the HXR9 peptide (an inhibitor of the HOXA9-PBX3 interaction). Supporting our hypothesis, we observed an inhibitory effect on hHP-NH viability after HXR9 treatment. Finally, by combining the expression profile data from hHP-NH and the ChIP-seq results using GSEA analysis, we show that NH is able to induce both overexpression and down-regulation of its target genes. To provide evidences of the activator-repressor role of NH, we performed different Co-IPs that demonstrated its direct interaction with both p300 (transcriptional activator) and HDAC1 (transcriptional inhibitor). Taken together, we show that the direct overexpression of the complex MEIS1-HOXA9-PBX3 is one of the pathogenic mechanisms induced by NH. As expected, the disruption of this complex with the HXR9 peptide in the hHP-NH model has a direct effect on cell viability. Furthermore, we show that NH interacts with this complex via PBX3 and also with p300 and HDAC1. The features and architecture of these interactions need to be further explored, but these findings allow us to consider the use of the HXR9 peptide or some HDAC inhibitors as possible treatments for these patients. Citation Format: Ana Rio-Machin, Alba Maiques-Diaz, Sandra Rodriguez-Perales, Sara Alvarez, Rocio N. Salgado, Álvaro Eguileor, Raul Torres, Juan C. Ramirez, Juan C. Cigudosa. Interactions of the fusion protein Nup98-Hoxa9 with Pbx3, p300 and HDAC1: widening the targeted therapy window in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2014-472

Identification of GSX2 and AF10 as NUP98 partner genes in myeloid malignancies

Identification of GSX2 and AF10 as NUP98 partner genes in myeloid malignancies

Epigenetic Therapy Inhibits NUP98-HOXA9-Mediated Myeloid Disease – Decitabine and Valproic Acid Work Synergistically to Rescue Normal Hematopoiesis in Transgenic Zebrafish.

AbstractAbstract 2391Acute myeloid leukemia (AML) results from multiple genetic lesions that alter white blood cell development, leading to hyperproliferation and a block in differentiation. A better understanding of these molecular pathways will enable the development of therapies that selectively target the specific abnormality in a leukemic cell with improved outcome and limited toxicity.Pertinent animal models serve as essential intermediaries between in vitro studies and the use of these new agents in clinical trials. The NUP98-HOXA9 (NHA9) fusion oncogene is found in high risk de novo AML, treatment related AML and chronic myeloid leukemia (CML) blast crisis. We previously generated a transgenic zebrafish model overexpressing human NHA 9 under the zebrafish pu.1 promoter (Forrester et al, BJH, 2012). Almost 25% of adult NHA9-transgenic fish develop a myeloproliferative neoplasm (MPN) in the kidney marrow, the site of adult hematopoiesis. Additionally, NHA9- transgenic embryos demonstrate an increase in immature myeloid cells (l-plastin1, 2.3-fold by in situ, P<0.005) at the expense of erythroid cells (gata1, 19.3-fold by qRT-PCR, P<0.00005). This embryonic phenotype in our NHA9 zebrafish provides an unprecedented opportunity to perform in vivo screens for collaborating genes in NHA9-induced leukemogenesis and evaluate the impact of molecularly targeted agents.Microarray analysis found high expression of DNA (cytosine-5-)-methyltransferase 1 (dnmt1) in NHA9 embryos, which was confirmed by qRT-PCR (3.4-fold increase). The methylating activity of the DNMT1 enzyme is part of the epigenetic machinery that represses genes needed for terminal myeloid differentiation. Overexpression of human DNMT1 has been found in some cases of AML, but has not been previously linked with NHA9-induced disease. The overexpression of the zebrafish dnmt1 homolog in NHA9 embryos may keep cells trapped in an immature state, a hallmark of AML.Injecting NHA9 embryos with a gene-blocking dnmt1 morpholino restored normal hematopoiesis with wild-type expression levels of both l-plastin and gata1. Similarly, a dose-dependent return to normal proportions of myeloid and erythroid cells was achieved by bathing NHA9 embryos in 50–100 μM decitabine (5-aza-2’-deoxycytidine), a demethylating agent that specifically targets the DNMT1 enzyme. However, decitabine use as a monotherapy carries the risk of genomic instability due to wide-spread DNA hypomethylation. We therefore considered treating our NHA9 zebrafish embryos with combination therapy against multiple molecular targets.DNMT1 is part of a larger epigenetic machinery and works in parallel with histone deacetylation complexes (HDACs). Similar to decitabine, exposing NHA9 embryos to 150–250 μM of the HDAC inhibitor, valproic acid (VPA), lead to a dose-dependent return of normal hematopoiesis. In vitro studies suggest that combined treatment with a demethylating agent and an HDAC inhibitor may synergize to be more effective than either compound alone in combating myeloid disease, permitting use of lower drug doses, thus avoiding unnecessary toxicity. Indeed, we found that combination doses as low as 10 μM each of decitabine and VPA restored normal hematopoiesis as effectively as significantly higher doses of each monotherapy. This drug synergy identified in our transgenic zebrafish directly links NHA9-induced leukemia and epigenetic regulation and has set the stage for a new and exciting therapeutic approach for high risk AML. True to our goal, translating this treatment to clinical studies may ultimately improve outcome and minimize toxic side-effects, thereby increasing the long-term survival of patients with high-risk AML. Disclosures:No relevant conflicts of interest to declare.

NUP98-HOXA9 drives High-Risk Myeloid Disease in Zebrafish – An In Vivo platform to Study Interacting Genes and Perform Drug Discovery,

Abstract 3622High-risk forms of acute myeloid leukemia (AML) remain fatal for 40% of patients, both due to refractory disease and toxicity from traditional chemotherapeutic agents. Using zebrafish, we aim to discover new or repurposed drugs to combat high-risk leukemia that are less toxic and achieve better survival for human patients.The NUP98-HOXA9 (NHA9) mutation is a hallmark of high-risk AML, characterized by a failure of granulocyte maturation and increased cell division. The zebrafish is a robust model to study blood development and leukemia, with the inherent capacity to easily perform drug screens in embryos. We created an AML disease model using transgenic zebrafish that harbor the NHA9 mutation (Forrester et al., Br J Haematol, Aug 2 Epub). We are using these living models of cancer to study the genetic interactions that promote high-risk leukemia.Our model has already provided novel insights into the direct mechanisms of NHA9 oncogenesis. In response to DNA-damage, NHA9 failed to initiate cell cycle arrest and apoptosis, likely through upregulation of bcl2 rather than direct suppression of tumor protein 53 (tp53) signaling. Upon exposure to 16 Gy ionizing radiation, wild-type embryos displayed a 4.16-fold decrease of phospho-histone-H3 (pH3)-labeled cells, whereas NHA9 embryos only suffered a 1.63-fold decrease (relative difference of 2.55-fold, P <0.005). We also found that NHA9 blocked the mitochondrial apoptosis response, with loss of caspase-3 activation (6.31-fold decrease, P <0.005). Both whole mount in situ hybridization (WISH) and qRT-PCR detected upregulation of proto-oncogenes, bcl2 and bcl2l1/bcl-xL in NHA9 embryos (3.79-fold, P <0.00005, and 2.07-fold, P <0.0005, respectively), particularly in the hematopoietic posterior blood island (PBI), but normal expression levels of canonical p53 target genes, bbc3/puma and cdkn1a/p21waf1/cip1.NHA9 also perturbs blood cell development. In embryos, NHA9 produced a loss of red blood cells (gata1a, decreased expression 19.34-fold by qRT-PCR, P <0.00005) and an increase of immature myeloid cells (spi1/pu.1, increased expression 2.79-fold by WISH, P <0.005), but no change in cell division (by BrdU incorporation and pH3 labeling). Between 19 to 23 months of age, nearly 25% of NHA9 fish developed a myeloproliferative neoplasm (MPN) in the kidney, which is the site of blood development in adult zebrafish. Affected kidneys show invasion of proliferating myeloid cells that stain positive with periodic acid-Schiff (PAS). This pathology is similar to NHA9 mice, which develop a long latency, polyclonal MPN, prior to onset of AML. However, frank leukemia was not observed in our NHA9 fish, suggesting the need for additional genetic “hits” to achieve malignant progression. We are investigating the interactions of NHA9 with β-catenin and Meis1.The cell division gene, β-catenin, has recently proven important for driving AML in mice, but co-operation with NHA9 is unknown. We are contrasting ‘stimulated’ fish (NHA9 + β-catenin, using 20 μM prostaglandin E2 [PGE2]) with ‘inhibited’ fish (NHA9 – β-catenin, using 20 μM indomethacin) to measure the combined effects of NHA9 and β-catenin on cell division and blood development. Similarly, overexpression of Meis1 accelerates the onset of NHA9 -induced leukemia in mice, but the underlying mechanism is unknown. We show that morpholino (MO) knockdown of zebrafish meis1 limits the myeloproliferative effects in NHA9 embryos by blocking myeloid cell development in the PBI (83% decreased expression of the myeloid marker, l-plastin /lcp1). Microarray analysis on these various cohorts of embryos will help to identify a subset of genes that are regulated by NHA9, β-catenin, and Meis1 together. These studies will help determine how normal blood cells are transformed into leukemia cells.Our ultimate goal is to use these NHA9 fish to discover new drugs that combat high-risk leukemia. Our model provides an excellent opportunity to perform chemical library screens for compounds that restore normal levels of red blood cells (measured by in situ for gata1a) in NHA9 embryos. We will present results of screening with compounds contained in the Biomol ICCB 480 library. Promising candidates may represent future therapies against NHA9 in human patients with AML. Disclosures:No relevant conflicts of interest to declare.

The Nup98-HoxA9 Leukemogenic Fusion Protein Blocks Xpo1 and Tap-Mediated Nuclear Export

Abstract 5228In leukemia Nup98 fusion genes are found as a product of the reciprocal translocation between Nucleoporin 98 (nup98) and homeobox-cluster genes. Nup98-HoxA9 (NHA9), the product of the t(7;11) translocation, is detected in acute myeloid leukemias (AMLs) and as a secondary abnormality in blast crisis of chronic myeloid leukemia (CML). The role of NHA9 in leukemogenesis is complex and incompletely understood. Here, we show an abrogation of nucleocytoplasmic shuttling of the nuclear export receptor Xpo1 and Tap in NHA9-expressing cells by using retroviral nuclear trafficking as a model. Lentiviral Rev, the prototype for nuclear export signal (NES)-containing proteins is shuttled through the nucleopore by Xpo1. NHA9 sequestered Xpo1 from the nuclear rim into nuclear aggregates resulting in deficient Xpo1-dependent nuclear exit of Rev and its mRNA substrates. Tap is involved in mRNA nucleocytoplasmic shuttling and is also responsible for the nuclear export of D-type retrovirus CTE-mRNAs. By using Tap/CTE-mRNA nuclear export as a model we also found that Tap colocalized in NHA9 nuclear aggregates leading to impaired Tap-mediated nuclear exit of CTE-mRNA substrates. Leukemogenicity of Nup98 fusion proteins may be accounted for in part by defects in Tap and Xpo1-mediated export of their substrates. Disclosures:No relevant conflicts of interest to declare.