Aberrant Expression Of HOXA9 Research Articles

A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Assess the Efficacy and Safety of Entospletinib in Combination with Intensive Induction and Consolidation Chemotherapy in Adults with Newly Diagnosed Nucleophosmin 1-mutated Acute Myeloid Leukemia

A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Assess the Efficacy and Safety of Entospletinib in Combination with Intensive Induction and Consolidation Chemotherapy in Adults with Newly Diagnosed Nucleophosmin 1-mutated Acute Myeloid Leukemia

Preclinical Activity of Selective SYK Inhibitors, Entospletinib and Lanraplenib, Alone or Combined with Targeted Agents in Ex Vivo AML Models with Diverse Mutational Backgrounds

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that mediates integrin and Fc receptor signaling in myeloid cells. SYK has been implicated as an oncogenic driver in acute myeloid leukemia (AML) with aberrant expression of HOXA9 and MEIS1 and cooperates with FLT3 internal tandem duplication to drive leukemogenesis. The oral SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity in HOXA9/MEIS1 driven AML and is currently being investigated in a phase 3 trial of previously untreated patients with nucleophosmin1-mutated (NPM1 mut) AML. Lanraplenib (LANRA) is a next generation oral SYK inhibitor with potency and selectivity comparable to ENTO. In healthy volunteers and patients with autoimmune disease, LANRA has shown pharmacokinetic properties that compare favorably with ENTO. To support the clinical development of LANRA for the treatment of AML, ex vivo treatment of patient-derived AML cells was used to compare its activity to that of ENTO, both as a single-agent and in combination with other AML therapies. First, ENTO and LANRA single-agent activities were evaluated in peripheral blood-derived blasts from 15 AML patients, representing different mutational backgrounds including NPM1, FLT3, PTPN11, and NRAS mutations. AML cells were seeded into 96 well plates and treated with ENTO and LANRA for 6 days. Comparable effects on viability were observed across the 15 models with the 2 compounds, and in 11 of the models, the half maximal inhibitory concentration (IC 50) values were within 2-fold of each other. ENTO had a slightly lower IC 50 value than LANRA in the FLT3-mutated models possibly due to the direct FLT3 inhibitory activity of ENTO. Next, we tested the activity of ENTO and LANRA ex vivo in bone marrow-derived AML blasts from 29 AML patients representing diverse mutational backgrounds, including NPM1, IDH1, FLT3, and RAS mutations as well as MLL rearrangements. The models were treated for 9 days with either ENTO or LANRA, and viability was assessed using Annexin V and 7-aminoactinomycin D staining. Again, ENTO and LANRA showed comparable effects on cell viability with no significant differences between the compounds when compared across the different mutational backgrounds. Both studies suggest the potential for anti-leukemic activity in several different genetically defined subsets of AML. Matrix combination assays were performed by combining ENTO or LANRA with either cytarabine (NPM1 mut), gilteritinib (FLT3 mut), or trametinib (RAS mut) with cell viability and death assessed after a 3-day incubation period. Increased cell death in an additive manner was observed in all combinations tested, with results for ENTO and LANRA being similar, indicating the utility of both compounds in combinatorial treatment paradigms. These results support the clinical evaluation of LANRA in genetically defined subsets of AML. A phase 1b/2 study of LANRA in combination with the selective FLT3 inhibitor gilteritinib, in patients with relapsed or refractory FLT3 mut AML is planned for the end of this year. DisclosuresDay: Cyteir Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding. Schinzel: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Obholzer: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lin: Kronos Bio, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Saffran: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company.

Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen.

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

HOXA9 Reprograms the Enhancer Landscape to Promote Leukemogenesis

Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis invivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.

The Adhesion Molecule “G-Protein Coupled Receptor 56” (Gpr56) Cooperates with the Homeobox Gene Hoxa9 to Induce Acute Myeloid Leukemia in Mice

Interactions between leukemic cells and the bone marrow microenvironment are essential for the maintenance and progression of myeloid leukemias. G-protein coupled receptor 56 (Gpr56) is an adhesion molecule which collaborates with the extracellular matrix through interaction with collagen III binding and transglutaminase 2, and by this activating the Rho A response pathway. In the hematopoietic hierarchy, GPR56 expression is highest in stem cells and decreasing in expression with differentiation. It is a poor prognostic factor in cytogenetically normal acute myeloid leukemia (AML), and was identified as a member of a signature expressed in functionally validated human AML stem cells (Eppert et al., Nature Medicine 2011). To test its functional relevance for AML, we first analyzed the expression of GPR56 in primary patient samples (n= 74) with different genotypes from the AML dataset available in the TCGA database. Normal karyotype patients showed significantly higher expression of GPR56 (3.1 fold p<0.05), when compared to patient samples with translocations. Analysis within the normal karyotype dataset showed high GPR56 expression independent of NPM1 or FLT3-ITD mutations. Quantification of GPR56 expression by TaqMan PCR confirmed high expression both in the CD34-positive and CD34-negative leukemic subpopulations in normal karyotype AML, indicating high expression of this protein in the compartment enriched for leukemic stem cells. As GPR56 expression was high in normal karyotype AML, known to be characterized by aberrant homeobox expression, we tested the functional relevance of Gpr56 expression in a murine Hoxa9 bone marrow (BM) transplantation model. Aberrant expression of Hoxa9 in 5-FU mobilized stem and progenitor cells increased GPR56 expression 1.3 fold (p<0.05), but failed to induce short latency AML up to an observation time of over 160 days post transplant. Retrovirally engineered aberrant overexpression of Gpr56 alone also did not induce any perturbations with regard to engraftment and cell differentiation over 90 days post transplant in mice. However, overexpression of Hoxa9 together with Gpr56 induced a significant and 1.8 fold increase in colony forming cell activity (CFC) (p<0.05), followed by a 1.6 fold (p<0.05) increase in secondary CFCs when compared to HoxA9 alone. Mice transplanted with BM cells co-transduced with HoxA9 and Gpr56 rapidly developed AML after a median latency of 139 days post transplant (HoxA9 MIEG3 N=8, HoxA9 Gpr56 N=10, p<0.05). The mice displayed splenomegaly (mean 0.47g ± 0.07), and a high expression of myeloid markers on leukemic cells (Mac-1: 31%-57%; Mac-1/Gr-1: 24%-53%; Gr-1: 0.5%-16%). Cells co-transduced with Gpr56 and HoxA9 showed a decrease in Cxcr4 expression, when compared to HoxA9 alone and a 6-fold reduction in CFCs (p<0.05) when cells were introduced to an in-vitro SDF-1 gradient migration assay. However, BM cells co-transduced with Hoxa9 and Gpr56 did not show any significant drop in their homing capacity in vivo. Of note, leukemogenicity of Hoxa9 BM cells co-transduced with Meis1 was dependent on Gpr56 expression, as knockdown of Gpr56 expression via shRNA doubled the latency time in transplanted mice (median latency 55 days compared to 95 days, respectively p<0.05). Knockdown of Gpr56 induced a decrease in cells capable of attaching to the stroma in the adhesion cobble-stone area forming cell assay (CAFC) (6.5 fold reduction). However, cells able to home to the BM in vivoincreased 2.3 fold (p<0.05), suggesting that the delay in disease progression was due to a decrease in attaching to the niche.In summary, our data characterize Gpr56 as an essential partner in Hoxa9 associated myeloid leukemogenesis, underlining the importance of members of the adhesion-GPCR family of receptors in myeloid leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Potential roles for Gfi1 in the pathogenesis and proliferation of glioma

Glioblastoma multiforme (GBM) is a major form of adult brain tumour with relatively poor prognosis and high mortality. Temozolomide (TMZ)-based chemotherapy following neurosurgery and radiotherapy has been suggested as the first line of treatment and is proven to effectively prolong overall survival and enhance patient quality of life. However, not all patients benefit from this treatment because of drug resistance. Even patients with TMZ-sensitive GBM may become resistant, which is partly due to the restoration of activity of the DNA repair enzyme O(6)-methylguanine-DNA-methyltransferase (MGMT); thus, patients cannot evade eventual tumour recurrence. The cellular activity of MGMT is the most important determinant of TMZ-resistance. However, some patients with a low level of activated MGMT are also TMZ-resistant. The aberrant expression of HOXA9, one of the 39 class I homeobox genes, is a marker of poor prognosis, and its level gradually increases with histologic malignant progression, shorter time to overall survival (OS) and free progression survival (FPS) in glioma patients, which further supports an oncogenic role for HOXA9 in gliomas. The HOXA9-PI3K signalling pathway is an important mechanism in GBM that is independent of MGMT promoter methylation status. The DNA binding sites of growth factor independent-1 (Gfi1) can overlap with the HOXA9 promoter through the “AATC” versus “GATT” core sequence. The competition for this binding site inhibits the expression of HOXA9 and induces different transcriptional outcomes, which suggests a new direction for investigation of the mechanism underlying targeted therapy of malignant gliomas.

Reversing HOXA9 Oncogene Activation by PI3K Inhibition: Epigenetic Mechanism and Prognostic Significance in Human Glioblastoma

HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using reverse transcription-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrently activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a phosphoinostide 3-kinase (PI3K) inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with tumor necrosis factor-related apoptosis-including ligand resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent, and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies.

HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Abstract 832We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures:No relevant conflicts of interest to declare.

DEK Proto-Oncogene Expression Interferes with the Normal Epithelial Differentiation Program

Overexpression of the DEK gene is associated with multiple human cancers, but its specific roles as a putative oncogene are not well defined. DEK transcription was previously shown to be induced by the high-risk human papillomavirus (HPV) E7 oncogene via E2F and Rb pathways. Transient DEK overexpression was able to inhibit both senescence and apoptosis in cultured cells. In at least the latter case, this mechanism involved the destabilization of p53 and the decreased expression of p53 target genes. We show here that DEK overexpression disrupts the normal differentiation program in a manner that is independent of either p53 or cell death. DEK expression was distinctly repressed upon the differentiation of cultured primary human keratinocytes, and stable DEK overexpression caused epidermal thickening in an organotypic raft model system. The observed hyperplasia involved a delay in keratinocyte differentiation toward a more undifferentiated state, and expansion of the basal cell compartment was due to increased proliferation, but not apoptosis. These phenotypes were accompanied by elevated p63 expression in the absence of p53 destabilization. In further support of bona fide oncogenic DEK activities, we report here up-regulated DEK protein levels in both human papilloma virus-positive hyperplastic murine skin and a subset of human squamous cell carcinomas. We suggest that DEK up-regulation may contribute to carcinoma development at least in part through increased proliferation and retardation of differentiation.

The Myocyte Enhancer Factor 2C (MEF2C) Is a Direct Transcriptional Target of HOXA9 and Mediates Leukemia Survival in Human Mixed-Lineage Leukemias.

Leukemias that harbor rearrangements of the mixed-lineage leukemia gene (MLL) possess unique clinical and biological characteristics. Aberrant high-level expression of select Homeobox (HOX) genes including HOXA9 is a consistent feature of MLL-rearranged leukemias, implicating an important role of dysregulated HOX gene expression in this entity. Applying HOXA9-directed RNAi, we have previously shown that aberrant HOXA9 expression is a prerequisite for leukemia survival in human MLL-rearranged leukemias and murine Mll-leukemia stem cells as HOXA9 suppression leads to a progressive induction of apoptosis in vitro and in vivo. In this study we aimed to identify potential direct HOXA9 target genes in human MLL-rearranged leukemias utilizing gene expression profiling after shRNA mediated HOXA9 knockdown and subsequent chromatin immunoprecipitation (ChIP) analysis. To establish efficient HOXA9 knockdown, 2 previously validated shRNA constructs targeting human HOXA9 with high efficiency (>80% mRNA suppression) were transduced into t(9;11) MOLM-14 AML cells. 72h after transduction, RNA was hybridized to Affymetrix HU 133A2.0 expression arrays. Supervised analysis identified a large group of genes concomitantly downregulated after HOXA9 suppression. Gene set enrichment analysis demonstrated significant enrichment of the top 300 genes in the HOXA9 suppression signature and genes more highly expressed in human MLL-AML as compared to other genetically defined AML subtypes (p=0.02). Interestingly, genes previously implicated in the pathogenesis of MLL-rearranged leukemias were among the most highly enriched (HOXA5, MEIS1, PBX3, MEF2C). To analyze if any of this genes might be a direct physiological HOXA9 target, we performed ChIP assays from FLAG-tagged HOXA9 expressing MOLM-14 cells. Whereas the promotor regions of HOXA5, MEIS1 and PBX3 did not show HOXA9 binding, the MEF2C promotor region DNA was highly enriched by ChIP. Furthermore HOXA9 binding was confined to the MEF2C promotor region as the regions -5kb 5'-upstream, +5kb and +10kb 3'-downstream of the MEF2C promotor region did not show any enrichment. Motive search analysis of the MEF2C promoter further revealed a perfect consensus binding site (TGATTTAT) for HOXA9 and its DNA binding partner PBX located at −721 to −714 from the transcriptional start site. We then analyzed if direct shRNA mediated targeting of MEF2C also abrogates leukemia survival in human MLL-rearranged leukemias similar to upstream HOXA9 suppression. Analysis of viability after MEF2C knockdown with two different shRNA constructs (>80% mRNA knockdown) in a panel of human 8 AML/ALL cell lines (4 MLL-rearranged; 4 MLL-wildtype) and primary AML patient cells (5 MLL-rearranged; 5 MLL-wildtype) revealed progressive induction of apoptosis which was significantly correlated with the presence of the MLL-fusion oncogene in the cell lines (mean viability at day 7: MLL-rearranged: 14.5% MLL-wildtype: 86.75%: p<0.001) and in the primary AML cells (p=0.006). Our data implicates MEF2C as a direct downstream target of HOXA9 and aberrant MEF2C expression is necessary for survival of MLL-rearranged leukemias. Targeting the HOXA9-MEF2C program may be a novel therapeutic approach.

Suppression of HOX A cluster genes inhibits proliferation and induces cell death in human mixed-lineage leukemias

14020 Background: Leukemias harboring translocations of the mixed lineage leukemia locus (MLL) are generally associated with poor clinical prognosis. Using gene expression profiling we and others have previously shown that Homeobox (HOX) A cluster genes are highly expressed in leukemias with MLL rearrangements. Methods: Here we studied the role of aberrant HOXA9 expression in human MLL- rearranged and non-rearranged leukemias utilizing an shRNA mediated knockdown approach. Results: Three different shRNA constructs targeting human HOXA9 were synthesized and stably introduced into t(9;11) MOLM14 cells utilizing a lentiviral vector system. 75–80% HOXA9 RNA knockdown was confirmed by quantitative PCR and Western Blot analysis. In a panel of 17 AML/ALL cell lines (7 MLL rearranged, 10 non rearranged), HOXA9 directed shRNA inhibited cell proliferation starting as early as 48h after transduction, and induced apoptosis beginning at 72h. Interestingly, impaired cell proliferation and induction of apoptosis was significantly higher in the MLL rearranged cell lines (mean viability: 51.88%) than in the non-rearranged cells (mean viability: 90.98%; p=0.007) and also significantly correlated with the baseline HOXA9 mRNA expression before knockdown (R= 0.8, p=0.00017). We then further analyzed the effect of HOXA9 knockdown in MLL rearranged and non-rearranged primary human AML cells. Similar to our findings in cell lines, a marked induction of cell death was observed, which was significantly higher in leukemias with an MLL translocation (p=0.005) and also significantly correlated with the baseline HOXA9 mRNA expression (R= 0.8, p=0.001). Next, the in vivo effect of HOXA9 knockdown was assessed by transplanting luciferase-expressing SEMK2 (t4;11) cells into SCID-beige mice followed by in vivo bioluminescent imaging. Leukemia burden was significantly reduced in HOXA9 shRNA treated mice (n=10) with a peak difference at day 15 (p=0.000059) shortly before mice of the control group (n=10) succumbed from overt leukemia. At this point all mice of the HOXA9 shRNA treated group were still healthy with no signs of leukemia. Conclusions: Taken together our data implicates that depletion of HOXA9 might be a novel approach for targeted therapy in human MLL rearranged leukemias. No significant financial relationships to disclose.

HoxA9 Knockdown Inhibits Proliferation and Induces Cell Death in Human MLL-Rearranged Leukemias.

Homeobox containing (Hox) genes are implicated in the regulation of normal and leukemic hematopoesis. Using gene expression profiling we and others have previously shown that HoxA9 is highly expressed in lymphoid and myeloid leukemias harboring MLL translocations and that high level HoxA9 expression is associated with poor clinical prognosis. Furthermore, HoxA9 plays variable roles in MLL-fusion induced murine leukemias. In this study we aimed to elucidate the role of aberrant HoxA9 expression in human MLL-rearranged and non-rearranged leukemia's utilizing an shRNA mediated knockdown approach. To establish an efficient knockdown assay three different shRNA constructs targeting human HoxA9 were synthesized and stably introduced into t(9;11) MOLM14 cells utilizing a lentiviral vector system. The shRNA construct which showed highest efficiency as measured by Taqman quantitative PCR (75–80% knockdown MLL-AF9 RNA) and Western Blot analysis was used for further experiments. In MOLM-14 cells, HoxA9 directed shRNA inhibited cell proliferation starting as early as 48h after transduction as determined by MTT assay, and at 72h demonstrated a markedly increased number of apoptotic cells as measured by Annexin V staining. This effect was rescued by introducing a non-targetable exogenous HoxA9 in MOLM-14 cells. To investigate if the HoxA9 knockdown related effects are specific for MLL rearranged cells we next analyzed cell growth and viability in 17 AML/ALL cell lines (7 MLL-rearranged, 10 non rearranged) after shRNA mediated HoxA9 knockdown. Interestingly, impaired cell proliferation and induction of apoptosis was significantly higher in the MLL rearranged cell lines (mean viability: 51.88%) than in the non-rearranged cells (mean viability: 90.98%; p=0.007). Moreover, the effect was also significantly correlated with the baseline HoxA9 mRNA expression before knockdown, with the greatest effect in cell lines expressing the highest HoxA9 levels (R= 0.8, p=0.00017). These findings prompted us to further analyze the effect of HoxA9 knockdown in MLL rearranged and non-rearranged primary human AML cells (6 MLL rearranged, 6 MLL germline). Similar to our findings in cell lines, we found a significantly higher effect on cell proliferation/viability in association with the presence of an MLL translocation (p=0.005) and a significant correlation with the baseline HoxA9 mRNA expression (R= 0.8, p=0.001). Next, we assessed the in vivo effect of HoxA9 knock down by transplanting luciferase-expressing SEMK2 (t4;11) cells and subsequent bioluminescent imaging. SEMK2 cells were transduced with either HoxA9 directed or control shRNA and intravenously injected into SCID-beige mice.Reconstitution was confirmed by in vivo bioluminescent imaging. 34 days after transplantation all mice in the HoxA9 shRNA group (n=4) are still alive with no signs of leukemia whereas all mice in the control group (n=3) have succumbed. Taken together our data implicates an important role for aberrant HoxA9 expression in human MLL rearranged leukemia cells.

Smad4 Inhibits Nup98-Hoxa9 Transcriptional Activity by Interacting at Hox Homeodomain.

Homeobox (Hox) genes play crucial roles in hematopoiesis. Alterations of Hox gene expressions are found in patients with hematological malignancy. Nup98-Hoxa9, a fusion protein combining the N-terminus of Nup98 and the C-terminus of Hoxa9 resulting from t (7; 11) chromosomal translocation, is the first evidence of Hox gene involvement in human leukemia. We have shown that BMP specific R-Smads and Smad4 interact with Hox proteins including Hoxa9, and the interaction blocks binding of Hox proteins to their DNA elements. Here, we show that Smad4, the common signal transducer of both TGF-β and BMP, interacts with the homeodomain of Nup98-Hoxa9 in a coimmunoprecipitation assay. Furthermore, this interaction abolished the DNA binding ability of Nup98-Hoxa9 to DNA in both EMSA using purified GST fusion protein and in in vivo chromatin immunoprecipitation assays. Subsequent mapping data revealed the MH1 domain of Smad4 mediated the interaction. These findings suggest that Smad4 could be used to inhibit Hox-induced leukemia.We then attempted to examine whether Smad4 could inhibit the downstream target gene by Nup98-Hoxa9. Although Nup98-Hoxa9 is believed to function as an aberrant DNA-binding transcriptional activator, downstream genes that contribute to leukemogenesis are largely unknown. It has been reported that Nup98-Hoxa9 induces Hoxa9 expression in murine primary bone marrow cells. Accumulating evidence suggests that Hoxa9 plays important roles in normal and malignant hematopoiesis. Enforced expression of Hoxa9 causes proliferative expansion of primitive hematopoietic stem/progenitor cells and subsequently inhibits their differentiation. Aberrant expression of HOXA9 due to retroviral integration induced acute myeloid leukemia (AML) in mice. By visually scanning the sequence of the Hoxa9 promoter, we found many TGAT and TTA (T/C) consensus sequences, which are preferentially recognized by the heterodimer of PBX protein and the proteins of the Abd-B-like gene family, including Hoxa9, respectively. To test whether Hoxa9 could be a direct transcriptional target for Nup98-Hoxa9, we used a Hoxa9 promoter/luciferase reporter construct along with the expression plasmid pcDNA-Nup98/Hoxa9 to transiently transfect NIH/3T3 fibroblast cells and Ba/F3 murine hematopoietic progenitor cells. Nup98-Hoxa9 showed substantial transcription activation on Hoxa9 promoter in these cell lines. This activation is further enhanced by cotransfection of PBX1a, which stabilizes the binding of Nup98-Hoxa9 to DNA. Nup98-Hoxa9 also bound to radiolabelled DNA oligo from the Hoxa9 promoter in vitro by EMSA assay. These data indicate that Nup98-Hoxa9 directly activates the Hoxa9 promoter. Interestingly, Smad4 removed the binding of Nup98-Hoxa9 to the Hoxa9 promoter. Co-overexpression of Smad4 inhibited the transactivation activity of Nup98-Hoxa9 on the Hoxa9 promoter regardless of the presence of PBX1a. In NIH/3T3 cells, Smad4 inhibited Nup98-Hoxa9-induced Hoxa9 expression both at the mRNA and protein levels. In summary, we show that Smad4 inhibited DNA binding activity of Nup98-Hoxa9 by interacting at Hox homeodomain. This interaction abolished the transcription activity of Nup98-Hoxa9 on its target gene, thus preventing Nup98-Hoxa9 from working as a transcription factor, which could lead to a new therapeutic target for the treatment of myeloid leukemia caused by Nup98-Hoxa9.

Aberrant Expression of HOXA9, DEK, CBL and CSF1R in Acute Myeloid Leukemia

Previous gene function analyses have indicated that HOXA9, DEK, CBL and CSF1R are aberrantly expressed in acute myeloid leukemia (AML). We analyzed the expression of these genes in a series of 41 adult patients with AML using quantitative real-time RT-PCR, and tested the association of the expression with the following hematologic and clinical parameters: age, FAB, immunophenotype and karyotype aberrations. A high proportion of the patients showed over- or underexpression of the analyzed genes. DEK was overexpressed in 98% of the cases, whereas CBL, CSF1R and HOXA9 were either overexpressed in 20%, 17% and 78% or underexpressed in 20%, 42% and 15% of the cases, respectively. Patients whose karyotype contained t(8;21)(q22;q22), showed lower relative expression of HOXA9 at a statistically significant level <formula>(<italic>p</italic><0.05).</formula> Bone marrow samples without expression of CD34 antigen were associated with either overexpression of DEK or HOXA9. Furthermore, an association was found between the AML-M2 subtype and lower expression of CBL, CSF1R or HOXA9, and between the AML-M5 subtype and CBL or CSF1R overexpression.