Human MLL-rearranged Leukemia Research Articles

MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia

Despite growing awareness of the biologic features underlying MLL-rearranged leukemia, targeted therapies for this leukemia have remained elusive and clinical outcomes remain dismal. MBNL1, a protein involved in alternative splicing, is consistently overexpressed in MLL-rearranged leukemias. We found that MBNL1 loss significantly impairs propagation of murine and human MLL-rearranged leukemia in vitro and in vivo. Through transcriptomic profiling of our experimental systems, we show that in leukemic cells, MBNL1 regulates alternative splicing (predominantly intron exclusion) of several genes including those essential for MLL-rearranged leukemogenesis, such as DOT1L and SETD1A. We finally show that selective leukemic cell death is achievable with a small molecule inhibitor of MBNL1. These findings provide the basis for a new therapeutic target in MLL-rearranged leukemia and act as further validation of a burgeoning paradigm in targeted therapy, namely the disruption of cancer-specific splicing programs through the targeting of selectively essential RNA binding proteins.

Leukemia Cell of Origin Influences p53 Activity and Therapeutic Sensitivity Via an Evi1-Dependent Mechanism

Leukemia Cell of Origin Influences p53 Activity and Therapeutic Sensitivity Via an Evi1-Dependent Mechanism

Selective Dependency of MLL-Rearranged Leukemia on Immunoproteasome Function

Several cellular pathways control the fine balance between self-renewal and differentiation to maintain leukemia-initiating cell (LIC) function. To identify cellular dependencies with relevance for oncogenic fusion proteins, we performed global proteome profiling. Acute myeloid leukemia (AML) was induced by retroviral expression of either MLL-AF9 (MA9) or AML1-ETO9a (AE) in murine hematopoietic stem and progenitor cells (HSPCs) (Lineage-Sca1+Kit+, LSK) which were subsequently transplanted into irradiated syngeneic recipients. After onset of leukemia, LIC-enriched (GFP+ Kithigh) cells isolated from 4 different primary recipients (per oncogene) were analyzed by in-depth quantitative proteomic analysis using high-resolution mass spectrometry (MS). More than 3,000 proteins were quantified with 868 proteins being differentially expressed between MA9 and AE LIC-enriched populations. In MLL-rearranged (MLLr) cells, gene set enrichment analysis (GSEA) revealed significant enrichment of cellular functions related to protein degradation and proteasome function. As this enrichment is present in MLLr-leukemia but not AE-driven LICs, may indicate an oncogene specific vulnerability. Expression of proteasome subunits is highly heterogeneous between different cell types and therefore may also be influenced by the underlying differentiation stage or oncogenic fusion. In published AML gene-expression datasets, immunoproteasome (IP) subunits PSMB8/LMP7 (p=0.0003***), PSMB9/LMP2 (p=0.0007***) and PSMB10/MECL1 (p<0.0001****) showed significantly higher expression in MLLr compared to non-MLLr-AML. IP is a proteasomal variant constitutively expressed in cells of hematopoietic origin, induced under stimulation with pro-inflammatory cytokines and relevant for mediating stress-responses during inflammation and infection. To assess for functional dependency of MLLr cells on IP subunits we performed an in vitro CRISPR/Cas9 dropout screen in MLLr MOLM-13 cells. Genetic inactivation of PSMB8/LMP7 resulted in outcompetition with 3/5 sgRNAs, while there was less dependency detectable for the other subunits. Specificity of this finding was confirmed in 5 different cell lines (4 MLLr; 1 non-MLLr) by RNAi using 2 shRNAs against PSMB8/LMP7 versus non-targeting control. To confirm these findings in primary cells, we used a previously published conventional LMP7 knockout mouse model (Fehling et al., Science, 1994). LSK cells sorted from the bone marrow (BM) of LMP7 knockout and wildtype mice were retrovirally transformed with either MA9, MLL-ENL (ME) or NUP98-HOXA9 (as non-MLLr control) to assess for disease development by serial plating in methylcellulose. Only in MA9 or ME transformed cells LMP7-deficiency limited re-plating capacity to 2-4 rounds. When we injected 2,5x 104 MA9-infected LSK cells into sublethally irradiated recipient mice, recipients of MA9-LMP7-/- cells (n=12) and MA9-LMP7+/+ (n=12) showed development of AML. However, recipients of MA9-LMP7-/- cells had a significant delay in AML development (median survival 63.0 days for LMP7+/+ versus 92.5 days for LMP7 -/- animals, p=0.0387*). Besides the significant delay in AML development, disease penetrance was reduced by 50%, indicating that deficiency for LMP7 impairs development of MA9 driven AML. In contrast, immunophenotypic abundance of HSPCs in LMP7-/- versus LMP7+/+ animals revealed comparable numbers in all relevant subpopulations. Competitive transplantation of LMP7-/- BM into recipient hosts showed no competitive disadvantage or lack of self-renewal capacity compared to LMP7+/+ controls. Pharmacologic inhibition of IP function using the specific LMP7-inhibitor PR-957 (ONX-0914) resulted in significant delay of disease development in secondary recipient hosts. To assess its effect on LIC frequency we performed limiting dilution assays of MA9 leukemic cells in sublethally irradiated recipient mice. PR-957 treatment reduced LIC frequency compared to DMSO control (1/57410 vs. 1/4450). Pharmacologic inhibition of PSMB8/LMP7 in human MLLr leukemia cell lines induced cellular differentiation. Likewise, cell cycle and metabolism appeared affected, functions which could be confirmed by global transcriptome analysis. Taken together, our studies uncover a selective dependency of MLLr-leukemia on IP function and identify PSMB8/LMP7 as a tractable target. Disclosures Heidel: Celgene: Consultancy; Novartis: Consultancy, Research Funding; CTI: Consultancy.

Discovery of Novel Inhibitors Targeting the Menin-Mixed Lineage Leukemia Interface Using Pharmacophore- and Docking-Based Virtual Screening.

Disrupting the interaction between mixed lineage leukemia (MLL) fusion protein and menin provides a therapeutic approach for MLL-mediated leukemia. Here, we aim to discover novel inhibitors targeting the menin-MLL interface with virtual screening. Both structure-based molecular docking and ligand-based pharmacophore models were established, and the models used for compound screening show a remarkable ability to retrieve known active ligands from decoy molecules. Verified by a fluorescence polarization assay, five hits with novel scaffolds were identified. Among them, DCZ_M123 exhibited potent inhibitory activity with an IC50 of 4.71 ± 0.12 μM and a KD of 14.70 ± 2.13 μM, and it can effectively inhibit the human MLL-rearranged leukemia cells MV4;11 and KOPN8 with GI50 values of 0.84 μM and 0.54 μM, respectively.

Flt3 Does Not Play a Critical Role in Murine Myeloid Leukemias Induced by MLL Fusion Genes

Leukemias harboring MLL translocations are frequent in children and adults, and respond poorly to therapies. The receptor tyrosine kinase FLT3 is highly expressed in these leukemias. In vitro studies have shown that pediatric MLL-rearranged ALL cells are sensitive to FLT3 inhibitors and clinical trials are ongoing to measure their therapeutic efficacy. We sought to determine the contribution of Flt3 in the pathogenesis of MLL-rearranged leukemias using a myeloid leukemia mouse model. Bone marrow from Flt3 null mice transduced with MLL-ENL or MLL-CBP was transplanted into host mice and Flt3 −/− leukemias were compared to their Flt3 wild type counterparts. Flt3 deficiency did not delay disease onset and had minimal impact on leukemia characteristics. To determine the anti-leukemic effect of FLT3 inhibition we studied the sensitivity of MLL-ENL leukemia cells to the FLT3 inhibitor PKC412 ex vivo. As previously reported for human MLL-rearranged leukemias, murine MLL-ENL leukemia cells with higher Flt3 levels were more sensitive to the cytotoxicity of PKC412. Interestingly, Flt3 deficient leukemia samples also displayed some sensitivity to PKC412. Our findings demonstrate that myeloid leukemias induced by MLL-rearranged genes are not dependent upon Flt3 signaling. They also highlight the discrepancy between the sensitivity of cells to Flt3 inhibition in vitro and the lack of contribution of Flt3 to the pathogenesis of MLL-rearranged leukemias in vivo.

Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Abstract 1311Mixed-lineage-leukemia (MLL) fusion oncogenes are involved in de novo and secondary therapy-related acute leukemia in humans. To understand the pathogenesis of MLL-rearranged leukemia, several murine models for this disease have been established. However, the leukemia derived from murine hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. In vivo studies, in which primary human leukemia cells were transplanted into immunodeficient mice, provided us significant advances to understand the pathogenesis of human leukemia. However, these models are not suitable for studying physiological leukemogenesis because the initiation and progression processes are hard to be evaluated. Therefore, a new in vivo experimental model for analyzing leukemogenesis derived from primary human HSCs and by genetic hits is required.Here we developed a humanized murine model for human leukemia by transplanting human cord blood-derived CD34-positive HSCs transduced with an MLL-AF10 oncogene and second hits into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice.Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, however, MLL-AF10 alone could not induce leukemic transformation. Accumulating evidence points to a multistep pathogenesis for leukemia development and progression. In the multi-genetic step models of leukemogenesis, particularly in the two-hit model of leukemia, the initial genetic hit often leads to abnormal cell differentiation (Type II mutation), while subsequent mutations may activate specific signaling pathways that are involved in cell growth, such as the Ras/MAP kinase pathway (Type I mutation). Because active mutations in Ras genes are often found in MLL-related leukemia, we next transduced constitutively active form of K-Ras mutant along with the MLL-AF10 oncogene. Enforced expression of the mutant K-Ras alone was insufficient to induce leukemia. Eight weeks after transplantation, all of the recipient mice transduced both MLL-AF10 and mutant K-Ras developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We could also observe organ infiltration in liver and spleen in vivo that mimicked clinical symptoms observed in human leukemia.We thus successfully established a murine model for human MLL-rearranged leukemia derived from human HSCs. The present model is a useful experimental platform for the multi-step leukemogenesis of human leukemia and for new therapeutic approaches that should be evaluated in vivo. Disclosures:No relevant conflicts of interest to declare.

Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.

Two isoforms of HOXA9 function differently but work synergistically in human MLL-rearranged leukemia

HOXA9 plays a critical role in both normal hematopoiesis and leukemogenesis, particularly in the development and maintenance of mixed lineage leukemia (MLL)-rearranged leukemia. Through reverse transcription-polymerase chain reaction (RT-PCR) analysis of HOXA9 transcripts in human leukemia and normal bone marrow samples, we identified a truncated isoform of HOXA9, namely HOXA9T, and found that both HOXA9T and canonical HOXA9 were highly expressed in leukemia cell lines bearing MLL rearrangements, relative to human normal bone marrow cells or other subtypes of leukemia cells. A frameshift in HOXA9T in exon I causes a premature stop codon upstream of the PBX-binding domain and the homeodomain, which leads to the generation of a non-homeodomain-containing protein. Unlike the canonical HOXA9, HOXA9T alone cannot transform normal bone marrow progenitor cells. Moreover, HOXA9T cannot cooperate with MEIS1 to transform cells, despite the presence of a MEIS1-binding domain. Remarkably, although the truncated isoforms of many proteins function as dominant-negative competitors or inhibitors of their full-length counterparts, this is not the case for HOXA9T; instead, HOXA9T synergized with HOXA9 in transforming mouse normal bone marrow progenitor cells through promoting self-renewal and proliferation of the cells. Collectively, our data indicate that both truncated and full-length forms of HOXA9 are highly expressed in human MLL-rearranged leukemia, and the truncated isoform of HOXA9 might also play an oncogenic role by cooperating with canonical HOXA9 in cell transformation and leukemogenesis.

Abstract SY02-01: Targeting the histone methyltransferase DOT1L in MLL-rearranged leukemia

Abstract Protein methyltransferases (PMTs) are enzymes that catalyze the addition of methyl groups to the lysine and arginine residues of proteins, including histones. Recent evidence shows that PMTs are misregulated in a variety of cancers and thus may serve as therapeutic targets. A subset of acute leukemias have reciprocal translocations at 11q23 involving the PMT gene mixed lineage leukemia (MLL). MLL translocations occur in approximately 5% of adult acute lymphocytic leukemias (ALL) and 5 to 10% of adult acute myeloid leukemias (AML) including therapy-related leukemias. Multiple studies have demonstrated that patients with leukemias bearing the MLL translocation have a poor prognosis and are in need of new therapies. Translocations of the MLL gene result in the loss of the catalytic domain of the protein. The most common translocation partners, AF4, AF9 and ENL, recruit another PMT, DOT1L that methylates histone H3K79. Recruitment of DOT1L to the MLL fusion protein results in increased H3K79 methylation at MLL fusion protein target genes and elevated expression of the MLL target genes resulting in leukemogenesis. Mistargeted DOT1L enzymatic activity has therefore been proposed to be required for the development and maintenance of leukemias bearing the MLL translocation. To evaluate the potential of DOT1L as a therapeutic target in MLL- rearranged leukemia, we designed and characterized the DOT1L inhibitor EPZ004777, a small molecule with sub-nanomolar affinity for this enzyme and >1000 fold selectivity against other PMTs. EPZ004777 selectively inhibits intracellular histone H3K79 methylation in a concentration and time dependent manner. Significant changes in genes expression were observed in MLL-rearranged cell lines following the decrease in histone H3K79 methylation. Gene set enrichment analysis (GSEA) of genes downregulated following EPZ004777 treatment demonstrated strong enrichment for genes overexpressed in MLL-rearranged human acute leukemias as compared with MLL-germline acute leukemias. Phenotypically, EPZ004777 inhibits proliferation and induces apoptosis in human MLL-rearranged leukemia cell lines, with little effect on non-MLL-translocated cells. In vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These studies provide compelling validation for the development of DOT1L inhibitors as targeted therapeutics for MLL-rearranged leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY02-01. doi:1538-7445.AM2012-SY02-01

HOXA9 is required for survival in human MLL-rearranged acute leukemias

Leukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biologic characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression of select Homeobox genes, including HOXA9. Here, we investigated the effects of HOXA9 suppression in MLL-rearranged and MLL-germline leukemias using RNA interference. Gene expression profiling after HOXA9 suppression demonstrated co-down-regulation of a program highly expressed in human MLL-AML and murine MLL-leukemia stem cells, including HOXA10, MEIS1, PBX3, and MEF2C. We demonstrate that HOXA9 depletion in 17 human AML/ALL cell lines (7 MLL-rearranged, 10 MLL-germline) induces proliferation arrest and apoptosis specifically in MLL-rearranged cells (P = .007). Similarly, assessment of primary AMLs demonstrated that HOXA9 suppression induces apoptosis to a greater extent in MLL-rearranged samples (P = .01). Moreover, mice transplanted with HOXA9-depleted t(4;11) SEMK2 cells revealed a significantly lower leukemia burden, thus identifying a role for HOXA9 in leukemia survival in vivo. Our data indicate an important role for HOXA9 in human MLL-rearranged leukemias and suggest that targeting HOXA9 or downstream programs may be a novel therapeutic option.

H3K79 Methylation Profiles Define Murine and Human MLL-AF4 Leukemias

We created a mouse model wherein conditional expression of an Mll-AF4 fusion oncogene induces B precursor acute lymphoblastic (ALL) or acute myeloid leukemias (AML). Gene expression profile analysis of the ALL cells demonstrated significant overlap with human MLL-rearranged ALL. ChIP-chip analysis demonstrated histone H3 lysine 79 (H3K79) methylation profiles that correlated with Mll-AF4-associated gene expression profiles in murine ALLs and in human MLL-rearranged leukemias. Human MLL-rearranged ALLs could be distinguished from other ALLs by their H3K79 profiles, and suppression of the H3K79 methyltransferase DOT1L inhibited expression of critical MLL-AF4 target genes. We thus demonstrate that ectopic H3K79 methylation is a distinguishing feature of murine and human MLL-AF4 ALLs and is important for maintenance of MLL-AF4-driven gene expression.

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.

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.