MLL Fusion Proteins Research Articles

The Role of MLL1 and MLL2 in MLL Fusion Oncoprotein-Initiated Leukemia

The MLL1 histone methyltransferase gene at 11q23 undergoes many distinct chromosomal translocations to yield poor prognosis leukemia. In these MLL-rearranged (MLL-r) leukemias, the remaining wild-type allele is typically expressed, and the fusion oncoprotein lacks the C terminal histone methyltransferase domain. The role of the wild-type, endogenous MLL1 allele in MLL-fusion protein mediated leukemogenesis is not fully understood, with some evidence suggesting an important functional role. On the other hand, the wild-type allele is lost in some patients and in the MLL-r ML2 cell line, suggesting that the wild-type allele is not absolutely required. Here we rigorously address the role of endogenous MLL1 in MLL-r leukemia. Using precise and independent animal models, we show that MLL1 is dispensable for MLL-AF9- or MLL-AF6-mediated acute myelogenous leukemia (AML). In parallel experiments, deletion of Menin, encoding a chromatin targeting cofactor for MLL-fusion proteins, has severe effects on the proliferation and survival of MLL-r leukemia, as previously published. Potential redundancy between MLL family methyltransferases was addressed by co-deleting the closest paralog, Mll2. Surprisingly, deletion of Mll2 by itself had a significant impact on survival of MLL-AF9 leukemia cells, and co-deletion of Mll1 and Mll2 more effectively killed these cells, suggesting a redundant or synthetic lethal relationship between the two genes. A comparison of gene expression and histone modification changes in Mll1, Mll2 and Mll1;Mll2 double knockout leukemia cells showed that 1) global defects in histone H3, lysine 4 methyation can be exclusively attributed to MLL2, 2) MLL1- versus MLL2-dependent genes are largely distinct, and 3) Mll1;Mll2 double knockout leukemia cells exhibit deficiencies in NFκB and integrin ß3 pathways, but exhibit little evidence of deregulation of either MLL1- or MLL-fusion protein target genes. These findings reshape our thinking about the role of endogenous MLL family proteins in MLL-fusion protein leukemogenesis. The data presented here, in conjunction with the minimal effect of Mll2 deletion on hematopoietic stem and progenitor cells make MLL2 an attractive candidate for targeted therapy in MLL-r leukemia. DisclosuresErnst:Amgen: Other: stocks. Yokoyama:Sumitomo Dainippon Pharma: Research Funding. Neff:Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel; Epizyme: Patents & Royalties: patent filed.

Proteomic/Transcriptomic Signatures of Infant MLL-r Rearranged B-ALL at Diagnosis and Relapse Reveal Lineage Plasticity and Diagnostic Heterogeneity

Infant leukemia, defined as leukemia before 1 year of age, is an aggressive set of diseases with dramatically poorer outcome when compared to childhood leukemia. A particularly intractable subset of infant leukemia patients, who are prone to relapse, are those with B-cell acute lymphoblastic leukemia (B-ALL) harboring a chromosomal translocations involving the epigenetic modifier gene mixed lineage leukemia (MLL), referred hereafter as MLL rearranged (MLL-r) B-ALL. Rearrangements in MLL occur in 70-80% of all infant B-ALL patients (compared to 5% in pediatric leukemia) and this cohort is characterized by a record setting dearth of cooperating lesions beyond MLL-r.To better understand the mechanism of relapse in this patient cohort we employed RNA sequencing and deep mass spectrometry based proteomics to characterize infant MLL-R B-ALL patient derived xenografts. Five infant MLL-r B-ALL patients with matched diagnosis and relapse primary sample were individually injected into NSG immunocompromised mice. For all patients >95% human engraftment purity was achieved in mouse bone marrow and spleen. Fresh cells were submitted for RNA sequencing and nuclear fractionated as well as whole cell lysates were generated for mass spec protein analysis. Protein and RNA quantitation at relapse and diagnosis across all patients was achieved on molecules that correspond to about 8,000 genes for protein and 17,000 genes for RNA.Hierarchal clustering as well as multi-dimensional scaling analysis of RNA sequencing data revealed a closer intra-patient relationship at the two treatment time points (diagnosis and relapse) than an inter-patient relationship at diagnosis or at relapse. A common relapse signature was simply non-existent according to the data, however this could be attributed to the small sample size. The integration of proteomic and transcriptomic data revealed a strong correlation of relapse/diagnosis fold changes at the level of RNA and protein. Intriguingly, a plasticity at relapse was observed in key developmental and differentiation marker genes associated with the myeloid, T, and B cell lineages. For example, in Patient 1 up-regulation at the level of protein and RNA of myeloid/acute myeloid leukemia (AML) associated genes; RUNX2, MPO, and CD33, and down-regulation of T and B cell associated genes; CD1A, CD2, and BCL6 where observed at relapse. Patient 2 showed an RNA/protein up-regulation of myeloid and AML markers CEBPB and MPO while a concurrent down-regulation of the Ikaros family member Helios, B/T cell attenuator BTLA, and the oncogene ETV6 at relapse. Patient 3 showed an RNA/protein up-regulation of Helios, the oft mutated in B-ALL; ARID5B, and the pre-B-cell leukemia associated transcription factor PBX3 at relapse. Patient 1 and 2 appear to show signs that myeloid programs are being greater utilized at relapse while in Patient 3 seems to intensify its commitment to the B-cell lineage. All patients showed some degree of lineage marker infidelity at relapse and interestingly, this plasticity was not reflected in the traditional flow cytometric markers used to differentiate AML from B-ALL suggesting that this lineage infidelity might be more common than previously thought.Despite the mutational uniformity of infant MLL-R B-ALL genomes it is surprising that such a diversity of RNA expression and subsequent protein levels is observed. This data suggests that MLL fusion proteins have great flexibility to control of the transcriptional regulation/protein expression and could in part help explain why these patients do not rely upon newly acquired mutations to evade treatment protocols. Furthermore, lineage switching in leukemia is rarely observed but as this data suggests that this is more common in MLL-r B-ALL than previously thought. DisclosuresNo relevant conflicts of interest to declare.

LAMP5 - a Novel Target of MLL-Fusion Proteins Is Required for the Propagation of Leukemia

Acute leukemias with Mixed Lineage Leukemia gene (MLL, also called KMT2A) translocations are associated with poor outcomes. These leukemias are most frequently encountered in infants and as secondary malignancies, and can be either lymphoid or myeloid. In spite of aggressive treatments, including bone marrow transplantation, infants with MLL-leukemias face a grim prognosis, with predicted survival of only 20%. Novel, effective therapies are thus urgently needed. In search for molecular targets, we observed that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5, also known as C20orf103). This observation was consistent across several gene-expression-profiling studies and occurred in both ALL and AML. Moreover, data from the TCGA study on AML showed that patients with LAMP5 expression suffered worse prognosis compared to those lacking LAMP5. We first confirmed LAMP5 expression in human leukemia cell lines by immunoblot and RT-qPCR assays. We readily detected LAMP5 mRNA and protein in MLL-fusion leukemia cell lines (MV4;11, MOLM13, THP1, RS4;11, KOPN8), while no expression of LAMP5 was found in the non-MLL-cell lines (Kasumi, K562, REH). Published ChIP-seq studies on leukemia cell lines show the MLL-fusion protein directly bound at the promotor region of LAMP5. To validate that the MLL-fusion protein activates the expression of LAMP5, we transformed human CD34+ cord blood cells with an inducible (Tet-off) retrovirus carrying the MLL-AF9 fusion cDNA. In this system, addition of Doxycycline represses expression of the MLL-AF9 oncogene. We found that LAMP5 expression directly correlated with MLL-AF9 levels, with levels of both decreasing upon addition of Doxycycline. To investigate the role of LAMP5 in MLL-fusion leukemia, we studied the effect of shRNA-mediated knockdown. By screening several hairpin sequences, we identified one construct that efficiently inhibited LAMP5 expression in MLL-fusion leukemia cells but had no effect on LAMP5-negative cells, implying specificity. All MLL-fusion leukemia cell lines tested showed growth inhibition with LAMP5 knockdown. Specifically, growth of MV4;11, THP1 and MOLM13 cells was decreased by 69%, 73% and 80% respectively compared to controls (non-targeting shRNA). When cultured in semi-solid methylcellulose media for 10 days, LAMP-5-depleted MV4;11 cells formed significantly fewer colonies than control cells (64.3 ± 25.98 and 245.3 ± 27.42 colonies per 1000 cells respectively). To investigate the role of LAMP5 in leukemia-propagation in vivo, we transplanted control and LAMP5-depleted MV4;11 cells into Busulfan-conditioned immune-deficient (NRGS) mice (2x105cells/mouse). Preliminary results from bone marrow aspirates of transplanted mice at weeks 4 post-transplant showed abundant human leukemia cells in mice receiving control cells while the mice receiving LAMP5-knockdown cells showed near-absence of human leukemia cells. Collectively, these results demonstrate that LAMP5, a novel target of MLL-fusion proteins is required for the propagation of leukemia. In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it localizes to the ER-Golgi intermediate compartment (ERGIC). In leukemia cells, using immunofluorescent confocal microscopy we detected LAMP5 in the perinuclear zones where it co-localized with ERGIC-53, a marker of the ERGIC compartment. While little is known about the functions of LAMP5 in normal pDC, studies suggest that it functions as a co-chaperone with UNC93B1, a known Toll-like Receptor (TLR) chaperone, to shuttle the TLRs to their respective locations in the plasma membrane or endosomes. In ongoing experiments, we are determining the functions of LAMP5 in leukemia, including its association with UNC93B1 and with the TLR-NFKB signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we postulate that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias. DisclosuresNo relevant conflicts of interest to declare.

The Transcriptome Heterogeneity of MLL-Fusion ALL Is Driven By Fusion Partners Via Distinct Chromatin Binding

Chromosome rearrangements involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 account for 15-20% of acute lymphoid leukemia (ALL) and confer poor prognosis. Such rearrangements generate the MLL-fusion proteins, in which the N-terminus of the MLL protein mediating chromatin interactions is fused with one of more than 70 different partner proteins. Proteins that are frequently involved in MLL translocations, including AF4, ENL, AF9 and AF10, were identified as components of the super elongation complex (SEC) or DOT1L complex (an H3K79 histone methyltransferase). Based on these observations, a consensus model of MLL-fusion leukemogenesis has been proposed, which suggests that all fusion proteins bind to the targets of wildtype MLL and lead to the aberrant transcriptional elongation and H3K79 methylation via the recruitment of SEC or DOT1L complex and thus the uncontrolled activation of the target genes. Therefore, regardless of the nature of fusion partners, all MLL-fusion proteins work in a similar fashion by dysregulating the same pathways.Our group has successfully established xenograft models of MLL-AF4 and MLL-AF9 B-ALL using human CD34+ hematopoietic stem and progenitor cells transduced with FLAG-tagged MLL-Af4 or MLL-AF9, which faithfully recapitulate the clinical features of the disease. We generated MLL-Af4 and -AF9 ALL using matched units of human CD34+ cells to directly test the consensus model. Interestingly, although having the same genetic background, the immunophenotype of the two ALL are unique, with CD34 expressed only in MLL-Af4 but not -AF9 cells. The transcriptomes of the two ALL were analyzed by RNAseq and dysregulated genes were defined by comparison with the transcriptome of normal pro-B cells (p≤0.05, fold-change≥2). Strikingly, only 40% of MLL-Af4-regulated genes overlap with those of MLL-AF9. This transcriptome heterogeneity is mirrored in clinical samples, where the gene signature generated from our model leukemia can be utilized to accurately classify patient samples in unsupervised hierarchical clustering analysis, with MLL-AF4 patient samples readily distinguishable from MLL-AF9 samples.To identify the mechanisms accounting for this heterogeneity, we performed ChIP-seq analysis using anti-FLAG antibody to compare the chromatin occupancy of MLL-Af4 and MLL-AF9 in our model ALL cells. The MLL-Af4 ChIP-seq signal displayed a clear correlation with those of published MLL-AF4 ChIP-seq datasets from patient-derived cell lines, in the range of 70-90%, highlighting the faithfulness of our model. Surprisingly, MLL-Af4 shows a distinct genome-wide distribution compared to MLL-AF9, with only 20% of MLL-Af4 peaks and 35% of MLL-AF9 peaks overlapping. In contrast to MLL-Af4 which predominantly binds to promoter regions, MLL-AF9 has a relatively greater enrichment at intra- and inter-genic regions. Intriguingly, MLL-AF9 tends to bind at repetitive sequences in introns, suggesting these repetitive sequences may serve as regulatory elements for gene expression. Integration with RNAseq data reveals a significant association between differentially-expressed MLL-Af4 and -AF9 targets and specific chromatin binding of different MLL-fusion proteins. This data demonstrates that chromatin binding is not solely controlled by the MLL portion of the fusion protein and that differential target recognition of different fusion proteins is one molecular mechanism driving gene expression heterogeneity.To test whether distinct co-factor recruitment by MLL-fusions adds another layer regulating gene expression heterogeneity beyond DNA binding, we purified the core complexes of MLL-Af4 and -AF9 from ALL cells by anti-FLAG immunoprecipitation and analyzed by mass spectrometry. These experiments identified both common and fusion-specific interacting proteins. MLL-Af4 showed a higher affinity with SEC component EAF2 but a lower affinity with DOT1L compared to MLL-AF9, suggesting that MLL-fusions have distinct associations with complex components which may achieve differential gene regulation.In summary, our data question the consensus model of MLL-fusion leukemia and emphasize that MLL-fusion ALL is a heterogeneous disease. These findings have important implications for therapy development as each MLL-fusion leukemia could have its own Achilles' heel and customized therapy may need to be introduced for each type of disease. DisclosuresNo relevant conflicts of interest to declare.

The PAF Complex Regulation of Prmt5 Facilitates Leukemic Progression

Acute myeloid leukemias have been linked with dysregulated epigenetic landscapes sometimes attributed to altered functions of epigenetic regulators. The Polymerase-Associated Factor complex (PAFc) is an epigenetic regulator involved in transcriptional initiation, elongation and termination and directly interacts with the CTD of RNA Pol II. The complex is comprised of 6 subunits in human cells, Paf1, Cdc73, Ctr9, Leo1, Rtf1 and Ski8. Many of these subunits have key roles in a variety of cancers including acute myeloid leukemia (AML). We have previously shown the relevance of the PAFc in MLL-rearranged leukemias where its interaction with MLL fusion-proteins is required for leukemic progression in vitro and in vivo (Muntean et al. 2013 Blood, Muntean et al. 2010 Cancer Cell). However, little is known about the gene programs controlled by the PAFc and how these contribute to leukemogenesis.Here we identify Prmt5, an arginine methyltransferase, as a direct downstream target gene of the PAFc. Prmt5 is upregulated in variety of cancers and has been linked to cell cycle progression and activation of known oncoproteins. In addition, Prmt5 has been implicated in AML and is essential for normal hematopoiesis where loss of Prmt5 induces bone marrow aplasia due to impaired cytokine signaling (Tarighat et al. 2015 Leukemia, Liu et al. 2015 J Clin Invest). Our work establishes a major role for the PAFc in regulating Prmt5 expression in AML. We observe that excision of the Cdc73 subunit of the PAFc results in reduced proliferation, the induction of differentiation, cell cycle arrest, and a mild increase in apoptosis. Several key epigenetic marks are reduced globally upon loss of Cdc73 including H4R3me2s, a modification catalyzed by Prmt5. RNA sequencing and bioinformatics analysis using GSEA, revealed that loss of Cdc73 led to increased expression of a gene program associated with hematopoietic differentiation, in agreement with our cellular characterization. In addition, the downregulation of a methyltransferase gene program was detected upon Cdc73 excision. Included in this signature were several members of the Prmt family. Analysis of changes in expression following loss of Cdc73 and functional relevance in MLL-AF9 leukemic cells led us to Prmt5 as a gene critically important in AML cells and modulated by the PAFc. To interrogate the function of Prmt5 in AML cells, we performed shRNA knockdown experiments which resulted in reduced proliferation, reduced cell fitness, G1 cell cycle arrest and global reduction H4R3me2s. ChIP experiments revealed that the PAFc localizes to the Prmt5 locus in mouse and human derived leukemic cells. Further, preliminary data suggests the MLL-AF9 fusion protein also localizes to the Prmt5 locus and may enhance its transcriptional output. The enzymatic activity of Prmt5 is necessary for AML cell growth as wild type PRMT5 can rescue proliferation of Prmt5 knock-down cells while a catalytic dead mutant cannot. Furthermore, we have observed that knockdown of Prmt5 increases the disease latency of Hoxa9/Meis1 induced leukemia in vivo. Utilizing a commercially available inhibitor for Prmt5, EPZ015666 (Chan-Pembre et al. 2015 Nat Chem Bio), we show pharmacologic inhibition of PRMT5 reduces the growth of a spectrum of human leukemic cell lines, suggesting PRMT5 is important for multiple subtypes of AML. Overall, our findings elucidate the PAFc as a regulator of Prmt5 expression that is necessary for the maintenance of AML. DisclosuresNo relevant conflicts of interest to declare.

High Order Chromatin Structure Regulates Gene Expression in Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation

High order chromatin structure is implicated in multiple developmental processes and disease. However, a global picture of chromosomal looping interaction alterations during stem cell self-renewal and differentiation is lacking. Hematopoietic stem cell (HSCs) and their differentiated progenitors (HSPCs) offer a system in which to examine this. Of the key differentiated lineages, the erythroid lineage undergoes a unique nuclear condensation process during a well-characterized differentiation process which can be induced in vitro from CD34+ HSPCs. Thus erythroid differentiation offers an ideal model system to study differentiation-associated changes in high order chromatin structure.We have thus generated the in situ Hi-C contact map for human cord blood CD34+ CD38- HSPC (CD34+) and erythroid progenitors undergoing differentiation in vitro at day 7 from CD34+ HSPCs (EryD7). In our 5kb resolution map, we identified over 2000 chromosomal loop interactions in both CD34+ and Day 7 erythroid respectively . The EryD7 sample exhibited higher random intra-chromosomal interactions in comparison with CD34+, presumably due to nuclear condensation.By comparing the chromosomal loop interactions in the 2 cell types. We identified self-renewal and erythroid differentiation-specific looping patterns in the two cell types. Strikingly, we found that a gene depleted region (GDR) 2MB upstream of the HOXA cluster forms a strong chromosome loop with the HOXA cluster exclusively in the HSPCs (Fig1A). Within this GDR site, we identified two conserved CTCF sites, which are thought to organized chromosome looping. Utilizing the CRISPR-mediated deletion of each of the two CTCF sites, we found that deletion of either site reduce the colony forming ability of CD34+, indicating a loss of stem cell self-renewal. (Fig 1B) Gene expression analysis showed that HOXA9 expression was compromised the CTCF site deletion. These data suggest that the GDR is forming a distant regulatory loop which controls the expression of HOXA9 in HSPCs. Because the GDR is implicated in controlling HOXA9 expression, a key gene in leukemogenesis, we then tested the importance of this looping site in different leukemia cell lines that are dependent on HOXA9. Of those cell lines, we found the deletion of the CTCF sites inhibit the growth of DNMT3A and NPM1 mutated OCI-AML3 and promote the apoptosis. In contrast, growth of the MLL translocation cell line MV 4:11 was not abrogated by their deletion (Fig 1C). As a control cell line which doesn't express HOXA9, HL60 cells were not sensitive to the deletion of the GDR CTCF sites. Together, these data indicate leukemic cells may adopt different strategies to activate HOXA9. MLL translocation leukemias activates HOXA9 by the direct binding of the MLL fusion protein, while the NPM1 mutated leukemia is more likely to utilize the stem cell looping to activate HOXA9 expression.Among EryD7 specific interactions, we found the β-globin locus specifically forms chromatin loops at Day7 that are not evident in the CD34+ HSPCs. Detailed examination showed that Dnase I hypersensitivity sites HS5 and 3'HS1 both contains CTCF site and form chromosomal loops. Two other loop-forming CTCF sites, both on the telomeric and centromeric side of β-globin locus were also identified. Interestingly, we found a CTCF binding site adjacent to OR52A5 gene which forms a chromosomal loop with HS5 and is not well studied. To test the role of the chromosomal looping in the regulation of hemoglobin gene expression in β-globin locus, we deleted the OR52A5-CTCF site and the 3'HS1 CTCF site in K562 and adult CD34+ HSPCs. We found the deletion of OR52A5-CTCF resulted in a decrease of HBE and increase of HBB expression in K562 cells, which suggest the OR52A5 CTCF also plays a role in regulating hemoglobin gene expression in the β-globin locus (Fig 1D). Furthermore, we found the deletion of 3'HS1 CTCF resulted in a 4-fold increase of HBG2 expression in adult CD34+ HSPC during erythroid differentiation (Fig1 E). Thus this indicating the 3'HS1 and OR52A5-CTCF CTCF sites in β-globin locus are forming loops that regulate the β-globin locus gene expression.In summary, we have mapped the higher order chromatin structure alterations during stem cell differentiation and identified the critical looping interaction essential for the self-renewal and differentiation specific functions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Protease-Activated Receptor 1 (PAR-1) Inhibits Proliferation but Enhances Leukemia Stem Cell Activity in Acute Myeloid Leukemia

Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. DisclosuresNo relevant conflicts of interest to declare.

An Epigenetic Regulator Screen Identifies Novel Targets That Sensitize MLL-Rearranged Leukemia to DOT1L Inhibition

Mixed Lineage Leukemia gene rearrangements (MLL-r) account for nearly 10% of human acute leukemia cases and are generally associated with poor prognosis. Previous studies have revealed an essential role of the histone H3K79 methyltransferase Disruptor of Telomeric Silencing-1 Like (DOT1L) in MLL-r leukemogenesis. Our recent report (Chen et al. 2015 Nature Medicine) further identified a role for histone acetylation in DOT1L dependent gene expression driven by MLL-fusion proteins including MEIS1 and HOXA cluster genes. A first-in-human Phase I clinical trial demonstrated clinical activity of DOT1L inhibition in MLL-r leukemia patients, thus providing a potential opportunity for treating these malignant diseases. Nevertheless, the incomplete silencing of the leukemic program by only targeting DOT1L motivates the need for additional and perhaps combinational approaches to improve therapies against MLL-r leukemias.To enhance the efficacy of DOT1L inhibition, we sought to identify genes whose suppression would synergize with the DOT1L inhibitors to suppress the proliferation of mouse bone marrow progenitors transformed with MLL-AF9. We conducted a pooled RNAi screen using a customized library composed of 2,252 shRNA targeting 468 epigenetic regulators (i.e. writers, readers, and erasers of chromatin modifications; Fig 1). The integrated shRNA sequences were assessed using high-throughput sequencing. By comparing the change in frequency of each shRNA construct cultured in control vs. an IC50 DOT1L inhibitor EPZ4777, we identified several candidate modulators of DOT1L dependency, which had multiple shRNAs selectivity depleted only in the DOT1L suppressed condition. Notably, using a network correlation study, we found that one of the top candidate genes Plant Homeodomain Finger Protein 20 (PHF20) is highly associated with histone acetylation in the mammalian epigenome. Knockdown of PHF20 drastically increased the sensitivity of MLL-AF9 leukemic blasts to DOT1L inhibitors through enhanced myeloid differentiation and reduced cell proliferation, colony formation, and re-plating capacity. Similar phenotypes were also observed in PHF20-deficient MLL-AF9 cells generated by CRISPR/Cas9-mediated gene knockout.PHF20 is an epigenetic adaptor protein that has no predicted enzymatic activity. To investigate the role of PHF20, we conducted a CRISPR functional domain screen and identified the requirement of the chromatin reader domains in PHF20, including the Tudor domains and the PHD-finger, in supporting the survival of MLL-r leukemic cells upon DOT1L inhibition. We also performed RNA-seq and found that suppression of PHF20 facilitated the silencing of the MLL-AF9 leukemic program induced by DOT1L inhibitor treatment. Chromatin immunoprecipitation and sequencing (ChIP-seq) analyses validated that PHF20 contributes to the maintenance of histone acetylation including H3K9ac and H4K16ac at MLL-AF9 target loci. In line with the profound loss of histone acetylation at MLL-AF9 target loci in PHF20-depleted cells, we found that knockdown of a known PHF20 interacting partner KAT8 (a histone acetyltransferase; also known as MOF or MYST1) phenocopies the effects observed in PHF20-knockdown cells. Finally, we showed that pharmacological inhibition of DOT1L and KAT8 synergistically suppresses the proliferation and survival of MLL-AF9 leukemic cells. These data collectively highlight the involvement of a novel DOT1L-PHF20-KAT8 axis in mammalian gene regulation and MLL-r leukemogenesis.In summary, our studies show that MLL-rearrangements may drive leukemic transformation by coordinating an epigenetic network involving several histone modifications associated with gene transcription (e.g. H3K79 methylation and H3K9/H4K16 acetylation). Our results also suggest that simultaneous targeting of multiple components of this epigenetic feed-forward loop including DOT1L and PHF20/KAT8 may provide a novel and more effective approach against MLL-r leukemia. [Display omitted] DisclosuresBradner:Novartis Institutes for BioMedical Research: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Transcriptional activation by MLL fusion proteins in leukemogenesis.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause aggressive leukemia. Fusion proteins of MLL and a component of the AF4 family/ENL family/P-TEFb complex (AEP) are responsible for two-thirds of MLL-associated leukemia cases. MLL-AEP fusion proteins trigger aberrant self-renewal of hematopoietic progenitors by constitutively activating self-renewal-related genes. MLL-AEP fusion proteins activate transcription initiation by loading the TATA-binding protein (TBP) to the TATA element via selectivity factor 1. Although AEP retains transcription elongation and mediator recruiting activities, the rate-limiting step activated by MLL-AEP fusion proteins appears to be the TBP-loading step. This is contrary to prevailing views, in which the recruitment of transcription elongation activities are emphasized. Here, I review recent advances towards elucidating the mechanisms underlying gene activation by MLL-AEP fusion proteins in leukemogenesis.

Complementary activities of DOT1L and Menin inhibitors in MLL-rearranged leukemia.

Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.

Instructive Role of MLL-Fusion Proteins Revealed by a Model of t(4;11) Pro-B Acute Lymphoblastic Leukemia

The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.

Autophagy mediates proteolysis of NPM1 and HEXIM1 and sensitivity to BET inhibition in AML cells.

The mechanisms underlying activation of the BET pathway in AML cells remain poorly understood. We have discovered that autophagy is activated in acute leukemia cells expressing mutant nucleophosmin 1 (NPMc+) or MLL-fusion proteins. Autophagy activation results in the degradation of NPM1 and HEXIM1, two negative regulators of BET pathway activation. Inhibition of autophagy with pharmacologic inhibitors or through knocking down autophagy-related gene 5 (Atg5) expression increases the expression of both NPM1 and HEXIM1. The Brd4 inhibitors JQ1 and I-BET-151 also inhibit autophagy and increase NPM1 and HEXIM1 expression. We conclude that the degradation of NPM1 and HEXIM1 through autophagy in certain AML subsets contributes to the activation of the BET pathway in these cells.

High-affinity small molecular blockers of mixed lineage leukemia 1 (MLL1)-WDR5 interaction inhibit MLL1 complex H3K4 methyltransferase activity

MLL1-WDR5 protein-protein interaction is essential for MLL1 H3K4 methyltransferase activity. Targeting MLL1 enzymatic activity to regulate expression level of MLL-dependent genes represents a therapeutic strategy for acute leukemia harboring MLL fusion proteins. Herein we reported a series of biphenyl compounds disturbed MLL1-WDR5 interaction. These compounds effectively inhibited MLL1 histone methyltransferase (HMT) activity in vitro and in MV4-11 cell line. The representative compound 30 (DDO-2084) inhibited proliferation and induced apoptosis of MV4-11 cells through deregulating expression level of Hoxa9 and Meis-1 genes, which emphasized our compounds were on-target. Optimization of compound 30 led to high-affinity inhibitors. Especially, compound 42 (DDO-2117, IC50 = 7.6 nM) bearing an amino and a 4-aminobutanamido group was the most potent inhibitor reported to-date, and showed the most potent inhibitory activity (IC50 = 0.19 μM) in HMT assay.

Targeting MLL-CXXC Domain with Synthetic CpG Dinucleotides: Docking and Molecular Dynamics Simulation Based Approach

Mixed lineage leukemia (MLL) is an aggressive type of childhood leukemia characterized by the presence of MLL fusion proteins resultant of chromosomal translocations affecting the MLL gene. These resulting chimeric proteins act as transcriptional regulators that take control of MLL targets. MLL fuses with more than 60 different partner genes, these fusion proteins retain the CXXC domain which binds to nonmethylated CpG DNA. The Molecular docking of CXXC domain and CpG dinucleotides were performed, large scale and long time (10 ns each) molecular dynamics simulation on CXXC domain wild type and AF9-MLL Fusion Protein have been performed to study the mechanism at atomic level. The effects of point mutation on DNA bindings were also studied through Molecular Docking. The results confirm that this point mutation have efficacy of interrupt the DNA bindings. Moreover, in-silico studies suggest external synthetic CpG DNA efficiently binds to CXXC domain and may be potential lead molecule for further in-vitro studies.

Abstract 3898: Structure-based optimization of small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia (MLL)

Abstract Chromosomal translocations that affect the Mixed Lineage Leukemia (MLL) gene result in acute myeloid and lymphoid leukemias. Fusion of MLL with one of 60 different partner genes generates MLL fusion proteins which lead to enhanced cell proliferation, up-regulation of Hoxa9 and Meis1 genes and block hematopoietic differentiation, ultimately leading to acute leukemia. Importantly, the N-terminal region of MLL is retained in all MLL fusion proteins and represents the interaction site of menin, a critical oncogenic cofactor for MLL fusion proteins. As a result, inhibition of the menin-MLL interaction should abrogate the development and progression of MLL leukemia. Therefore, disruption of the menin-MLL interaction with small molecule inhibitors might represent a therapeutic strategy for patients harboring MLL-rearrangements. The development of potent small molecule inhibitors of protein-protein interactions with optimized drug-like properties represents a challenging task in the lead optimization process. Here, we employed structure-based design with extensive medicinal chemistry efforts to optimize the thienopyrimidine class of menin-MLL inhibitors represented by the MI-136 compound. Our efforts resulted in development of MI-538, which represents the most potent small molecule inhibitor of the menin-MLL interaction developed to date. MI-538, which binds to menin with a Kd of 6.5 nM demonstrated superior cellular activity in MLL-AF9 transformed leukemic cells with a GI50 = 83 nM. In a mouse xenograft model utilizing MV4:11 human MLL leukemia cells, treatment with MI-538 significantly reduced tumor growth. Overall, systematic exploration of substituents on MI-136 led to the identification of MI-538 with improved activity, selectivity, polarity and pharmacokinetic profile, making it suitable for in vivo studies. Interestingly, we found that simultaneous incorporation of multiple substituents optimized for each site on the cyanoindole ring of MI-136 does not always positively affect the activity or drug-like properties of these compounds. This study demonstrates challenges in optimizing inhibitors of protein-protein interaction for potential therapeutic applications. Citation Format: Jonathan Pollock, Dmitry Borkin, Katarzyna Kempinska, Trupta Purohit, Xiaoqin Li, Bo Wen, Ting Zhao, Hongzhi Miao, Shirish Shukla, Miao He, Duxin Sun, Tomasz Cierpicki, Jolanta Grembecka. Structure-based optimization of small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia (MLL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3898.

Abstract 3803: Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis

Abstract Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangements of the MLL1 gene generate fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. To gain insights into the unique functions of DOT1L in MLL-driven leukemia, we elucidated the mechanisms of DOT1L recruitment to the MLL fusion partners. The binding site was mapped to a short segment of 10 amino acids in DOT1L and peptides derived from this region disrupted the interaction between DOT1L and MLL-AF9. DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. This discovery has established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias. Applying high throughput screening approach several different chemical classes of small molecules that disrupt the protein-protein interactions between DOT1L and oncogenic MLL-fusion proteins were identified and validated. To evaluate if the AF9-binding domain of DOT1L is critical for its functions in normal hematopoietic stem cells as opposed to leukemias driven by MLL fusion proteins, genetic tools were developed to functionally investigate the importance of the DOT1L AF9-binding domain in MLL-AF9-driven leukemia and its role in the physiological functions of DOT1L in normal hematopoiesis. Our findings demonstrate that pharmacological inhibition of the DOT1L complex through disrupting the AF9-DOT1L interactions may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression. Citation Format: Sierrah Grigsby, Jennifer Chase, James Ropa, Justin Serio, Chenxi Shen, Martha Larsen, Preston Donover, Melvin Reichman, Andrew Muntean, Ivan Maillard, Zaneta Nikolovska-Coleska. Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3803.

Deregulation of the HOXA9/MEIS1 axis in acute leukemia.

HOXA9 is a homeodomain transcription factor that plays an essential role in normal hematopoiesis and acute leukemia, in which its overexpression is strongly correlated with poor prognosis. The present review highlights recent advances in the understanding of genetic alterations leading to deregulation of HOXA9 and the downstream mechanisms of HOXA9-mediated transformation. A variety of genetic alterations including MLL translocations, NUP98-fusions, NPM1 mutations, CDX deregulation, and MOZ-fusions lead to high-level HOXA9 expression in acute leukemias. The mechanisms resulting in HOXA9 overexpression are beginning to be defined and represent attractive therapeutic targets. Small molecules targeting MLL-fusion protein complex members, such as DOT1L and menin, have shown promising results in animal models, and a DOT1L inhibitor is currently being tested in clinical trials. Essential HOXA9 cofactors and collaborators are also being identified, including transcription factors PU.1 and C/EBPα, which are required for HOXA9-driven leukemia. HOXA9 targets including IGF1, CDX4, INK4A/INK4B/ARF, mir-21, and mir-196b and many others provide another avenue for potential drug development. HOXA9 deregulation underlies a large subset of aggressive acute leukemias. Understanding the mechanisms regulating the expression and activity of HOXA9, along with its critical downstream targets, shows promise for the development of more selective and effective leukemia therapies.

Cooperation between AlphavBeta3 integrin and the fibroblast growth factor receptor enhances proliferation of Hox-overexpressing acute myeloid leukemia cells.

A poor prognosis subtype of acute myeloid leukemia (AML) is characterized by increased expression of a set of homeodomain (HD) transcription factors, including HoxA9, HoxA10 and Cdx4. This encompasses AML with MLL1 gene translocations, because Mll1-fusion proteins aberrantly activate HOX transcription. We previously identified FGF2 (Fibroblast Growth Factor 2) as a target gene for HoxA9 and HoxA10 that was indirectly activated by Mll-Ell (an Mll1-fusion protein). Autocrine stimulation of Mll-Ell+ myeloid progenitor cells by Fgf2 stabilized βcatenin and increased expression of βcatenin target genes, including CDX4. Since HOXA9 and HOXA10 are Cdx4 target genes, Fgf2 indirectly augmented direct effects of Mll-Ell on these genes. ITGB3, encoding β3 integrin, is another HoxA10 target gene. In the current studies, we found activation of ITGB3 transcription in Mll-Ell+ myeloid progenitor cells via HoxA9 and HoxA10. Increased expression of αvβ3 integrin increased Syk-activation; contributing to cytokine hypersensitivity. However, inhibiting Fgf-R partly reversed αvβ3 activity in Mll-Ell+ progenitor cells by decreasing ITGB3 promoter activity in a βcatenin- and Cdx4-dependent manner. Inhibitors of Fgf-R or Syk impaired proliferation of CD34+ bone marrow cells from AML subjects with increased Hox-expression; with a greater combined effect. These studies identified a rational therapeutic approach to this AML subtype.

Discovery of Novel Dot1L Inhibitors through a Structure-Based Fragmentation Approach.

Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to repression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket binding mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morphing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors.

MLL1 and MLL1 fusion proteins have distinct functions in regulating leukemic transcription program.

Mixed lineage leukemia protein-1 (MLL1) has a critical role in human MLL1 rearranged leukemia (MLLr) and is a validated therapeutic target. However, its role in regulating global gene expression in MLLr cells, as well as its interplay with MLL1 fusion proteins remains unclear. Here we show that despite shared DNA-binding and cofactor interacting domains at the N terminus, MLL1 and MLL-AF9 are recruited to distinct chromatin regions and have divergent functions in regulating the leukemic transcription program. We demonstrate that MLL1, probably through C-terminal interaction with WDR5, is recruited to regulatory enhancers that are enriched for binding sites of E-twenty-six (ETS) family transcription factors, whereas MLL-AF9 binds to chromatin regions that have no H3K4me1 enrichment. Transcriptome-wide changes induced by different small molecule inhibitors also highlight the distinct functions of MLL1 and MLL-AF9. Taken together, our studies provide novel insights on how MLL1 and MLL fusion proteins contribute to leukemic gene expression, which have implications for developing effective therapies in the future.