MLL-AF6 Mediated Transformation Is Dependent On the H3K79 Methyl-transferase Dot1l

Abstract Abstract 3502 The t(6;11)(q27;q23) produces a chimeric MLL-AF6 oncogene, and is a recurrent chromosomal rearrangement observed in patients with diverse hematologic malignancies such as acute myelogenous leukemia (AML), as well as both B-cell and T-cell acute lymphoblastic leukemias (ALL). The presence of an MLL-AF6 translocation predicts a particularly poor prognosis. Of particular biological interest, the MLL-AF6 translocation is the most common fusion event in which MLL fuses to a predominantly cytoplasmic protein. Very little is known about the molecular mechanisms of transformation mediated by the MLL-AF6 fusion oncogene, forestalling the development of specific therapeutic strategies for t(6;11)(q27;q23) positive leukemias. Recent studies suggest that the histone methyltransferase DOT1L could be an important therapeutic target in MLL-rearranged leukemias. We sought to assess whether MLL-AF6 mediated transformation is also dependent on aberrant H3K79 methylation using genomic, genetic and pharmacological approaches. First, we performed chromatin immuno-precipitation using H3K79me2 specific antibodies followed by next generation sequencing (ChIP-seq) on murine MLL-AF6 leukemias as well as on ML2, the human myelomonocytic leukemia cell line bearing the MLL-AF6 fusion gene. We observed that in both murine and human MLL-AF6 leukemia cells, MLL-fusion target genes display markedly high levels of H3K79 dimethylation as compared to other highly expressed genes. We then investigated whether MLL-AF6-induced transformation was dependent on aberrant H3K79 methylation through genetic or pharmacologic inhibition of the Dot1l histone methyltransferase. Lineage negative/Sca-1 positive/Kit positive (LSK) cells from mice bearing homozygous Dot1l floxed alleles were immortalized by retroviral expression of the MLL-AF6 fusion gene. Cre-recombinase mediated excision of Dot1l from MLL-AF6 transformed bone marrow cells resulted in a significant reduction in H3K79 dimethylation at the promoters of the MLL-target genes Hoxa9, Hoxa10 and Meis1, with a concomitant decrease in their expression. Dot1l excision significantly diminished the clonogenic capacity, abrogated blast colony formation in methylcellulose based medium, and enhanced differentiation of MLL-AF6 transformed cells. We then sought to assess whether EPZ004777, a recently described specific small molecule inhibitor of DOT1L could show efficacy against murine and human MLL-AF6 transformed cells. Dot1l inhibition using EPZ004777 significantly diminished H3K79 dimethylation globally (as assessed by immunoblotting) as well as on MLL-target genes (as assessed by ChIP-qPCR) using H3K79me2 specific antibodies. Importantly, EPZ004777 treatment significantly impaired the proliferation of both murine MLL-AF6 transformed cells as well as the ML2 cell line, whereas the proliferation rates of Hoxa9-Meis1 transformed cells as well as the human MLL-germline cell line HL60 were unaffected despite a similar decrease in H3K79me2 levels. EPZ004777 treatment induced cell cycle arrest as well as increased apoptosis in MLL-AF6 positive, but not control leukemia cells, demonstrating a selective activity of the DOT1L inhibitor EPZ004777 on MLL-AF6 transformed cells. In summary, we demonstrate that the MLL-AF6 oncoprotein requires continued activity of the histone methyltransferase DOT1L for aberrant epigenetic activation of downstream target oncogenes. More studies are needed to understand the mechanisms by which DOT1L is recruited to MLL-target genes by the MLL-AF6 fusion, since AF6 is not believed to normally associate with DOT1L. Nevertheless, the demonstration that H3K79 methylation is important for MLL-AF6 mediated transformation indicates that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation. Disclosures: Olhava: Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

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The Interaction Between DOT1L and AF10 Is Required for H3K79 Dimethylation and MLL-AF9 Leukemia

Blood ◽

10.1182/blood.v120.21.401.401 ◽

2012 ◽

Vol 120 (21) ◽

pp. 401-401

Author(s):

Aniruddha J Deshpande ◽

Liying Chen ◽

Kathrin M Bernt ◽

Stuart Dias ◽

Deepti Banka ◽

...

Keyword(s):

Gene Expression ◽

Mass Spectrometry ◽

Target Gene ◽

Leucine Zipper ◽

Target Genes ◽

Fusion Gene ◽

Transformed Cells ◽

Target Gene Expression ◽

Transforming Activity ◽

H3k79 Methylation

Abstract Abstract 401 MLL-fusion proteins induce changes in histone modifications that result in the abnormal and sustained expression of downstream oncogenic target genes. A number of recent studies have identified aberrant histone 3 lysine 79 (H3K79) methylation by the chromatin modifying enzyme DOT1L as an important epigenetic modification that sustains MLL-target gene expression. Aberrant H3K79 methylation has been shown to be necessary for oncogenic transformation mediated by a number of MLL-fusions. These recent findings have generated tremendous interest in H3K79 methylation as a therapeutic target in the MLL rearranged leukemias. The plant-homeodomain (PHD) and leucine zipper-containing protein AF10 biochemically interacts with DOT1L and is believed to influence H3K79 methylation. We generated conditional knockout mice in which the Dot1l-interacting octapeptide-motif leucine zipper (OM-LZ) domain of Af10 was flanked by LoxP sites. Deletion of the Af10OM-LZ domain with the Cre recombinase is predicted to abrogate the Af10-Dot1l interaction. Deletion of the Af10OM-LZ domain greatly reduced global H3K79 dimethylation as assessed by immunoblotting as well as mass spectrometry in Af10OM-LZ deleted HoxA9/Meis1a transformed cells. Given the importance of H3K79 methylation in MLL-rearranged leukemias, we sought to assess whether the transforming activity of the MLL-AF9 fusion gene was dependent on the Af10-Dot1l interaction. Using an MLL-AF9-IRES-GFP encoding retrovirus, we established immortalized blast-colony forming cultures from mouse lineage negative Sca-1 positive/Kit positive (LSK) bone marrow cells bearing floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain with Cre-recombinase dramatically reduced H3K79me2 on the MLL-target genes Hoxa5-10 and Meis1, leading to downregulation of these transcripts. We performed colony-forming cell (CFC) assays from MLL-AF9 transformed cells in the presence or absence of the Af10OM-LZ allele. In the first week, Af10OM-LZ deletion profoundly impaired the blast-colony forming potential of MLL-AF9 transformed LSKs and the only clones that could serially replate in subsequent passages had escaped Af10OM-LZ excision. Af10OM-LZ deleted colonies were very small and spread-out and showed morphological features of terminal myeloid differentiation. In contrast, HoxA9/Meis1 transformed LSK cells expanded normally in the absence of the Af10OM-LZ domain. These results demonstrate that the Af10OM-LZ, much like Dot1l, is critical for the in vitro transforming activity of the MLL-AF9 fusion gene, but does not non-specifically inhibit cellular proliferation. We then sought to investigate the potential role of the Af10OM-LZ domain in the in vivo leukemogenic activity of MLL-AF9. We generated primary MLL-AF9 leukemias from LSKs harboring floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain in cells explanted from the MLL-AF9 primary leukemias led to a significant increase in the disease latency in secondary recipient mice. Moreover, limiting dilution analysis of MLL-AF9 leukemias with or without the Af10OM-LZ domain demonstrated a >100 fold decrease in the frequency of leukemia initiating cells in the absence of the Af10OM-LZ domain. Microarray analysis showed that a vast majority of MLL-AF9 target genes were significantly downregulated in Af10OM-LZ deleted as compared to Af10OM-LZ wildtype MLL-AF9 leukemias. However, the Af10OM-LZ deleted cells could still eventually cause leukemia. This is intriguing given that Af10OM-LZ deletion, similar to Dot1l deletion, leads to a significant reduction in H3K79 dimethylation as well as MLL-target gene expression. A more detailed analysis of H3K79 methylation using mass spectrometry revealed that in contrast to H3K79 dimethylation, global levels of H3K79 mono-methylation were largely unchanged in Af10OM-LZ deleted cells. This suggests the residual MLL-AF9 target gene expression seen in Af10OM-LZ deleted cells is maintained by H3K79 monomethylation. Our results demonstrate a surprising role for Af10 in the conversion of H3K79 monomethylation to dimethylation and reveal the AF10-DOT1L interaction as an attractive therapeutic target in MLL-rearranged leukemias. Disclosures: Armstrong: Epizyme: Consultancy.

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Optimized Fully-Synthetic Salicylamide Heparin Antagonists Have Greater Efficacy Versus LMWHs and Display Improved Hemodynamic Responses as Compared to Protamine

Blood ◽

10.1182/blood.v116.21.191.191 ◽

2010 ◽

Vol 116 (21) ◽

pp. 191-191

Author(s):

Richard W. Scott ◽

Michael J. Costanzo ◽

Katie B. Freeman ◽

Robert W. Kavash ◽

Trevor M. Young ◽

...

Keyword(s):

Blood Pressure ◽

Cell Line ◽

Factor Xa ◽

In Vivo Model ◽

Hemodynamic Effects ◽

Employment Equity ◽

Human Liver Cell ◽

Equity Ownership

Abstract Abstract 191 A series of salicylamides, fully synthetic cationic foldamers designed to disrupt the binding of the pentasaccharide unit of heparin to antithrombin III, were found to be potent neutralizers of the activity of unfractionated heparin (UFH) and low molecular weight heparins (LMWHs). A compound from this series, PMX-60056, is currently in human clinical trials for neutralization of UFH and LMWHs. PMX-60056 potently neutralizes UFH and LMWHs but is not as efficacious versus fondaparinux (FPX). The goal of the present research was to 1) identify back-up compounds to optimize activity against the LMWHs and FPX and 2) mitigate the hemodynamic effects commonly associated with protamine and observed clinically with PMX-60056 in the absence of heparin. Compounds were first tested for their ability to neutralize the anticoagulant activity of enoxaparin (ENX), tinzaparin or FPX in an in vitro amidolytic assay for factor Xa activity. While only minor improvements were observed in the neutralization of ENX and tinzaparin, compounds were identified which had 6 to 40 fold increase in activity against FPX (EC50s of 0.09 – 0.58 uM) in comparison to PMX-60056 (EC50 3.64 uM). Activated partial thromboplastin time (aPTT) assays demonstrated that these compounds maintained activity against heparin in a plasma based clotting assay. Rotation thromboelastometry (ROTEM) was used to show that these compounds are able to neutralize heparin and ENX in human whole blood, restoring normal coagulation profiles. As an initial test for safety, compounds were tested in hemolysis and cytotoxicity assays using isolated human erythrocytes, a transformed human liver cell line (HepG2 cells) and a mouse fibroblast cell line (NIH3T3). Lead back-up compounds were not cytotoxic (or hemolytic) at >100 fold concentrations over their EC50 concentrations in the anti-coagulation assays, indicating a high selectivity index between toxicity and efficacy. Five compounds were selected for further studies based on their in vitro profiles. The in vivo efficacy of these compounds was evaluated in a rat coagulation model for neutralization of ENX (2 mg/kg). Three minutes following IV dosing with ENX, either saline, protamine or one of the five salicylamide test compounds was administered. Blood was collected before dosing with ENX, and at 1, 3, 10, and 60 min after dosing, for aPTT and factor Xa analysis. Three of the five salicylamides (PMX640, PMX686 and PMX747) were more efficacious than protamine; with PMX640 and PMX686 neutralizing 91 – 100% and PMX747 neutralizing 78–100% of the ENX anti-factor Xa activity over the entire 60 minute time course. In a second in vivo model, PMX747 and PMX686 (2 mg/kg) completely neutralized the prolonged bleeding times in a rat tail bleeding model caused by treatment with 2 mg/kg ENX. Significantly, with protamine at a 5 mg/kg dosage, only partial restoration was obtained. Protamine routinely causes a transient decrease in blood pressure upon dosing, and hemodynamic effects have also been observed with PMX-60056 in human subjects in the absence of heparin. To address this issue, structural features that have successfully reduced hemodynamic liabilities in other cationic compounds were incorporated into the design of the back-up salicylamides. The effect of compounds on blood pressure and heart rate was measured via arterial catheters in rats following IV administration of protamine, PMX-60056, or test agents. As expected, in rats treated with a low dose of UFH (50 u/kg) and high dosages of antagonist, both protamine and PMX-60056 displayed transient or prolonged blood pressure reductions at 8 and 16 mg/kg, respectively. However, the lead back-up salicylamides, PMX640, PMX686 and PMX747 had little to no effect on blood pressure at these same dosages. In conclusion, we have discovered compounds in the salicylamide series that have greater efficacy versus LMWHs and that have significantly reduced hemodynamic liabilities in rats as compared to protamine. Furthermore, these compounds potently neutralize FPX activity in vitro; exceeding the activity of protamine and our clinical lead salicylamide, PMX-60056, by up to 40 fold. Thus we have been able to optimize the salicylamide series, identifying compounds that offer the potential to greatly improve upon the current clinical heparin antagonist, protamine, in respect to both activity against LMWHs and side effect profile. Disclosures: Scott: PolyMedix Inc.: Employment, Equity Ownership. Costanzo:PolyMedix Inc.: Employment, Equity Ownership. Freeman:PolyMedix Inc.: Employment, Equity Ownership. Kavash:PolyMedix Inc.: Employment, Equity Ownership. Young:PolyMedix, Inc.: Employment, Equity Ownership. DeGrado:PolyMedix, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jeske:PolyMedix, Inc.: Research Funding.

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DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs or DNA Hypomethylating Agents in MLL-Rearranged Leukemia Cells

Blood ◽

10.1182/blood.v122.21.3930.3930 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3930-3930 ◽

Cited By ~ 3

Author(s):

Christine R Klaus ◽

Scott R. Daigle ◽

Dorothy Iwanowics ◽

L. Danielle Johnston ◽

Carly A Therkelsen ◽

...

Keyword(s):

Cell Line ◽

Stock Options ◽

Leukemia Cell ◽

Standard Of Care ◽

Treatment Model ◽

Hypomethylating Agents ◽

Employment Equity ◽

Acute Leukemias ◽

Equity Ownership ◽

Pre Treatment

Abstract EPZ-5676 is a small molecule inhibitor of the histone methyltransferase DOT1L that is currently under clinical investigation as a potential therapy for acute leukemias bearing MLL-rearrangements. Gene knockout and small molecule inhibitor studies have demonstrated that DOT1L is required for MLL-fusion protein–mediated leukemogenesis in model systems. In preclinical studies EPZ-5676 promoted cell killing of acute leukemia lines bearing MLL translocations in vitro while sparing those without MLL gene translocations and also caused sustained tumor regressions in a rat xenograft model of MLL-rearranged leukemia [Daigle et al. Blood 2013]. To support potential future clinical scenarios, we evaluated the activity of EPZ-5676 in combination with current standard of care agents for acute leukemias as well as other chromatin modifying drugs in cell proliferation assays with three human acute leukemia cell lines; Molm-13 (MLL-AF9 expressing acute myeloid leukemia (AML)), MV4-11 (MLL-AF4 expressing acute biphenotypic leukemia cell line) and SKM-1 (non-MLL-rearranged AML). We established a high density combination platform suitable for testing the anti-proliferative activity of a complete titration matrix of two agents with multiple replicate points to enable generation of statistically meaningful results. This platform was used to evaluate the anti-proliferative effects of EPZ-5676 combinations tested in a co-treatment model in which the second agent was added along with EPZ-5676 at the beginning of the assay, or in a pre-treatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. The drug combination analysis was performed using the Chou-Talalay method [Chou TC Pharmacological Reviews 2006]. Graphs representing values of combination index (CI) versus Fractional effect (Fa) known as Fa-CI plots were generated and synergy was evaluated. Drug synergy was statistically defined by CI values less than 1, antagonism by CI >1 and additive effect by CI equal to 1. We found that EPZ-5676 acts synergistically with the AML standard of care agents cytarabine or daunorubicin in Molm-13 and MV4-11 MLL-rearranged cell lines. However, in the non-rearranged SKM-1 cell line EPZ-5676 had no effect alone and did not act synergistically with cytarabine or daunorubicin. Moreover, a persistent combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the standard of care agents (Figure 1), suggesting that EPZ-5676 sets up a durable altered chromatin state that enhances the effect of chemotherapeutic agents in MLL-rearranged cells. We are currently exploring the mechanism of action of this synergy in more detail.Figure 1. Fa-CI plots show that EPZ-5676 and cytarabine act synergistically to induce an antiproliferative effect in the Molm-13 cell line in a pre-treatment model. (A) Ten-day continuous dosing of EPZ-5676 with addition of cytarabine at day 7 showed a range of fractional effects with CI values <1 denoting synergy. (B) EPZ-5676 was removed at day 7 prior to the addition of cytarabine showing durable combination benefit.Figure 1. Fa-CI plots show that EPZ-5676 and cytarabine act synergistically to induce an antiproliferative effect in the Molm-13 cell line in a pre-treatment model. (A) Ten-day continuous dosing of EPZ-5676 with addition of cytarabine at day 7 showed a range of fractional effects with CI values <1 denoting synergy. (B) EPZ-5676 was removed at day 7 prior to the addition of cytarabine showing durable combination benefit. Our evaluation of EPZ-5676 in conjunction with other chromatin modifying drugs also revealed a consistent combination benefit including synergy with DNA hypomethylating agents. In summary, our results indicate that EPZ-5676 is highly efficacious as a single agent and is synergistic with other anticancer agents including AML standard of care drugs and DNA hypomethylating agents in MLL-rearranged cells. Disclosures: Klaus: Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Daigle:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Iwanowics:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Johnston:Epizyme, Inc: Employment, Equity Ownership, Stock Options Other. Therkelsen:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Smith:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Moyer:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Copeland:Epizyme Inc. : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Mersana: Membership on an entity’s Board of Directors or advisory committees. Olhava:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Porter Scott:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Raimondi:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other.

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Early Different Downstream Target of Glucocorticoid Receptor Contributing to Glucocorticoids Sensitivity in Kasumi-1 Cells

Blood ◽

10.1182/blood.v128.22.5132.5132 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5132-5132

Author(s):

Wenbin Gu ◽

Meng Li ◽

Liang Liang ◽

Jian Zhang ◽

Chongye Guo ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Fusion Protein ◽

Cell Line ◽

Myeloid Leukemia ◽

Target Genes ◽

Conflicts Of Interest ◽

Critical Role ◽

Leukemia Cell ◽

Leukemia Cells ◽

Non Coding Rnas

Abstract The t(8;21) chromosome translocation frequently occurs in acute myeloid leukemia (AML), resulting in an in-frame fusion between the DNA-binding domain of AML1 and almost the entire of ETO gene. The fusion AML1-ETO protein is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells, such as Kasumi-1 and SKNO-1 cells. Glucocorticoids (GC) can induce apoptosis in these cells at low concentrations, whereas most other myeloid leukemia cell lines are resistant to glucocorticoid-induced apoptosis. To experimentally address possible sensitive mechanisms in leukemia cells with AML1-ETO translocation, we generated aGC-resistant Kasumi-1 cell line by induction of 10-6 M dexamethasone (Dex) for three weeks. The IC50 of Dex to cells is increased from 2.5×10-8 M for original GC-sensitive Kasumi-1 cell line ( K-S cell line) to more than 1×10-5 M for induced GC-resistant Kasumi-1 cell line (K-R cell line). Since GC resistance often results from mutations in the glucocorticoid receptor (GR), all the exons of GR gene were sequenced and no mutation was found in K-R cells. Comparing to those in K-S cells, the GR protein level didn't decrease in K-R cells after 2h, 4h, 8h, 12h and 24h exposure to dexamethasone. Given that the difference of direct GR downstream genes between K-S and K-R cells may play a key role in the GC sensitivity, we systematically analyzed the changes of gene expression induced by Dex versus ethanol vehicle for 8h in K-S and K-R cells by high throughput RNA sequencing. The time point of 8h was selected according to the expression peaks of several foregone GR target genes after Dex induction. There were found 32 genes conversely regulated in K-S and K-R cells, including 14 mRNAs and 18 long non-coding RNAs. Pathway analysis indicated that the upregulated genes in K-S cells might promote the AML1-ETO fusion protein degradation by proteasomes, while the component genes of this pathway were downregulated in K-R cells. Further validation and function studies of these mRNAs and long non-coding RNAs are ongoing. Our data suggested that the downstream targets of GR among GC-sensitive and -resistant Kasumi-1 cells were significant different and they may contribute to the GC sensitivity and resistance by degradation or reservation of AML-ETO fusion protein and the regulation of apoptosis in t(8;21) leukemia cell subtype. Disclosures No relevant conflicts of interest to declare.

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MBNL1 As a New Therapeutic Target in MLL-Fusion Gene Leukemia

Blood ◽

10.1182/blood.v126.23.462.462 ◽

2015 ◽

Vol 126 (23) ◽

pp. 462-462 ◽

Cited By ~ 2

Author(s):

Svetlana S Itskovich ◽

Jason Clark ◽

James C. Mulloy ◽

Matthew D Disney ◽

Ashish R Kumar

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Fusion Gene ◽

Leukemia Cell ◽

Leukemia Cells ◽

Transformed Cells ◽

Wild Type

Abstract Translocations of the Mixed Lineage Leukemia (MLL) gene located on chromosome 11 are commonly found in infants with AML or ALL and in secondary leukemia at all ages. A majority of patients with these translocations have a poor prognosis. Gene expression profiling studies demonstrate that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is muscleblind-like 1 (MBNL1). Further, MBNL1 was also identified as a direct transcriptional target of MLL-fusion proteins. An RNA-binding protein, MBNL1 is known to be a key factor in the pathophysiology of Myotonic Dystrophy Type I (DM), where sequestration of MBNL1 leads to splicing defects in muscle and neuronal cells. However, the role of MBNL1 in hematopoiesis and leukemogenesis is unknown. To determine the role of MBNL1 in normal hematopoiesis we studied MBNL1-/- mice. Compared to littermate controls, MBNL1-/- mice showed no differences in peripheral blood counts or bone marrow cellularity. When challenged with 5-FU, both MBNL1-/- and wild type mice displayed similar kinetics of peripheral blood cytopenia and recovery. Next we examined the role of MBNL1 in hematopoietic stem cell function using a competitive transplantation assay. Lethally irradiated mice were transplanted with a 1:1 mix of CD45.1 and CD45.2 bone marrow, with the latter being wild-type or MBNL1-/-. Flow cytometry analysis of peripheral blood at 4 weeks post-transplant showed donor chimerism being 53±4.14% in recipients of wild type marrow and 25±5.41 % in the MBNL1-/- recipients. Successive analyses every 4 weeks showed the chimerism to be stable over the next 16 weeks. To determine the role of MBNL1 in leukemia, we transformed MBNL1-/- or wild type bone marrow cells with various oncogenes delivered via retroviral transduction and compared them in methylcellulose colony replating assays. Absence of MBNL1 significantly reduced colony formation in MLL-AF9 and E2A-HLF transformed cells by 59.5% (± 27.1) and 50.7% (± 23) respectively, compared to controls. To assess the role of MBNL1 in leukemia in vivo, we transplanted MLL-AF9-transformed wild type or MBNL1-/- cells into irradiated mice. All recipients injected with wild-type MLL-AF9-transformed cells succumbed to leukemia with a median time of 106 days. In contrast, the majority of recipients of MBNL1-/- cells survived leukemia-free for at least 140 days post-transplantation (p=0.0017, log rank test). We next assessed the role of MBNL1 in human leukemia cells. Lentiviral-shRNA knockdown of MBNL1 in leukemia cell lines (MV4;11, THP-1) significantly inhibited cell growth, both in liquid culture and methylcellulose colony forming assays. To determine the requirement of MBNL1 for leukemia propagation in vivo, we used cord blood-derived leukemia cells bearing the MLL-AF9 fusion gene and mutant NRAS (MA9NRAS). MA9NRAS cells transduced with MBNL1-specific or control (non-targeting, NT) shRNA were transplanted into immunodeficient mice. Six weeks after transplant, bone marrow aspirates showed persistence of lentiviral-transduced cells in 85% of the NT-group. On the other hand, MBNL1-shRNA transduced cells were not detected in any of the recipient mice. These results suggest that MBNL1 is essential for leukemia cell propagation in vivo. Finally, we tested therapeutic targeting of MBNL1 in MLL-fusion gene leukemia. A lead inhibitor that prevents binding of MBNL1 to its targets was recently identified. Treatment of MA9NRAS cells with the inhibitor for 48 hours led to significant apoptosis whereas normal cord blood CD34+ cells were relatively less sensitive. Blockade of MBNL1 in leukemia cells either by shRNA-knockdown or by the inhibitor showed identical changes in splicing patterns of known MBNL1 target genes. Collectively, our data suggest that MBNL1 is required for the initiation and propagation of MLL-fusion gene leukemia while it appears relatively dispensable for normal hematopoiesis. Further, we have identified a promising lead inhibitor that could be developed for novel treatments for therapy-resistant leukemias. Disclosures No relevant conflicts of interest to declare.

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AG-221, a Small Molecule Mutant IDH2 Inhibitor, Remodels the Epigenetic State of IDH2-Mutant Cells and Induces Alterations in Self-Renewal/Differentiation in IDH2-Mutant AML Model in Vivo

Blood ◽

10.1182/blood.v124.21.437.437 ◽

2014 ◽

Vol 124 (21) ◽

pp. 437-437 ◽

Cited By ~ 12

Author(s):

Alan H. Shih ◽

Kaitlyn R Shank ◽

Cem Meydan ◽

Andrew M. Intlekofer ◽

Patrick Ward ◽

...

Keyword(s):

Dna Methylation ◽

Clinical Trials ◽

Small Molecule ◽

Leukemia Cells ◽

Employment Equity ◽

Advisory Committees ◽

Self Renewal ◽

Equity Ownership ◽

The Impact

Abstract Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are observed in patients with acute myeloid leukemia (AML). Leukemia-associated IDH1/2 mutations result in aberrant accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). The observation that IDH1/2 mutations are mutually exclusive with TET2 mutations led to the finding that IDH1/2-mutant production of 2-HG inhibits TET2 function and induces changes in DNA methylation. These data suggested that small molecule inhibition of mutant IDH enzymes might reverse the aberrant epigenetic remodeling of IDH-mutant leukemia cells and restore normal hematopoietic differentiation. We therefore investigated the in vivo efficacy of AG-221, a potent and selective mutant IDH2 inhibitor in early-phase clinical trials, in murine models of IDH2-mutant leukemia. We first assessed the impact of AG-221 on 2-HG production in hematopoietic cells expressing mutant IDH2-R140Q. AG-221 treatment (10mg/kg or 100mg/kg bid) led to a reduction in 2-HG in vivo (96.7% below pre-treatment levels). Moreover, AG-221 treatment restored megakaryocyte-erythroid progenitor (MEP) differentiation that is suppressed by mutant IDH2 expression in vivo (mean MEP% mean, 39% Veh vs 50% AG-221). We next investigated the impact of mutant IDH2 inhibition with AG-221 on DNA methylation in vivo. We used eRRBS, a bisulfite-based next-generation sequencing platform, to assess the effect of AG-221 therapy on DNA methylation. AG-221 or vehicle therapy treated LSK stem cells (lin- Sca+ c-Kit+) were sorted from mice expressing IDH2-R140Q and evaluated by eRRBS. AG-221 therapy reversed the effects of mutant IDH2; we observed a significant reduction in DNA methylation, including 180 genes that had 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 84 of these genes had reduced methylation at 10 or more DMCs in the gene promoter with AG-221 therapy compared to vehicle. Mutant IDH2 inhibition with AG-221 reversed aberrant methylation at many genes with a known role in hematopoietic proliferation and differentiation, including the master transcriptional factor RUNX1. We next assessed in vivo effects of the small-molecule IDH2-R140Q inhibitor in a mouse model of IDH2-mutant leukemia. We generated mice that simultaneously expressed a constitutive Flt3ITD knock-in allele and a conditional mutant IDH2R140Q knock-in allele. As reported recently using retroviral/transgenic models, Mx1-Cre IDH2R140QFlt3ITD developed fully penetrant, transplantable AML with expansion of c-Kit+ positive blasts in the peripheral blood, and widespread leukemic infiltration. AG-221 inhibited the serial replating capacity of IDH2R140QFlt3ITD expressing cells in vitro. We competitively transplanted IDH2R140QFlt3ITD AML cells and normal bone marrow cells into secondary recipients, and then assessed the effect of AG-221 therapy on leukemia in vivo and on disease burden. AG-221 (100mg/kg bid) treatment of mice engrafted with Mx1-Cre IDH2R140QFlt3ITD AML cells markedly reduced 2HG levels consistent with on target inhibition in vivo. AG-221 therapy induced differentiation of leukemic cells, with an increase in the CD11b+ population and a decrease in the c-Kit+ population in the peripheral blood at 2wks. We next assessed the impact of treatment with both AG-221 therapy with AC220, a potent, specific Flt3 inhibitor in late phase clinical trials. Combined IDH2R140Q and Flt3ITD inhibition resulted in a marked decrease in leukemic burden to vehicle-treated mice, with a significant reduction in leukemic cell chimerism in vivo in the setting of combined inhibition at 2 wks, (mean 45.2 fraction 88% veh, 73% AG-221, p<.01). These data demonstrate that AG-221 inhibits mutant IDH2-mediated 2-HG production in vivo and reverses the effects of mutant IDH2 on DNA methylation in mutant stem/progenitor cells. AG-221 induces differentiation and impairs self-renewal of IDH2-mutant leukemia cells, effects that are further enhanced by simultaneous inhibition of Flt3ITD. Clinical trials combining IDH2 inhibitors with other targeted AML therapies are warranted in order to increase therapeutic efficacy. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Thompson:Agios: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Straley:Agios: Employment, Equity Ownership. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. Yen:Agios: Employment, Equity Ownership. Levine:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Single Cell-Level Signaling Profiling of Acute Myeloid Leukemia Following Treatment with Axl Kinase Inhibitor BGB324

Blood ◽

10.1182/blood.v126.23.4931.4931 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4931-4931

Author(s):

Monica Hellesøy ◽

Katarzyna Wnuk-Lipinska ◽

Anna Boniecka ◽

Eline Milde Nævdal ◽

Hakon Reikvam ◽

...

Keyword(s):

Signal Transduction ◽

Overall Survival ◽

Cell Line ◽

Research Funding ◽

Xenograft Model ◽

Employment Equity ◽

In Vitro Response ◽

Equity Ownership ◽

Dose Dependent

Abstract Axl is a member of the Tyro3, Axl, Mer (TAM) receptor tyrosine kinase family that regulate a wide range of cellular functions, including cell survival, proliferation, migration/invasion and adhesion. Axl has been shown to play a key role in the survival and metastasis of many tumors, and has also been found to be upregulated and constitutively active in human AML. Indeed, Axl has been reported as an independent prognostic marker and a potential novel therapeutic target in AML. BGB324 is a first-in-class highly selective small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in clinical safety studies in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in phase I b clinical trials for AML and non-small cell lung cancer. In this study, we use phosphoflow cytometry to measure changes in signal transduction nodes in single AML cells treated with BGB324. We are applying this approach to monitor signaling profiles in primary AML cells harvested from patients undergoing BGB324 treatment. Results: The human AML cell line MOLM13 was treated in vitro with BGB324 (0.5 and 1µM for 1 hour) and analyzed for signal transduction changes by phosphoflow cytometry. We found a significant reduction in phosphorylation of Axl (pY779), Akt(pS473), Erk1/2(pT202/Y204) and PLCɣ1(pY783). Next we established a systemic MOLM13 preclinical AML model in NOD/SCID mice. The mice were treated with 25 or 50 mg/kg BGB324 until moribund (up to 16 days). We found a dose-dependent and significant increase in overall survival in BGB324-treated mice. We further investigated intracellular signaling in BGB324-treated cells in vivo. Mice carrying systemic AML disease (MOLM13) were treated with BGB324 at 50mg/kg for 4 days, and we monitored CD33/45-positive MOLM13 cells harvested from spleen and bone marrow by flow cytometry. BGB324-treated mice showed a significant reduction in pErk and pPLCɣ1 relative to mice in the control group. PBMCs from peripheral blood of AML patients treated with BGB324 400 mg x1 at day 1 and 2, and thereafter 100 mg daily were collected for single cell signal profiling of signal transduction changes by conventional flow cytometry (phospho-flow) and mass cytometry (CyTOF). Preliminary phopho-flow analyses show decrease of pAkt(T308) and pPLCgamma1(Y783) in one patient. Further analyses are ongoing and will be presented. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Disclosures Hellesøy: BerGenBio AS: Other: Previous employee. Stock option holder. Wnuk-Lipinska:BerGenBio AS: Employment. Boniecka:BerGenBio AS: Employment. Nævdal:BerGenBio AS: Employment. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding. Cortes:Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Lorens:BerGenBio AS: Employment, Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Gausdal:BerGenBio AS: Employment. Gjertsen:Haukeland University Hospital: Research Funding.

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Inecalcitol and Decitabine Synergize to Inhibit Cell Proliferation and to Induce Differentiation of Human Acute Myeloid Leukemia Cell Lines

Blood ◽

10.1182/blood-2018-99-118125 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5234-5234

Author(s):

Enguerran Mouly ◽

Emilie Rousseau ◽

Cecile Planquette ◽

Remi Delansorne

Keyword(s):

Cell Proliferation ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Employment Equity ◽

Elderly Aml ◽

Equity Ownership ◽

Inhibit Cell Proliferation ◽

Stimulation Of

Abstract Decitabine (DAC) is a hypomethylating agent indicated as front-line therapy for de novo or secondary acute myeloid leukemia (AML) in newly diagnosed patients aged 65 years or older unfit for standard induction chemotherapy (Kantarjian et al., 2012, Malik & Cashen, 2014, Nieto et al., 2016, He et al., 2017). Its mechanism of action at the DNA level mostly results in inhibition of cell proliferation. Cellular differentiation can also be involved in some extent in a fraction of the leukemic cell population, as reported in initial pharmacological studies (Creusot et al., 1982; Pinto et al., 1984). Overall survival advantage is nevertheless limited to several more months and the next challenge is to combine DAC with other drugs to improve it further (Kubasch & Platzbecker, 2018). Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol) is a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating general properties on cancer cells and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), and especially on AML cell lines (AACR 2017, 2018). INE is currently being tested in combination with DAC in this category of elderly AML patients unfit for standard chemotherapy. The aim of the present report was to look for synergies in vitro between DAC and INE on four non-APL human AML cell lines (MOLM-13, U-937, THP-1, OCI-AML2) both on inhibition of proliferation and induction of differentiation. After 72 hours of incubation, cells were counted and labeled for CD11b and CD14 at the cell surface as biomarkers of monocytic/macrophagic differentiation. The range of DAC concentrations had to adapted to each cell line to avoid maximal cytotoxicity: 1.2 nM to 100 nM on MOLM-13, 3 nM to 250 nM on U-937 and THP-1, and 31 nM to 500 nM on OCI-AML2. The same range of 0.12 to 10 nM INE concentrations was tested on each cell line. Each concentration of INE was tested in combination with each concentration of DAC. Synergy was calculated as the excess over the highest single agent (HSA) using the open source Combenefit software (Di Veroli et al., 2016). The highest concentration of DAC alone (MOLM-13: 100 nM, U-937 and THP-1: 250 nM; OCI-AML2: 500 nM) induced a decrease in cell count of 30% of THP-1 cells, 50% of OCI-AML2 cells, 65% of U-937 cells and 80% of MOLM-13 cells. The highest concentration of INE alone (10 nM) induced a decrease in cell count of 20% of U-937 and THP-1 cells, 60% of OCI-AML2 cells and 70% of MOLM-13 cells. The antiproliferative effects of DAC and INE were at least additive in all combinations tested. Significant HSA synergy indexes were found for the decrease in cell number in all four cell lines, ranging from 12% to 23% depending on cell lines and combinations of concentrations. The highest concentration of DAC alone had no (U-937, THP-1) or limited activity (<+12% of labeled MOLM-13 or OCI-AML2 cells) to induce either CD11b or CD14 on the cell surface. By contrast, the highest concentration of INE alone (10 nM) stimulated the expression of CD11b and CD14 in up to 70% to 95% of the cells depending on the cell line (except the CD14 labeling of U-937 cells which remained < 8%). The respective EC50 of INE for CD11b and CD14 induction was 1 and 3 nM on THP-1 cells, 4 and 3 nM on MOLM-13 cells, 3 and 3 nM on OCI-AML2 cells and 1 nM on U-937 cells (50% not reached for CD14). There was no antagonistic effect of DAC towards the pro-differentiating properties of INE. A significant HSA synergy index in the 16% to 26% range was observed for both CD11b and CD14 in MOLM-13 cells and for CD14 in OCI-AML2 cells. A very high HSA synergy index of 75% was observed for the stimulation of CD14 in U-937 cells. In summary, DAC exerted more antiproliferative activity than INE which was more potent to induce monocytic/macrophagic differentiation of four non-APL human AML cell lines. The combination of DAC and INE systematically resulted in a synergy to inhibit cell proliferation, and the strong stimulation of cell differentiation induced by INE alone was in some cases boosted by DAC. These in vitro results provide the mechanistic basis for the potential interest of treating elderly AML patients with INE in addition to DAC in the ongoing double-blind placebo-controlled Phase II clinical trial (NCT02802267). Disclosures Mouly: Hybrigenics: Employment. Rousseau:Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.

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Establishment of a NOD/SCID/γc−/−-Dependent Lymphoid Leukemia Cell Line, D593, with Novel RUNX1-LAF4 Fusion Gene.

Blood ◽

10.1182/blood.v108.11.4276.4276 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4276-4276

Author(s):

Akihiro Abe ◽

Manabu Ninomiya ◽

Shizuka Imagama ◽

Momoko Suzuki ◽

Fumihiko Hayakawa ◽

...

Keyword(s):

T Cell ◽

Cell Line ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Cell Receptor ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Pcr Analysis ◽

Lymphoid Leukemia

Abstract We established a NOD/SCID/γc−/−(NOG mouse)-dependent human lymphoid leukemia cell line, D593, by repeated xenotransplantation of pediatric T-cell acute lymphoblastic leukemia cells with the translocation t(2;21). The cell line, D-593, could be serially transplanted from mouse to mouse over a 2-year period. D593 had the same immuno-phenotype as the original leukemia cells: positive for CD2, 5, 7, 14, and 34, and negative for CD3, 4, 8, 19, and 41a. Cytoplasmic CD3 was positive and the rearrangement of T-cell receptor was detected by Southern blot analysis. A previously unreported translocation of t(2;21)(q11;q22) was observed in both the original patient sample and D593. The split signal of RUNX1 was detected by fluorescence in site hybridization in D593 indicating the involvement of RUNX1. Using 3′-RACE and RT-PCR analysis, we identified novel chimeric transcripts of RUNX1-LAF4 joining exon 7 of RUNX1 to exon 4 of LAF4. In the transplanted NOG mice, D593 homed into the trabecular endosteal region of bone marrow (BM), and proliferated from the endosteum to medulla. At the late stage of engraftment, the BM was filled with human lymphoblasts and metastases into the trabecular of the spleen and Glisson’s sheath of the liver were also observed. These findings suggest that D593 is a useful cell line to study not only the leukemia-related biology of RUNX1-LAF4 but also the novel therapeutic model against core-binding factor (CBF) leukemia.

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A NUP98-NSD1 Fusion Gene Can Be Detected in a Small Subset of Adult AML Patients with Normal Karyotype

Blood ◽

10.1182/blood.v118.21.2531.2531 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2531-2531

Author(s):

Annette Fasan ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

...

Keyword(s):

Fusion Gene ◽

Normal Karyotype ◽

Fusion Transcript ◽

Chromosomal Band ◽

Event Free Survival ◽

Employment Equity ◽

Aberrant Expression ◽

Free Survival ◽

Platelet Counts ◽

Equity Ownership

Abstract Abstract 2531 Introduction: Fusion genes can be detected in approximately 30–35% of all AML cases and usually are the result of a cytogenetically detectable chromosomal rearrangement. Very recently, a novel fusion gene has been described in AML with normal karyotype (Hollink et al, Blood, 2011). This cryptic fusion involves nucleophosmin 98kD (NUP98) in chromosomal band 11p15 and the non homeobox gene NSD1 in chromosomal band 5q35. NUP98-NSD1 has been described in this single study with a frequency of 16.1% in pediatric and 2.3% in adult AML patients with distinct characteristics (e.g. mutual exclusivity with NPM1) and dismal prognosis. Aim: The aim of this study was to further evaluate NUP98-NSD1 rearrangements in adult AML with normal karyotype (NK) for frequency, association with other mutations and impact on outcome. Patients and Methods: Screening for NUP98-NSD1 fusion gene was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) in a total cohort of 148 de novo AML patients with NK and NPM1 wildtype status. The NUP98-NSD1 positive cases were verified by direct Sanger Sequencing of the PCR products. The cohort was composed of 84 males and 64 females. Median age was 55.4 years (range: 15.7 to 85.8 years). Further mutation analysis was available in subcohorts: FLT3-ITD (n=32 mut/117 screened), CEBPA (n=22 mut/124 screened), MLL-PTD (n=32 mut/117 screened) and RUNX1 (n=26 mut/83 screened). Results: In total, in 8/148 (5.4%) patients a NUP98-NDS1 fusion transcript was detected. NUP98-NDS1-positive cases had significantly higher platelet counts (median 221 vs 87 × 10e9/L; p=0.001). Patients with NUP98-NDS1 were younger than the NUP98-NDS1-negative patients (median: 43.5 years vs 55.4 years, p=0.067). Sex (5 male vs. 3 female), white blood cell count and hemoglobin levels at diagnosis were not different compared to NUP98-NDS1-negative cases. Cytomorphology revealed AML with minimal differential differentiation (n=4), with maturation (n=1), and myelomonocytic AML (n=3). In 3 NUP98-NDS1-positive cases immunophenotyping data was available and all 3 cases aberrantly expressed CD7. NUP98-NDS1-positive cases have a higher frequency of FLT3-ITD compared to NUP98-NDS1-negative cases (5/8, 62.5% vs. 27/140, 19.3%; p=0.015) and were mutually exclusive of CEBPA and RUNX1 mutations. With respect to survival the NUP98-NDS1-positive cases had a worse event free survival compared to NUP98-NDS1-negative cases (median 5.1 months vs. 25.2 months; p=0.054). Conclusions: A NUP98-NSD1 fusion transcript was detected in 5.4% of normal karyotype adult AML patients without NPM1 mutation. NUP98-NSD1-positive cases are characterized by younger age, high coincidence of FLT3-ITD, aberrant expression of CD7, relatively high platelet counts, and a short event free survival. Thus NUP98-NSD1 translocations seem to define a new subgroup of NK-AML. Importantly, in this prognostically adverse and so far cytogenetically undetectable group close and sensitive PCR based monitoring for minimal residual disease is available. Thus, this data suggests to perform PCR based screening for NUP98-NSD1 in AML with normal karyotype that lack NPM1 and CEBPA mutations. Disclosures: Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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