Mixed Lineage Leukemia Cells Research Articles

MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells

Mixed lineage leukemia-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce MLL through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here, we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II in HEL, a human erythroleukemia cell line without MLL1 rearrangement. MLL1 and AF9 only coregulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely believed elongation.

EPCO-28. DOT1L AND PRC2 REGULATE A SHARED EPIGENETIC MECHANISM IN GLIOBLASTOMA AND MIXED LINEAGE LEUKEMIA

Abstract Glioblastoma (GBM) is the most aggressive adult brain tumor, with a median survival of 15 months despite current treatments. We recently established that the epigenetic regulator Disruptor of Telomeric Silencing-1-Like (DOT1L) was essential for the growth of brain tumor stem cells (BTSCs), which are thought to underlie GBM tumor initiation and treatment resistance. Given the previously recognized importance of DOT1L histone methylation for the regulation of the similarly rare and aggressive childhood cancer, Mixed Lineage Leukemia (MLL), we interrogated for common mechanisms in both BTSCs and MLL cells, that overlap due to the epigenetic role of DOT1L. To gain a more detailed perspective of the importance of the DOT1L epigenetic mark in BTSCs, we performed a chemogenomic screen using the DOT1L inhibitor, EPZ-5676. Results from this screen revealed genes from transcriptional and epigenetic complexes required for a therapeutic response to DOT1L inhibition in BTSCs. Gene targeting approaches and growth assays further identified the Polycomb Repressive Complex 2 (PRC2) as a common determining factor for the growth response of both BTSCs and MLL cells following DOT1L inhibition. Furthermore, analysis of the chromatin accessibility changes regulated by DOT1L and PRC2 histone methylation identified both shared and unique epigenetic characteristics of GBM and MLL. The extent to which these shared mechanisms underpin the pathogenic process in these distinct diseases is being further investigated by assessing the divergence in transcriptional responses that emerge from this common epigenetic phenomenon. The findings from this study will provide insight into the importance of shared epigenetic mechanisms that underlie the tumorigenesis of unique cancers affecting the brain and blood.

Deubiquitinating enzyme inhibitor alleviates cyclin A1-mediated proteasome inhibitor tolerance in mixed-lineage leukemia.

Drug resistance is a significant obstacle to effective cancer treatment. Drug resistance develops from initially reversible drug‐tolerant cancer cells, which offer therapeutic opportunities to impede cancer relapse. The mechanisms of resistance to proteasome inhibitor (PI) therapy have been investigated intensively, however the ways by which drug‐tolerant cancer cells orchestrate their adaptive responses to drug challenges remain largely unknown. Here, we demonstrated that cyclin A1 suppression elicited the development of transient PI tolerance in mixed‐lineage leukemia (MLL) cells. This adaptive process involved reversible downregulation of cyclin A1, which promoted PI resistance through cell‐cycle arrest. PI‐tolerant MLL cells acquired cyclin A1 dependency, regulated directly by MLL protein. Loss of cyclin A1 function resulted in the emergence of drug tolerance, which was associated with patient relapse and reduced survival. Combination treatment with PI and deubiquitinating enzyme (DUB) inhibitors overcame this drug resistance by restoring cyclin A1 expression through chromatin crosstalk between histone H2B monoubiquitination and MLL‐mediated histone H3 lysine 4 methylation. These results reveal the importance of cyclin A1‐engaged cell‐cycle regulation in PI resistance in MLL cells, and suggest that cell‐cycle re‐entry by DUB inhibitors may represent a promising epigenetic therapeutic strategy to prevent acquired drug resistance.

Exosomes mediate intercellular transfer of non-autonomous tolerance to proteasome inhibitors in mixed-lineage leukemia.

Proteasome inhibitors significantly improve cancer outcomes, but their use is eventually followed by proteasome inhibitor resistance and relapse. Current understanding of proteasome inhibitor resistance is limited to cell‐autonomous mechanisms; whether non–autonomous mechanisms can be implicated in the development of proteasome inhibitor resistance is unclear. Here, we show that proteasome inhibitor tolerance can be transmitted non–autonomously through exosome‐mediated intercellular interactions. We revealed that reversible proteasome inhibitor resistance can be transmitted from cells under therapy stress to naïve sensitive cells through exosome‐mediated cell cycle arrest and enhanced stemness in mixed‐lineage leukemia cells. Integrated multi‐omics analysis using the Tied Diffusion through Interacting Events algorithm identified several candidate exosomal proteins that may serve as predictors for proteasome inhibitor resistance and potential therapeutic targets for treating refractory mixed‐lineage leukemia. Furthermore, inhibiting the secretion of exosomes is a promising strategy for reversing proteasome inhibitor resistance in vivo, which provides a novel proof of principle for the treatment of other refractory or relapsed cancers.

PS1000 POST‐TRANSLATIONAL MODIFICATIONS OF PROTEIN PHOSPHATASE 2A (PP2A) IN MIXED LINEAGE LEUKEMIA (MLL)

Background:Mixed lineage leukemia (MLL) is the most common leukemia in infant patients. This leukemia is due to chromosomal translocations at locus 11q23 and is associated with poor clinical outcome. Novel, targeted therapeutic approaches are needed. Protein phosphatase 2A (PP2A) is a serine threonine phosphatase which regulates the phosphorylation of several kinases, including Erk, Akt, GSK3 which are fundamental for MLL cells’ survival. PP2A is a trimeric protein complex in which a core dimer formed between the scaffold subunit and the catalytic subunit is associated with one of the many regulatory subunits that facilitate and direct the interaction of the trimer with substrate proteins. PP2A activity is regulated by interaction with PP2A inhibitor proteins (PIPs), as well as by post‐translational modifications of PP2A complex components.Aims:In this context, our aim is to understand how PP2A post‐translational modifications affect the activity of PP2A on its downstream targets and to investigate whether PP2A re‐activation, by preventing phosphorylation and activation of collateral pathways, might represent a valid therapeutic strategy for MLL.Methods:12 different leukemic cell lines and 8 primary samples from MLL patients were included in this study. We measured the PP2A phosphatase activity by a colorimetric assay using a synthetic phospho‐peptide and malachite green reagent. The protein levels of PP2A, phospho‐PP2A, demethyl‐PP2A, LCMT‐1, PME‐1, Akt, phospho‐Akt, Erk, and phospho‐Erk were determined by western blot using specific antibodies.Results:Compared to healthy bone marrow, we found an increase in the phosphorylation at Tyrosine 307 (Tyr307) and a decrease in the methylation at Leucine 309 (Leu309) of the catalytic subunit of PP2A in MLL samples. These changes in the post‐translational modifications of the catalytic subunit correlated with a decrease in the phosphatase activity of PP2A. Methylation at Leu309 has been described as an absolute requirement for the binding of the regulatory subunit B55α to PP2A core. B55α was found up‐regulated in MLL cell lines and primary samples compared to healthy bone marrow. The methylation of the catalytic subunit of PP2A is catalyzed by leucine carboxyl methyltransferase (LCMT‐1), which we found down‐regulated in MLL samples, whereas demethylation is catalyzed by the phosphatase methylesterase (PME‐1) that was found up‐regulated in MLL. In line with the inactivation of PP2A in the MLL samples, PP2A downstream targets such as Akt and Erk were found hyper‐phosphorylated in MLL samples.Summary/Conclusion:Our results suggest that PP2A phosphatase activity inhibition might sustain in MLL samples a persistent serine/threonine phosphorylation of PP2A substrates, such as Akt and Erk that mediate pro‐survival and anti‐apoptotic signals. This inhibition might be explained, at least in part, by phosphorylation and demethylation of PP2A catalytic subunit. A better understanding of the mechanisms that regulate PP2A activity could provide new strategies to rescue PP2A phosphatase activity and target fundamental mechanisms of leukemic survival and chemotherapy resistance.

PF241 TARGETING SET‐PP2A INTERACTION AS A NOVEL THERAPEUTIC APPROACH FOR MIXED LINEAGE LEUKEMIA (MLL)

Background:Mixed Lineage Leukemia (MLL) is an aggressive form of acute leukemia caused by chromosomal translocation involving the MLL gene at locus 11q23. Despite novel therapeutic approaches, there are no efficient targets for MLL treatments and patients present a very poor prognosis. PP2A is a serine‐threonine phosphatase that downregulates crucial target proteins such as Akt, Erk, and c‐Myc, responsible for proliferation and survival of MLL leukemic cells. De‐regulation of PP2A is a recurring event in many cancers.SET is an endogenous binding partner of PP2A mostly localised in the nucleus. In many diseases including cancer, PP2A inactivation is attributed to cytoplasmic accumulation of SET which inhibits PP2A through direct interaction with PP2A catalytic subunit.FTY720, a sphingosine analogue drug, prevents the formation of SET‐PP2A complex, binding SET and rescuing PP2A activity (Pippa et al., 2014).Aims:To determine the role of SET‐mediated inactivation of PP2A in MLL, in order to define whether this might be an axis amenable to therapeutic target.Methods:The study is performed in vitro using a set of MLL cell lines (n = 8) and primary samples (n = 8). SET gene expression and protein level profile were investigated by real time PCR and Western blot.We analysed SET localization by nucleus‐ cytoplasm separation assay and SET‐PP2A interaction by co‐immunoprecipitation assay. The effect of FTY720 on human cell lines was evaluated by analysis of survival by Trypan Blue exclusion and phosphorylation of PP2A targets by Western blot.Results:Analysis of SET gene expression did not show any significant difference between MLL cell lines and primary samples compared to healthy bone marrow samples. In contrast, SET protein levels were selectively increased in MLL primary samples and cell lines. Analysis of nuclear and cytosolic extracts in MLL cell lines showed that SET was mostly localised into the cytoplasm. Furthermore, immunoprecipitation experiments showed that in MLL cell lines SET is phosphorylated on a serine residue. This modification has been previously shown to be crucial for PP2A binding and inhibition. We evaluated the effect of FTY720 on MLL survival and growth of BCR‐ABL+ cell line K562 by trypan blue exclusion. The results indicate an IC50 ranging from 1.6 to 4.2 μM. The effects of FTY720 on PP2A was evaluated by analysing the phosphorylation of PP2A targets (Akt, Erk, GSK3) in MLL cell lines 24 and 48 hours after treatment. Preliminary data showed no significant changes in phosphorylation of the above targets. These results may suggest that targeting SET alone might not be sufficient to rescue the activity of PP2A in MLL and that redundant mechanisms might occur. The effect of potential combination of FTY720 and standard chemotherapy treatment will be evaluated in future studies.Summary/Conclusion:We showed for the first time that SET is overexpressed in MLL samples and mostly localised into the cytoplasm. Given the implication of SET as an endogenous PP2A inhibitor, we aim to understand the mechanism by which it interacts with PP2A in MLL to target this complex with the aim of designing novel therapeutic approaches for this challenging disease.

Discovery of Novel Disruptor of Silencing Telomeric 1-Like (DOT1L) Inhibitors using a Target-Specific Scoring Function for the (S)-Adenosyl-l-methionine (SAM)-Dependent Methyltransferase Family.

The disruptor of telomeric silencing 1-like (DOT1L) protein is a histone H3K79 methyltransferase that plays a key role in transcriptional elongation and cell cycle regulation and is required for the development and maintenance of MLL-rearranged mixed lineage leukemia. Much effort has been dedicated toward discovering novel scaffold DOT1L inhibitors using different strategies. Here, we report the development and application of a target-specific scoring function, the SAM score, for (S)-adenosyl-l-methionine (SAM)-dependent methyltransferases, for the discovery of novel DOT1L inhibitors. On the basis of the SAM score, we successfully identified a novel class of DOT1L inhibitors with a scaffold of [1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazole, in which compound 6 exhibits an IC50 value of 8.3 μM with selectivity versus other tested SAM-dependent methyltransferases. In cellular studies, 6 selectively targets DOT1L, blocks the proliferation of mixed lineage leukemia cell lines, and causes cell cycle arrest and apoptosis. Moreover, we analyzed the putative binding modes of 6 and its analogues obtained by molecular docking, which may assist with the future development of DOT1L inhibitors with improved potency and selectivity profiles.

An Evaluation of the Cytotoxic Effect of the Natural Product Aaptamine Against t(4;11) Leukemias

The marine environment has been shown to be a rich source of pharmacologically-active secondary metabolites. Three marine- compounds have FDA approval for cancer indications. Aaptamine is a sponge-derived alkaloid that exhibits multiple pharmacological activities including proapoptotic/antiproliferative effects on leukemia cell lines. The effect of the aaptamine class has not been previously studied for high risk leukemias with mixed-lineage leukemia (MLL) gene rearrangements. Using the CellTiter-Glo cell viability assay we evaluated the cytotoxic effect of aaptamine against a panel of leukemia cell lines. We observed that cell lines containing t(4;11) are the most sensitive to aaptamine. Translocation (4;11) is associated with mixed-lineage leukemia and responsible for a very aggressive and refractory pediatric leukemia. Specifically, infants less than one year with t(4;11) have poor survival rates (≈ 19%) and new therapies are urgently needed. Interestingly other MLL cell lines that contain t(9;11) are comparatively less susceptible to aaptamine-mediated cytotoxicity. Jurkat cells overexpressing MLL-AF4 fusion protein are also more sensitive to aaptamine-induced cytotoxicity than wild type or MLL-AF9 overexpressing Jurkat cells indicating the specificity of aaptamine for t(4;11). To further confirm the specificity we conducted a flow based apoptosis assay and observed that aaptamine induces significant apoptosis and necrosis in RS4;11 and MV4;11 cell lines starting at 10µM but not in the t(9;11) containing THP1 cell line. We also found that aaptamine treatment induced G0/G1 arrest specifically in t(4;11) containing cell lines but not in THP1. Additionally we observed that aaptamine did not induce any resistance to the sensitive cell lines after 27 days of chronic exposure. Importantly the compound was well tolerated by healthy activated PBMCs and mice at high concentrations.In order to decipher the mechanism of specificity, we conducted a global proteomic study with treated and untreated RS4;11 and THP1 cell lines. Our proteomic data revealed a significant upregulation of p21 and p27 in aaptamine treated RS4;11 cells but not in THP1. In agreement with the proteomic data, we observed a dose-dependent upregulation of p21 and p27 in both protein and mRNA levels in RS4;11 and MV4;11 cells but not in resistant THP1 cells. Using p21 and p27 promoter-driven luciferase reporter constructs, we observed a significant upregulation of luminescence signal in the RS4;11 cell line at much lower concentration of aaptamine (1µM) whereas the THP1 cell line required 50µM of aaptamine for significant increase in luminescence signal.Cyclin-dependent kinase regulates the G1/S cell cycle transition by phosphorylating retinoblastoma protein (RB). Upregulation of cyclin-dependent kinase inhibitors, such as p27 and p21, promote RB hypophosphorylation and induce G0/G1 arrest. To confirm that this molecular mechanism is responsible for aaptamine induced G0/G1 arrest, we investigated the effect of aaptamine on Rb phosphorylation. We observed a dose dependent downregulation of Rb phosphorylation by aaptamine in sensitive cell lines and predicted it as a major cause of cell cycle arrest. Previous studies have shown that translocation (4;11) is associated with p27 upregulation; thus we hypothesize by further upregulating p27, aaptamine may induce G0/G1 arrest specifically in t(4;11) containing cell lines. To validate the efficacy of aaptamine in vivo, we xenografted 10 NSG mice with 1 million luciferase expressing RS4;11 cells. Four days after leukemia induction we treated half of the mice with subcutaneous injection of aaptamine (100mg/kg, daily) and the other half received vehicle treatment. Bioluminescence imaging (BLI) data revealed a significantly lower disease (p< 0.03) burden in the aaptamine treated group compared to vehicle treated group after 2 weeks. These findings are being confirmed in patient samples. Additional aaptamine analogs are being designed and will be evaluated for improved therapeutic efficacy.Together our in vitro and in vivo findings suggest that by inducing p21 and p27 aaptamine can induce cell cycle arrest and eventually apoptosis specifically in leukemia cells that contain t(4;11) with relatively low toxicity . Therefore the aaptamine class of drug may provide additional therapeutic options for t(4;11) containing high-risk MLL leukemia patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract B23: Selective and rapid killing of mixed lineage leukemia and CALM-AF10 leukemia by SM7, a novel small molecule identified by chemical library screening

Abstract Translocations involving the Mixed Lineage Leukemia (MLL) gene define the major proportion of infant leukemia, a disease that is characterised by a dismal prognosis due to treatment toxicity and therapy resistance. There is an urgent need for the development of less toxic, more selective and targeted therapies for this disease. In this study, we performed a cell-based chemical library screen on an infant MLL cell line, PER-485, in order to identify selective inhibitors of MLL leukemia. After screening initial hits for viability against a panel of 25 cell lines including MLL rearranged and wild-type MLL leukemia, solid tumors and control cells, SM7 was identified as a compound that significantly and selectively decreased the viability of MLL-rearranged (four out of seven) and related CALM-AF10 (two out of two) leukemia cell lines, without killing cell lines derived from wild-type MLL leukemia, solid tumours or normal cells. In contrast to currently developed MLL-inhibitors, the effect of SM7 in suppressing the characteristic MLL-driven gene expression signature in MLL-rearranged cells, with downregulation of leukemogenic HoxA9 and Meis1 expression, occurred within an hour after treatment. The transcript levels of cMyc and Bcl2, important survival proteins regulated by HoxA9, were also rapidly decreased in response to SM7. SM7 induced a rapid caspase-dependent apoptosis, with mitochondrial depolarization within twenty four hours of treatment. This is distinct from the action of other previously described MLL small molecule inhibitors. The rapid and selective toxicity of SM7 towards MLL-rearranged and CALM-AF10 leukemia cell lines suggests an important new avenue in the development of targeted therapies for this disease. Citation Format: Daria Chudakova, Klaartje Somers, Shiloh Middlemiss, Victoria Wen, Alan Kwek, Bing Liu, Molly Clifton, Sukey Pan, Sarah Cruikshank, Marissa Scandlyn, Tim Failes, Wendy Hoang, Ursula Kees, Olga Chernova, Andrei V. Gudkov, Michelle Haber, Murray Norris, Michelle Henderson. Selective and rapid killing of mixed lineage leukemia and CALM-AF10 leukemia by SM7, a novel small molecule identified by chemical library screening. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B23.

Targeting MLL1 H3K4 Methyltransferase Activity in Mixed-Lineage Leukemia

Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.

Anticancer Peptidylarginine Deiminase (PAD) Inhibitors Regulate the Autophagy Flux and the Mammalian Target of Rapamycin Complex 1 Activity

Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.

Abstract 4093: Investigating a role for menin and MLL-AF9 in suppressing C/EBPβ-mediated mixed lineage leukemia cell differentiation

Abstract The Mixed Lineage Leukemia (MLL) gene forms chromosomal translocations with one of multiple partner genes, leading to the expression of MLL fusion proteins (MLL-FPs), which cause acute leukemias with particularly poor prognoses. MLL-FPs contain an N-terminal portion the MLL protein, which is responsible for MLL-FP recruitment to MLL target genes, through interactions with various proteins, including the MEN1 gene product menin. The ability of menin to recruit MLL-FPs to target genes is critical for leukemic transformation. A central hallmark of leukemia is oncogene-mediated suppression of differentiation. Many leukemic oncogenes, including Bcr-Abl and AML-ETO suppress differentiation through inhibiting the C/EBPα transcription factor, a driver of myeloid differentiation. However, a mechanism for MLL-FP-mediated inhibition of C/EBPα has not been reported. We have found that disruption of the MLL-AF9 pathway by acute depletion of its recruiting factor menin causes differentiation of MLL-AF9 leukemia cells in vivo. These results suggest that a major function of menin/MLL-AF9 is to maintain leukemia cells in a de-differentiated state. Further, cDNA microarray analysis of primary MLL-AF9 splenocytes with acute menin depletion showed induction of genes involved in hematopoietic differentiation, including transcriptional targets of C/EBPβ. These results suggest a mechanism for MLL-FP-mediated inhibition of C/EBPα and suppression of leukemic progenitor cell differentiation. 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 4093. doi:1538-7445.AM2012-4093

Selective Killing of Mixed Lineage Leukemia Cells by a Potent Small-Molecule DOT1L Inhibitor

Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.

A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting

A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combina-tion are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

AbstractAbstract 4174Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures:No relevant conflicts of interest to declare.

Selective Killing of Mixed Lineage Leukemia Cells by a Potent Small-Molecule DOT1L Inhibitor

AbstractAbstract 780Rearrangements of the mixed lineage leukemia (MLL) gene on chromosome 11q23 are found in over 70 % of infant leukemias, and approximately 10% of adult acute myeloid leukemias (AML). Patients with MLL-rearranged leukemias have aggressive disease with a poor prognosis. Recent studies suggest that DOT1L, a histone methyltransferase that methylates lysine 79 of histone H3 (H3K79), plays a fundamental role in the development and maintenance of this genetically defined subset of leukemia. Rearrangements of the MLL gene result in the expression of MLL-fusion proteins that gain the ability to recruit DOT1L to chromatin. This leads to aberrantly high levels of H3K79 methylation and gene expression at specific genomic loci, including HOXA9 and MEIS1 that are thought to promote leukemogenesis. These findings, together with studies demonstrating a key role for DOT1L in propagating the transforming activity of MLL-fusion proteins in model systems, support the development of inhibitors of this enzyme as targeted therapeutics for patients bearing MLL-rearranged leukemias. To this end, we have used mechanism-guided design to identify EPZ01, the first small molecule DOT1L inhibitor. This compound is a potent and specific inhibitor of DOT1L methyltransferase activity with a Ki of ~ 400 pM in biochemical assays. EPZ01 acts as a competitive inhibitor with the co-factor S-adenosyl-methionine (SAM), and demonstrates greater than 500-fold selectivity for DOT1L over other lysine and arginine histone methyltransferases. Incubation of MLL-rearranged leukemic cell lines with EPZ01 leads to a dramatic decrease in cellular H3K79 methylation but does not affect the methylation of other histone residues, including H3K4, H3K27, H3K36 and H3K9. Analysis of the effects of EPZ01 on the proliferation of a panel of acute lymphoid leukemia (ALL) or AML-derived human MLL-rearranged cell lines including SEMK2, MV4-11, RS4;11, MOLM-13 and THP-1, and non-rearranged leukemia cell lines including HL-60, Jurkat and U937, reveals anti-proliferative activity that is remarkably selective for cell lines bearing the MLL-rearrangement. EC50 values for inhibition of proliferation by EPZ01 are in the nanomolar to low micromolar range for all MLL-rearranged lines tested. In contrast, EPZ01 shows little or no effect on the proliferation of cells lacking an MLL-rearrangement despite an equal decrease in cellular H3K79 methylation. A more detailed analysis of the cellular effects of EPZ01 in MLL-rearranged cell lines reveals that treatment with the inhibitor causes a decrease in mRNA expression of known MLL-fusion target genes including HOXA9 and MEIS1, cell cycle arrest in G0/G1, an increase in expression of differentiation markers in MLL-rearranged AML cells and death by apoptosis. We are currently evaluating the effects of EPZ01 and related compounds in in vivo models of MLL-rearranged leukemia where preliminary results indicate that we are able to achieve inhibition of DOT1L activity. EPZ01 therefore represents the first example of a histone methyltransferase inhibitor that selectively kills tumor cells bearing a defined genetic lesion. These data provide compelling validation for the development of DOT1L inhibitors as targeted therapeutics for MLL-rearranged leukemias and we are currently working towards this goal. Disclosures:Pollock:Epizyme, Inc: Employment. Daigle:Epizyme, Inc: Employment. Olhava:Epizyme, Inc: Employment. Therkelsen:Epizyme, Inc: Employment. Majer:Epizyme, Inc: Employment. Song:Epizyme, Inc: Employment. Allain:Epizyme, Inc: Employment. Sneeringer:Epizyme, Inc: Employment. Johnston:Epizyme, Inc: Employment. Porter Scott:Epizyme, Inc: Employment. Jin:Epizyme, Inc: Employment. Kuntz:Epizyme, Inc: Employment. Chesworth:Epizyme, Inc: Employment. Moyer:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy. Copeland:Epizyme, Inc: Employment. Richon:Epizyme, Inc: Employment.

The Leukemogenic Properties of MLL Fusion Genes Do Not Require FLT3 Signaling.

Leukemias harboring chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene express high levels of the receptor tyrosine kinase FLT3. Activation of FLT3 signaling is very frequent in human acute myeloid leukemias and its role in the transformation of myeloid cells was demonstrated experimentally. To study what role FLT3 activation played in the cellular transformation leading to MLL-rearranged leukemias we used retroviral vectors to express MLL fusion genes in flt3+/+ and flt3−/− bone marrow cells. Cohorts of mice transplanted with flt3+/+ and flt3−/− bone marrow expressing either MLL-ENL or MLL-CBP were established. All mice succumbed to AML within 60 days (MLL-ENL) or 120 days (MLL-CBP) regardless of flt3 status.Parameters reflecting cellular proliferation (leukocytosis, spleen weights) or maturation (morphology, immunophenotype) were identical in flt3−/− versus flt3+/+ leukemic blasts. Transplantability and clonality of the leukemias were also unchanged. We showed that the FLT3 protein was expressed in flt3+/+ MLL-ENL leukemic blasts and to explore its role in cell growth we studied the sensitivity of the cells to various FLT3 inhibitors ex vivo. We found that PKC412, AG1296 and Herbymicin A had growth inhibitory activity on MLL-ENL leukemic cells in culture. However the IC50 of these drugs was much higher than those determined on cell lines expressing FLT3 mutants that constitutively activate the FLT3 signaling pathway. Furthermore, flt3−/− MLL-ENL leukemic cells were equally sensitive to the growth inhibitory activity of these drugs than flt3+/+ leukemic cells. This demonstrates that PKC412, AG1296 and Herbymicin A impair the growth of MLL cells through the inhibition of tyrosine kinases other than FLT3. Altogether our data question the value of FLT3 as a therapeutic target to fight MLL-rearranged leukemias and suggest that the efficacy of FLT3 inhibitors is in fact mediated by the interference with another pathway.

Targeting FLT3 in primary MLL-gene–rearranged infant acute lymphoblastic leukemia

AbstractAcute lymphoblastic leukemia (ALL) in infants is characterized by rearrangements of the mixed lineage leukemia (MLL) gene, drug resistance, and a poor treatment outcome. Therefore, novel therapeutic strategies are needed to improve prognosis. Recently, we showed that FLT3 is highly expressed in MLL rearranged ALL (MLL). Here we demonstrate FLT3 expression in infants with MLL (n = 41) to be significantly higher compared to both infant (n = 8; P < .001) and noninfant patients with ALL (n = 23; P = .001) carrying germline MLL genes. Furthermore, leukemic cells from infants with MLL were significantly more sensitive to the Fms-like tyrosine kinase 3 (FLT3) inhibitor PKC412 (N-benzoyl staurosporine) than noninfant ALL cells, and at least as sensitive as internal tandem duplication-positive (ITD+) AML cells. Surprisingly, activation loop mutations only occurred in about 3% (1 of 36) of the cases and no FLT3/ITDs were observed. However, measuring FLT3 phosphorylation in infants with MLL expressing varying levels of wild-type FLT3 revealed that high-level FLT3 expression is associated with ligand-independent FLT3 activation. This suggests that infant MLL cells displaying activated FLT3 as a result of overexpression can be targeted by FLT3 inhibitors such as PKC412. However, at concentrations of PKC412 minimally required to fully inhibit FLT3 phosphorylation, the cytotoxic effects were only fractional. Thus, PKC412-induced apoptosis in infant MLL cells is unlikely to be a consequence of FLT3 inhibition alone but may involve inhibition of multiple other kinases by this drug. (Blood. 2005;106: 2484-2490)

Granulocyte-Macrophage colony-Stimulating factor receptor-Targeted therapy of chemotherapy- and radiation-Resistant human myeloid leukemias

Contemporary therapies for acute myeloid leukemia (AML) commonly fail to cure patients because of the emergence of drug resistance. Drug resistance in AML is multifactorial but can be associated with the overexpression of transmembrane transporter molecules, including P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP), or associated with inactivation of the p53 tumor suppressor gene, as well as overexpression of the anti-apoptotic protein bcl-2. We are investigating if novel recombinant biotherapeutics can circumvent these resistance mechanisms to effectively treat refractory AML. To target the lethal action of diphtheria toxin (DT) to high affinity granulocyte-macrophage colony-stimulating factor (GMCSF) receptors on AML blasts, we have produced a recombinant chimeric fusion toxin, DTctGMCSF. Since DTctGMCSF enters and kills its target cells by unique mechanisms (GMCSF-receptor binding and protein synthesis inhibition) and is not similar in structure to Pgp or MRP substrates, we postulated that it would be an active agent against therapy-resistant AML. DTctGMCSF was selectively cytotoxic (IC50 1–10ng/ml) to GMCSF-receptor positive AML cells expressing the Pgp- or MRP-associated multi-drug resistant phenotypes, despite high level resistance to conventional chemotherapeutic agents. DTctGMCSF also efficiently killed AML cells deficient in p53 expression, as well as radiation-resistant AML cells and mixed lineage leukemia cells expressing high levels of bcl-2. In addition, DTctGMCSF killed>99% of primary leukemic progenitor cells from therapy-refractory AML patients under conditions that we have previously found to not adversely affect the proliferative capacity or differentiation of pluripotent normal hematopoietic progenitor cells. DTctGMCSF may prove useful in treating myeloid leukemias that are otherwise resistant to a wide range of conventional therapies.