Mixed Lineage Leukemia Fusion Research Articles

In vivo Leukemogenesis Model Using Retrovirus Transduction.

Various genetic alterations such as chromosomal translocation cause leukemia. For examples, gene rearrangements of the mixed-lineage leukemia (MLL) gene generate MLL fusion genes, whose products are potent oncogenic drivers in acute leukemia. To better understand the mechanism of disease onset, several murine leukemia models using retroviral gene transduction, xenograft, or Cre-mediated chromosomal translocation have been developed over the past twenty years. Particularly, a retroviral gene transduction-mediated murine leukemia model has been frequently used in the leukemia research field. Here, we describe the detailed protocol for this model.

Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate.The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML.In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs.The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells.To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells.Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo.Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation.Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. DisclosuresNo relevant conflicts of interest to declare.

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.

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.

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 PR01: A novel MLL-AF4 in vivo model phenocopies t(4;11) pro-B ALL and reveals a lymphoid lineage bias of the fusion protein in human cells

Abstract The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes in both acute myeloid and acute lymphoid leukemia (AML, ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently found in both AML and ALL, MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It accounts for 10-15% of ALL cases and confers a poor prognosis. Although many MLL-fusion leukemia models have been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Human hematopoietic CD34+ cells transduced with a modified MLL-AF4 fusion gene successfully initiate ALL in NSG mice with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNA-Seq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusion leukemia samples in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to lineage redirection in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11). This in vivo model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. This abstract is also presented as Poster A06. Citation Format: Shan Lin, Roger T. Luo, Mark Wunderlich, Joseph J. Kaberlein, Ahmad Rayes, John Anastasi, Maureen M. O'Brien, James C. Mulloy, Michael J. Thirman. A novel MLL-AF4 in vivo model phenocopies t(4;11) pro-B ALL and reveals a lymphoid lineage bias of the fusion protein in human cells. [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 PR01.

The CDK9 Inhibitor Dinaciclib Exerts Potent Apoptotic and Antitumor Effects in Preclinical Models of MLL-Rearranged Acute Myeloid Leukemia.

Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins.

PBX3 and MEIS1 Cooperate in Hematopoietic Cells to Drive Acute Myeloid Leukemias Characterized by a Core Transcriptome of the MLL-Rearranged Disease.

Overexpression of HOXA/MEIS1/PBX3 homeobox genes is the hallmark of mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). HOXA9 and MEIS1 are considered to be the most critical targets of MLL fusions and their coexpression rapidly induces AML. MEIS1 and PBX3 are not individually able to transform cells and were therefore hypothesized to function as cofactors of HOXA9. However, in this study, we demonstrate that coexpression of PBX3 and MEIS1 (PBX3/MEIS1), without ectopic expression of a HOX gene, is sufficient for transformation of normal mouse hematopoietic stem/progenitor cells in vitro. Moreover, PBX3/MEIS1 overexpression also caused AML in vivo, with a leukemic latency similar to that caused by forced expression of MLL-AF9, the most common form of MLL fusions. Furthermore, gene expression profiling of hematopoietic cells demonstrated that PBX3/MEIS1 overexpression, but not HOXA9/MEIS1, HOXA9/PBX3, or HOXA9 overexpression, recapitulated the MLL-fusion-mediated core transcriptome, particularly upregulation of the endogenous Hoxa genes. Disruption of the binding between MEIS1 and PBX3 diminished PBX3/MEIS1-mediated cell transformation and HOX gene upregulation. Collectively, our studies strongly implicate the PBX3/MEIS1 interaction as a driver of cell transformation and leukemogenesis, and suggest that this axis may play a critical role in the regulation of the core transcriptional programs activated in MLL-rearranged and HOX-overexpressing AML. Therefore, targeting the MEIS1/PBX3 interaction may represent a promising therapeutic strategy to treat these AML subtypes.

Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein-Protein Interaction between Menin and Mixed Lineage Leukemia (MLL).

Development of potent small molecule inhibitors of protein-protein interactions with optimized druglike properties represents a challenging task in lead optimization process. Here, we report synthesis and structure-based optimization of new thienopyrimidine class of compounds, which block the protein-protein interaction between menin and MLL fusion proteins that plays an important role in acute leukemias with MLL translocations. We performed simultaneous optimization of both activity and druglike properties through systematic exploration of substituents introduced to the indole ring of lead compound 1 (MI-136) to identify compounds suitable for in vivo studies in mice. This work resulted in the identification of compound 27 (MI-538), which showed significantly increased activity, selectivity, polarity, and pharmacokinetic profile over 1 and demonstrated a pronounced effect in a mouse model of MLL leukemia. This study, which reports detailed structure-activity and structure-property relationships for the menin-MLL inhibitors, demonstrates challenges in optimizing inhibitors of protein-protein interactions for potential therapeutic applications.

Comparison of MLL Fusion Genes Expression among the Cytogenetics Abnormalities of Acute Myeloid Leukemia and Their Clinical Effects

Mixed-lineage leukemia (MLL) is a subtype of acute myeloid leukemia with more aggressive prognosis than other subtypes. Translocations of MLL gene with other partner genes, forming the MLL-fusion proteins (MLL-FPs), are the main characteristics of MLL leukemia. Many studies have demonstrated that MLL-FPs such as: MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ENL, MLL-ELL, MLL-EPS15, as well as partial tandem duplication are the most common abnormalities that play significant role in MLL-rearranged leukemia. Gene expression profiles from 197 patients and 180 clinical data were downloaded from TCGA database. R statistical program has classified clinical and genomic data simultaneously according to cytogenetic abnormalities. As a result of this analysis, the most frequent 47 MLLFPs genes expression have been detected and compared with other cytogenetic abnormalities such as t(4;11), t(9;11), t(8;21), t(15;17), complex, inversion 16, trisomy 8 and cytogenetically normal AML. 35 out of 46 MLL-FPs genes presented with abnormal gene expression profile. This study showed that MLL-FPs are not just active and related with MLL, but also with other subtypes of AML.

Systematic Classification of Mixed-Lineage Leukemia Fusion Partners Predicts Additional Cancer Pathways.

Chromosomal translocations of the human mixed-lineage leukemia (MLL) gene have been analyzed for more than 20 yr at the molecular level. So far, we have collected about 80 direct MLL fusions (MLL-X alleles) and about 120 reciprocal MLL fusions (X-MLL alleles). The reason for the higher amount of reciprocal MLL fusions is that the excess is caused by 3-way translocations with known direct fusion partners. This review is aiming to propose a solution for an obvious problem, namely why so many and completely different MLL fusion alleles are always leading to the same leukemia phenotypes (ALL, AML, or MLL). This review is aiming to explain the molecular consequences of MLL translocations, and secondly, the contribution of the different fusion partners. A new hypothesis will be posed that can be used for future research, aiming to find new avenues for the treatment of this particular leukemia entity.

Lymphoid Lineage Preference of MLL-AF4 Is Revealed in a Species-Specific Model

The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes that drive both acute myeloid and acute lymphoid leukemia (AML and ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (also referred to as AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently identified in AML, for example MLL-AF9 caused by t(9;11)(p22;q23), MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It is the most frequent MLL fusion in ALL and accounts for 10-15% of ALL cases. Patients with t(4;11) have a poor prognosis compared with other cytogenetically defined subsets. Although many MLL-fusion leukemia models have successfully been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful human cell based model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Transduced with a modified MLL-AF4 fusion gene, human hematopoietic CD34+ cells successfully initiate ALL in a xenograft system with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells with MLL-AF4 results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNAseq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusions in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to reprogramming in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Strikingly, this decreased BMI1 expression is evident in primary B-ALL patient samples as well, with MLL-AF9 B-ALL samples demonstrating increased BMI1 relative to MLL-AF4. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11) oncogene and highlight the limitations of using murine cells in human disease modeling. The model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. DisclosuresThirman:AbbVie: Research Funding; Pharmacyclics: Research Funding; Gilead: Research Funding.

Drug Induced MLL Fusion Degradation Via Hsp90 Inhibition

The survival rate for infants is less than 50%. The fast majority of infant acute leukemias are characterized cytogenetically by balanced chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic therapies that degrade the driver oncogene are associated with loss of cancer cell self-renewal and excellent cure rates. Therefore, therapy that degrades the MLL fusion gene would offer new hope to these patients.Recently it was shown that the Drosophila Trithorax gene, an analogue of the human MLL, is degraded by Radicicol. Radicicol is a natural compound and a well-known Hsp90 inhibitor.Here, we show that Radicicol is able to induce a dose dependent degradation of the MLL-fusion protein in a panel of human MLL rearranged cell lines and in human cord blood-derived MLL-AF9 immortalised myeloid cells. This drug induced degradation of the MLL-fusion gene results in down-regulation of the expression of MLL target genes, including HOXA9, MEIS1 and c-MYB. Functionally, this results in a loss of self-renewal of the leukemic stem cells, as shown by methylcellulose colony forming assays.Radicicol proved ineffective and too toxic for in vivo use. One of the best tolerated Hsp90 inhibitors is Ganetespib. It is currently in phase II/III clinical trials.We extended our Radicicol data to Ganetespib. Like Radicicol, Ganetespib induces MLL-fusion protein degradation and downregulation of MLL target genes. Treatment of MLL rearranged leukemia with Ganetespib results in a loss of leukemic stem cell activity.In conclusion, we show how the inactivation of the MLL-fusion and down-regulation of MLL target genes results in a block of leukemic stem cell self- renewal. We will validate these findings in a pre-clinical in vivo model in the near future. DisclosuresNo relevant conflicts of interest to declare.

The Molecular Mechanism of Transcriptional Activation By MLL-AEP Fusion Proteins

Chromosomal translocations generate a variety of mixed lineage leukemia (MLL) fusion genes, which cause aggressive leukemia. Although >70 different fusion partners have been identified, the majority of the cases are caused by the chimeric genes of MLL and a component of the AEP co-activator complex (hereafter referred to as AEP), which comprises of AF4 family proteins (e.g. AF4, AF5Q31), ENL family proteins (e.g. ENL, AF9), and the P-TEFb elongation factor. MLL-AEP fusion proteins constitutively activate their target genes by recruiting AEP components to their target chromatin, whereas wild-type MLL recruits AEP in a context-dependent manner. In the hematopoietic lineage, MLL fusion proteins aberrantly activate a subset of genes implicated in the hematopoietic stem cell (HSC) program, such as HOXA9 and MEIS1. Constitutive expression of these HSC program genes in hematopoietic progenitors has been shown to induce leukemia in a mouse model. It has been speculated that MLL-AEP activates transcription of those HSC program genes by aberrantly activating transcription elongation. However, it is largely unclear how AEP activates transcription.Using an extensive structure/function analysis, we revealed that a serine-rich domain of the AF4 family proteins, termed pSER, is an essential functional component of MLL-AEP-dependent gene activation and leukemic transformation. Through biochemical purification, we have identified Selectivity Factor 1 (SL1) as a novel factor associated with the pSER domain. SL1 comprises TBP and four TBP-associated factors (TAF1A, TAF1B, TAF1C, TAF1D), and is known as a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). In the presence of UBF, SL1 forms a PIC on the promoters of ribosomal RNA genes, to drive RNAP1-dependent transcription. However, its role in RNAP2-dependent transcription was unknown. The initiation of RNAP2-dependent transcription in eukaryotes occurs through the loading of TBP to the promoter, via a direct association with the TATA element or through as-yet-unidentified mechanisms. Our results demonstrate that AEP facilitates the initiation of RNAP2-dependent transcription via the loading of TBP onto the TATA element, through SL1 activity. MLL-AEP fusion proteins utilize this TBP-loading function to activate transcription initiation in leukemic transformation. The wild-type AEP complex activates gene expression in the same manner in the physiological conditions. Taken together, our results unveil a novel role of SL1 as a TBP-loading factor in RNAP2-dependent gene activation, and a previously unknown transcription initiation mechanism involving AEP, which is more important than its transcription elongation activities for leukemic transformation. These findings greatly advance our understanding of the molecular mechanism of MLL fusion-dependent leukemic transformation, which was previously interpreted simply as mis-regulated transcription elongation. DisclosuresYokoyama:Dainipon Sumitomo Pharma Co., Ltd.: Research Funding.

S6K1 Regulates Self-Renewal of Leukemia Initiating Cells and Normal Hematopoietic Stem Cells

The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult hematopoietic stem cells (HSCs) and regulation of progenitor population. Translocations in MLL have been detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. Various genes, including AF4, AF5, AF9, ELL and ENL have been identified as partners for translocation in MLL-rearranged leukemia. In hematopoietic cells, expression of MLL fusion proteins result in a block of differentiation. AML patients who successfully undergo treatment but relapse suggest the importance of targeting leukemia initiating cells (LICs) within the MLL leukemia. LICs remain in a quiescent state and are capable of survival despite treatment with chemotherapeutic agents or targeted molecular inhibitors. In mouse models, LICs are defined by the capability of successfully propagating disease upon serial transplantation. In order to prevent disease relapse, identification of molecules, which regulate the self-renewal of LICs, is essential.The phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway is a key regulator of self-renewal of both HSCs and LICs. Deletion of Raptor, a subunit of mTORC1, does not affect initiation and progression of acute myeloid leukemia (AML), but Raptor deficiency results in delayed propagation of AML. Upon its activation, mTORC1 phosphorylates and activates p70 ribosomal protein S6 kinase (S6K1) and inhibits the activity of eukaryote translation initiation factor 4E binding protein 1 (4E-BP1). S6K1 has been shown to be hyperactivated in hematopoietic cells expressing oncogenic MLL-AF9 fusion protein.In our study, we have assessed the role of S6K1 in the initiation, progression and propagation of AML using a genetic model of S6K1 knockout mice (S6K1-/-). We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and transplanted them into lethally irradiated recipients. Recipients of both WT and S6K1-/- HSC/Ps bearing MLL-AF9 displayed high white blood cell (WBC) count, splenomegaly and developed AML. There was no difference in survival between the WT and S6K1-/- recipients. In order to determine whether S6K1 regulates the self-renewal of LICs, we transplanted lethally irradiated mice with cells from WT and S6K1-/- primary recipients who developed AML. Recipients of S6K1 deficient AML cells survived significantly longer compared to controls (n=17/group, p<0.001). S6K1 deficient HSC/Ps expressing MLL-AF9 showed reduced activation of Akt as well as decreased mTORC1 activity, suggesting that deletion of S6K1 results in reduced activation of PI-3K-Akt-mTORC1 pathway both upstream and downstream of mTORC1 which indicates that S6K1 might be involve in a feedback loop within this pathway.To determine the role of S6K1 in normal HSC development and maintenance, we analyzed bone marrow derived HSCs in WT and S6K1-/- mice. S6K1 deficiency did not alter the frequency of long term HSCs (LT-HSCs) as defined by CD150+ CD48- Lin- Sca1+ c-Kit+ surface markers, but the absolute number of LT-HSCs were significantly reduced in S6K1 deficient mice (p<0.02). The absolute number of multipotent progenitors (MPPs) (p<0.001), common myeloid progenitors (CMPs) (p<0.01) and megakaryocyte-erythroid progenitors (MEPs) (p<0.01) were also significantly reduced in S6K1 deficient mice. Deficiency of S6K1 resulted in reduced quiescence of LT-HSCs (p<0.05). Expression level of p21, an inhibitor of cell cycle progression, was significantly decreased in LT-HSCs derived from S6K1-/- mice compared to LT-HSCs from control group. To study the role of S6K1 in HSCs' function, we performed competitive repopulation assay. Sorted LT-HSCs from bone marrow cells derived from either WT or S6K1-/- mice were transplanted with competitor cells into lethally irradiated recipients. S6K1 deficient LT-HSCs displayed reduced repopulating ability in secondary recipients (n=9-10/group, p<0.001). Expression level of p21 was downregulated in donor-derived HSCs isolated from secondary recipients of S6K1 deficient HSCs compared to control. Overall, our study establishes S6K1 as a critical regulator of self-renewal of both LICs and HSCs. Deficiency of S6K1 in AML cells results in delayed propagation of disease and deficiency of S6K1 in HSCs results in decreased self-renewal potential. DisclosuresNo relevant conflicts of interest to declare.

DNMT3A mutations mediate the epigenetic reactivation of the leukemogenic factor MEIS1 in acute myeloid leukemia.

Close to half of de novo acute myeloid leukemia (AML) cases do not exhibit any cytogenetic aberrations. In this regard, distortion of the DNA methylation setting and the presence of mutations in epigenetic modifier genes can also be molecular drivers of the disease. In recent years, somatic missense mutations of the DNA methyltransferase 3A (DNMT3A) have been reported in ~20% of AML patients; however, no obvious critical downstream gene has been identified that could explain the role of DNMT3A in the natural history of AML. Herein, using whole-genome bisulfite sequencing and DNA methylation microarrays, we have identified a key gene undergoing promoter hypomethylation-associated transcriptional reactivation in DNMT3 mutant patients, the leukemogenic HOX cofactor MEIS1. Our results indicate that, in the absence of mixed lineage leukemia fusions, an alternative pathway for engaging an oncogenic MEIS1-dependent transcriptional program can be mediated by DNMT3A mutations.

Recent progress in the treatment of infant acute lymphoblastic leukemia.

Treatment of infants with acute lymphoblastic leukemia (ALL), especially those with mixed lineage leukemia (MLL) rearrangement (MLL-r), which account for approximately 80% of cases, is still a major challenge for pediatric hematologists and oncologists worldwide. Continuing efforts by collaborative clinical study groups in Europe, North America, and Japan have rescued approximately half of the MLL-r ALL patients with intensive chemotherapy with or without allogeneic hematopoietic stem cell transplantation. Recent progress has clarified the unique mechanism of MLL-r ALL: the aberrant methylation and histone modifications via DOT1L and other related molecules by MLL fusion proteins lead to leukemogenetic gene expression, thus to overt leukemia. In order to overcome this dismal subtype of ALL, novel targeted therapy based on leukemia biology is urgently needed. Due to the extreme rarity of the disease, collaboration between the study groups in Europe (Interfant), North America (Children's Oncology Group), and Japan (Japanese Pediatric Leukemia/Lymphoma Study Group) is under way.

The Zinc Finger Transcription Factor Mds1‐Evi1 Forms a Novel Protein Complex in MLL leukemia

The dual transcription factor‐methyltransferase Mds1‐Evi1 is frequently overexpressed in high‐risk leukemias. Mds1‐Evi1 contributes to leukemias induced by the mixed lineage leukemia (MLL) fusion protein MLL‐AF9, although the mechanism is unknown. MLL contains chromatin‐targeting and histone lysine methyltransferase domains, and fusion to AF9 can cause aberrant protein recruitment to target genes. Although Mds1‐Evi1 contains a methyltransferase domain, it does not appear to act on histones. Therefore, we are interested in how protein‐protein interactions involving Mds1‐Evi1 contribute to leukemogenesis. Many known interactors of MLL and Mds1‐Evi1 include chromatin modifying enzymes, suggesting that coordination between epigenetic modulators is key in leukemia pathogenesis.Here, we report the biochemical purification of a novel Mds1‐Evi1‐containing protein complex using a unique murine model of leukemia. To isolate the complex, we induced acute MLL‐AF9 leukemia in mice containing a tandem affinity purification (TAP) tag knocked into the endogenous Mds1‐Evi1 gene. Leukemic spleen tissue from these mice was used to purify the TAP‐tagged protein complex. Proteomic analysis revealed several members of the SWI/SNF chromatin remodeling complex, which is involved in epigenetic control of a variety of gene programs. Association between Mds1‐Evi1 and SWI/SNF may allow the activation of cell‐cycle and differentiation programs, contributing to transformation. SWI/SNF also coordinates with MLL fusion proteins to activate the Hox family of leukemia oncogenes. Our results provide a link between Mds1‐Evi1 and the chromatin remodeling machinery, where abnormal coordination of Mds1‐Evi1, MLL‐AF9 and SWI/SNF may lead to gene activation and the development of leukemia.

Molecular mechanisms of MLL-associated leukemia.

Gene rearrangements of the mixed lineage leukemia (MLL) gene cause aggressive leukemia. The fusion of MLL and its partner genes generates various MLL fusion genes, and their gene products trigger aberrant self-renewal of hematopoietic progenitors leading to leukemia. Since the identification of the MLL gene two decades ago, a substantial amount of information has been obtained regarding the mechanisms by which MLL mutations cause leukemia. Wild-type MLL maintains the expression of Homeobox (HOX) genes during development. MLL activates the expression of posterior HOX-A genes in the hematopoietic lineage to stimulate the expansion of immature progenitors. MLL fusion proteins constitutively activate the HOX genes, causing aberrant self-renewal. The modes of transcriptional activation vary depending on the fusion partners and can be categorized into at least four groups. Here I review the recent progress in research related to the molecular mechanisms of MLL fusion-dependent leukemogenesis.