Meis1 supports leukemogenesis through stimulation of ribosomal biogenesis and Myc.

The homeobox transcription factors HoxA9 and Meis1 are causally involved in the etiology of acute myeloid leukemia. While HoxA9 alone immortalizes cells, cooperation with Meis1 is necessary to induce a full leukemic phenotype. Here, we applied degron techniques to elucidate the leukemogenic contribution of Meis1. Chromatin immunoprecipitation experiments revealed that Meis1 localized mainly to H3K27 acetylated and H3K4 mono-methylated enhancers preactivated by HoxA9. Chromatin association of Meis1 required physical presence of HoxA9 and all Meis1 DNA interactions were rapidly lost after HoxA9 degradation. Meis1 controlled a gene expression pattern dominated by Myc, ribosome biogenesis and ribosomal RNA synthesis genes. While Myc accounted for the cell cycle stimulating effect of Meis1, overexpression of this oncogene alone did not accelerate leukemogenesis. Besides its effect on Myc, Meis1 induced transcription of ribosomal biogenesis genes. This was accompanied by an elevated resistance against inhibition of ribosomal RNA synthesis and translation, but without affecting steady-state protein synthesis. Finally, we demonstrate that HoxA9 and Meis1 proteins are stabilized by post-translational modification. Mutation of HoxA9/Meis1 phosphorylation sites or inhibition of casein kinase 2 lead to rapid protein degradation suggesting a potential pathway for pharmacological intervention.

The interaction of ENL with PAF1 mitigates polycomb silencing and facilitates murine leukemogenesis.

Eleven-nineteen leukemia (ENL) is a chromatin reader present in complexes stimulating transcriptional elongation. It is fused to mixed-lineage leukemia (MLL) in leukemia, and missense mutations have been identified in Wilms tumor and acute myeloid leukemia. Here we demonstrate that ENL overcomes polycomb silencing through recruitment of PAF1 via the conserved YEATS domain, which recognizes acetylated histone H3. PAF1 was responsible for antirepressive activities of ENL in vitro, and it determined the transforming potential of MLL-ENL. MLL-ENL target loci showed supraphysiological PAF1 binding, hyperubiquitination of histone H2B and hypomodification with H2AUb, resulting in accelerated transcription rates. YEATS mutations induced a gain of function, transforming primary hematopoietic cells in vitro and in transplantation assays through aberrant transcription and H2B ubiquitination of Hoxa9 and Meis1 Mechanistically, H3 and PAF1 competed for ENL interaction, with activating mutations favoring PAF1 binding, whereas the MLL moiety provided a constitutive PAF1 tether allowing MLL fusions to circumvent H3 competition.

Meningioma 1 is indispensable for mixed lineage leukemia-rearranged acute myeloid leukemia.

Mixed lineage leukemia (MLL/KMT2A) rearrangements (MLL-r) are one of the most frequent chromosomal aberrations in acute myeloid leukemia. We evaluated the function of Meningioma 1 (MN1), a co-factor of HOXA9 and MEIS1, in human and murine MLL-rearranged leukemia by CRISPR-Cas9 mediated deletion of MN1. MN1 was required for in vivo leukemogenicity of MLL positive murine and human leukemia cells. Loss of MN1 inhibited cell cycle and proliferation, promoted apoptosis and induced differentiation of MLL-rearranged cells. Expression analysis and chromatin immunoprecipitation with sequencing from previously reported data sets demonstrated that MN1 primarily maintains active transcription of HOXA9 and HOXA10, which are critical downstream genes of MLL, and their target genes like BCL2, MCL1 and Survivin. Treatment of MLL-rearranged primary leukemia cells with anti-MN1 siRNA significantly reduced their clonogenic potential in contrast to normal CD34+ hematopoietic progenitor cells, suggesting a therapeutic window for MN1 targeting. In summary, our findings demonstrate that MN1 plays an essential role in MLL fusion leukemias and serve as a therapeutic target in MLL-rearranged acute myeloid leukemia.

Pbx3 and Meis1 cooperate through multiple mechanisms to support Hox-induced murine leukemia.

Hox homeobox transcription factors drive leukemogenesis efficiently only in the presence of Meis or Pbx proteins. Here we show that Pbx3 and Meis1 need to dimerize to support Hox-induced leukemia and we analyze the molecular details of this cooperation. In the absence of Pbx3, Meis1 was highly unstable. As shown by a deletion analysis Meis1 degradation was contingent on a motif coinciding with the Pbx-binding domain. Either deletion of this sequence or binding to Pbx3 prolonged the half-life of Meis1 by preventing its ubiquitination. Meis1 break-down could also be blocked by inhibition of the ubiquitin proteasome system, indicating tight post-transcriptional control. In addition, Meis1 and Pbx3 cooperated genetically as overexpression of Pbx3 induced endogenous Meis1 transcription. These functional interactions translated into in vivo activity. Blocking Meis1/Pbx3 dimerization abrogated the ability to enhance proliferation and colony-forming cell numbers in primary cells transformed by Hoxa9. Furthermore, expression of Meis1 target genes Flt3 and Trib2 was dependent on Pbx3/Meis1 dimerization. This correlated with the requirement of Meis1 to bind Pbx3 in order to form high affinity DNA/Hoxa9/Meis1/Pbx3 complexes in vitro. Finally, kinetics and severity of disease in transplantation assays indicated that Pbx3/Meis1 dimers are rate-limiting factors for Hoxa9-induced leukemia.

MiR-495 is a tumor-suppressor microRNA down-regulated in MLL-rearranged leukemia.

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies with variable response to treatment. AMLs bearing MLL (mixed lineage leukemia) rearrangements are associated with intermediate or poor survival. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been postulated to be important gene expression regulators virtually in all biological processes, including leukemogenesis. Through a large-scale, genome-wide miRNA expression profiling assay of 85 human AML and 15 normal control samples, we show that among 48 miRNAs that are significantly differentially expressed between MLL- and non-MLL-rearranged AML samples, only one (miR-495) is expressed at a lower level in MLL-rearranged AML than in non-MLL-rearranged AML; meanwhile, miR-495 is also significantly down-regulated in MLL-rearranged AML samples compared with normal control samples. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we show that forced expression of miR-495 significantly inhibits MLL-fusion-mediated cell transformation in vitro and leukemogenesis in vivo. In human leukemic cells carrying MLL rearrangements, ectopic expression of miR-495 greatly inhibits cell viability and increases cell apoptosis. Furthermore, our studies demonstrate that PBX3 and MEIS1 are two direct target genes of miR-495, and forced expression of either of them can reverse the effects of miR-495 overexpression on inhibiting cell viability and promoting apoptosis of human MLL-rearranged leukemic cells. Thus, our data indicate that miR-495 likely functions as a tumor suppressor in AML with MLL rearrangements by targeting essential leukemia-related genes.

The homeodomain region controls the phenotype of HOX-induced murine leukemia.

HOX proteins are widely involved in hematopoietic development. These transcription factors combine a conserved DNA-binding homeobox with a divergent N-terminus that mediates interaction with variable cofactors. The resulting combinatorial diversity is thought to be responsible for mammalian HOX specificity. Contrasting this proposed mechanism for normal HOX function, here we demonstrate that, in the context of hematopoietic immortalization and leukemogenesis, individual HOX properties are governed almost exclusively by the homeodomain. Swap experiments between HOXA1 and HOXA9, 2 members of nonrelated paralog groups, revealed that gene expression patterns of HOX transformed cells in vitro are determined by the nature of the homeodomain. Similar results were seen in vivo during HOX-mediated leukemogenesis. An exchange of the homeodomains was sufficient to convert the slow, low-penetrance phenotype of HOXA1-induced leukemia to the aggressive fast-acting disease elicited by HOXA9 and vice versa. Mutation and deletion studies identified several subregions within the DNA binding domain responsible for paralog specificity. Previously defined binding sites for PBX cofactors within the exchangeable, nonhomeobox segment were dispensable for in vitro oncogenic HOX activity but affected in vivo disease development. The transcriptional activator domain shared by HOXA1 and HOXA9 at the very N-terminus proved essential for all transformation.

A C/ebpα isoform specific differentiation program in immortalized myelocytes.

The transcription factor CCAAT-enhancer binding factor alpha (C/ebpα) is a master controller of myeloid differentiation that is expressed as long (p42) and short (p30) isoform. Mutations within the CEBPA gene selectively deleting p42 are frequent in human acute myeloid leukemia. Here we investigated the individual genomics and transcriptomics of p42 and p30. Both proteins bound to identical sites across the genome. For most targets, they induced a highly similar transcriptional response with the exception of a few isoform specific genes. Amongst those we identified early growth response 1 (Egr1) and tribbles1 (Trib1) as key targets selectively induced by p42 that are also underrepresented in CEBPA-mutated AML. Egr1 executed a program of myeloid differentiation and growth arrest. Oppositely, Trib1 established a negative feedback loop through activation of Erk1/2 kinase thus placing differentiation under control of signaling. Unexpectedly, differentiation elicited either by removal of an oncogenic input or by G-CSF did not peruse C/ebpα as mediator but rather directly affected the cell cycle core by upregulation of p21/p27 inhibitors. This points to functions downstream of C/ebpα as intersection point where transforming and differentiation stimuli converge and this finding offers a new perspective for therapeutic intervention.

Leukemogenic transformation by HOXA cluster genes.

HOX homeobox genes are important regulators of normal and malignant hematopoiesis. Abdominal-type HOXA genes like HOXA9 are highly leukemogenic. However, little is known about transformation by anterior HOXA genes. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5, all HOXA genes caused a block or delay of hematopoietic differentiation and cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all active HOXA genes immortalized mixed granulocytic/monocytic populations, HOXA13 preferentially specified monocytoid development. The anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells, generating permanent cell lines, although they did so less potently than HOXA9. Upon transplantation these lines induced myeloproliferation and acute myeloid leukemia in recipient animals. Kinetic studies with inducible HOX derivatives demonstrated that anterior HOXA genes autonomously contributed to cellular transformation. This function was not mediated by endogenous Hoxa9, which was persistently expressed in cells transformed by anterior HOX genes. In summary our results demonstrate a hitherto unexpected role of anterior HOXA genes in hematopoietic malignancy.

Misguided transcriptional elongation causes mixed lineage leukemia.

Fusion proteins composed of the histone methyltransferase mixed-lineage leukemia (MLL) and a variety of unrelated fusion partners are highly leukemogenic. Despite their prevalence, particularly in pediatric acute leukemia, many molecular details of their transforming mechanism are unknown. Here, we provide mechanistic insight into the function of MLL fusions, demonstrating that they capture a transcriptional elongation complex that has been previously found associated with the eleven-nineteen leukemia protein (ENL). We show that this complex consists of a tight core stabilized by recursive protein-protein interactions. This central part integrates histone H3 lysine 79 methylation, RNA Polymerase II (RNA Pol II) phosphorylation, and MLL fusion partners to stimulate transcriptional elongation as evidenced by RNA tethering assays. Coimmunoprecipitations indicated that MLL fusions are incorporated into this complex, causing a constitutive recruitment of elongation activity to MLL target loci. Chromatin immunoprecipitations (ChIP) of the homeobox gene A cluster confirmed a close relationship between binding of MLL fusions and transcript levels. A time-resolved ChIP utilizing a conditional MLL fusion singled out H3K79 methylation as the primary parameter correlated with target expression. The presence of MLL fusion proteins also kept RNA Pol II in an actively elongating state and prevented accumulation of inhibitory histone methylation on target chromatin. Hox loci remained open and productive in the presence of MLL fusion activity even under conditions of forced differentiation. Finally, MLL-transformed cells were particularly sensitive to pharmacological inhibition of RNA Pol II phosphorylation, pointing to a potential treatment for MLL. In summary, we show aberrant transcriptional elongation as a novel mechanism for oncogenic transformation.

Dimerization of MLL fusion proteins immortalizes hematopoietic cells.

MLL fusion proteins are leukemogenic, but their mechanism is unclear. Induced dimerization of a truncated MLL immortalizes bone marrow and imposes a reversible block on myeloid differentiation associated with upregulation of Hox a7, a9, and Meis1. Both dimerized MLL and exon-duplicated MLL are potent transcriptional activators, suggesting a link between dimerization and partial tandem duplication of DNA binding domains of MLL. Dimerized MLL binds with higher affinity than undimerized MLL to a CpG island within the Hox a9 locus. However, MLL-AF9 is not dimerized in vivo. The data support a model in which either MLL dimerization/exon duplication or fusion to a transcriptional activator results in Hox gene upregulation and ultimately transformation.

Homing and invasiveness of MLL/ENL leukemic cells is regulated by MEF2C.

Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis, we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8), which induces a myeloproliferation in vivo. Among the targeted genes, we identified Mef2c, encoding a MCM1-agamous-deficiens-serum response factor transcription factor, and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly, several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions, prompting an investigation of the causal interplay. Using a conditional mouse strain, we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however, its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors, providing a mechanistic link to increased homing and motility. Thus, MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype.

Myeloid transformation by MLL-ENL depends strictly on C/EBP.

Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL-transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL-transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPß deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPß expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPß, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

MLL fusion proteins and transcriptional control.

The highly leukemogenic MLL fusion proteins have a unique mechanism of action. This review summarizes the current knowledge of how MLL fusions interact with the transcriptional machinery and it proposes a hypothesis how these proteins modify transcriptional control to act as transcriptional amplifiers causing runaway production of certain RNAs that transform hematopoietic cells.

Exploitable metabolic dependencies in MLL-ENL-induced leukemia.

Mixed-lineage leukemia (MLL) fusions are transcriptional activators that induce leukemia, with a dismal prognosis that mandates further elucidation of their transformation mechanism. In this study, knockdown of the direct MLL-ENL target gene polypyrimidine tract binding protein-1 (PTBP1) was rate limiting for cell proliferation and caused a metabolic phenotype associated with reduced glucose consumption and lactate production. This effect was accompanied by a reduction of splice isoform-2 of pyruvate kinase M (PKM2). Because PKM2 restricts glycolytic outflow to provide anabolic intermediates, we tested the consequences of glucose, energy, and Ser/Gly starvation for cell physiology. Administration of deoxyglucose, energetic decoupling with rotenone, and inhibition of Ser biosynthesis by CBR5884 had a significantly stronger influence on self-renewal and survival of transformed cells than on normal controls. In particular, inhibition of Ser synthesis, which branches off glycolysis caused accumulation of reactive oxygen species, DNA damage, and apoptosis, predominantly in leukemic cells. Depletion of exogenous Ser/Gly affected proliferation and self-renewal of murine and human leukemia samples, even though they are classified as nonessential amino acids. Response to Ser/Gly starvation correlated with glucose transport, but did not involve activation of the AMPK energy homeostasis system. Finally, survival times in transplantation experiments were significantly extended by feeding recipients a Ser/Gly-free diet. These results suggest selective starvation as an option for supportive leukemia treatment.

Hypomethylation and expression of BEX2, IGSF4 and TIMP3 indicative of MLL translocations in acute myeloid leukemia.

BACKGROUND: Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines. RESULTS: Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLLmu AML cells, while 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu AML cell lines. MLLwt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. CONCLUSION: These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.

The molecular biology of mixed lineage leukemia.

Mixed lineage leukemia is a very aggressive blood cancer that predominantly occurs in pediatric patients. In contrast to other types of childhood acute leukemias, mixed lineage leukemia presents with a dismal prognosis and despite the availability of advanced treatment methods cure rates have stagnated over the last years. Mixed lineage leukemia is characterized by the presence of MLL fusion proteins that are the result of chromosomal translocations affecting the MLL gene at 11q23. These events juxtapose the amino-terminus of the histone methyltransferase MLL with a variety of different fusion partners that destroy normal histone methyltransferase function of MLL and replace it by heterologous functions contributed by the fusion partner. The resulting chimeras are transcriptional regulators that take control of targets normally controlled by MLL with the clustered HOX homeobox genes as prominent examples. Recent studies suggested that MLL fusion partners activate transcription by two different mechanisms. Some of these proteins are themselves chromatin modifiers that introduce histone acetylation whereas other fusion partners can recruit histone methyltransferases. In particular, histone H3 specific methylation at lysine 79 catalyzed by DOT1L has been recognized as a hallmark of chromatin activated by MLL fusion proteins. Interestingly, several frequent MLL fusion partners seem to coordinate DOT1L activity with a protein complex that stimulates the elongation phase of transcription by phosphorylating the carboxy-terminal repeat domain of RNA polymerase II. The discovery of these novel enzymatic activities that are essentially involved in MLL fusion protein function presents potential new targets for a rational drug development.

Identification of protein interaction partners by the yeast two-hybrid system.

The two-hybrid system is a genetic method to search for and to identify direct interaction partners of a protein of interest. This method is instrumental to elucidate the transformation mechanism of several oncogenes that play a role in childhood leukaemia. With respect to mixed lineage leukaemia gene (MLL) fusions, two-hybrid screening was applied to discover proteins that bind to various MLL fusion partners. Here we describe a streamlined protocol that enables any average molecular biology laboratory to conduct and evaluate a standard two-hybrid screen. Starting with a general explanation of the biological background of the two-hybrid method, this chapter covers the construction of bait vectors and two comprehensive protocols for screening either by yeast mating or yeast transformation. In addition, it also gives guidelines for the evaluation of two-hybrid results.

Molecular pathology of mixed-lineage leukemia.

Genomic rearrangements of the locus 11q23 are frequently observed in aggressive acute leukemias with poor prognosis. These chromosomal aberrations fuse the mixed-lineage leukemia (MLL) gene to one of more than 50 partners. The resulting mixed-lineage leukemia fusions often code for chimeric transcriptional activators, which are able to transform normal hematopoietic cells through the deregulation of leukemogenic target genes. This review provides a concise overview about the known functions encoded in MLL and the respective fusion partners. Additionally, the roles of some target genes, as well as co-factors of mixed-lineage leukemia fusion proteins, are described with an emphasis on recent advances potentially uncovering novel therapeutic targets.

Interaction of MLL amino terminal sequences with menin is required for transformation.

Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. Both wild-type MLL and MLL fusion proteins interact with the tumor suppressor menin and with the Hoxa9 locus in vivo. Here, we show that MLL sequences between amino acids 5 and 44 are required for interaction with menin and for the transformation of hematopoietic progenitors. Blocking the MLL-menin interaction by the expression of a dominant negative inhibitor composed of amino terminal MLL sequences down-regulates Meis1 expression and inhibits cell proliferation, suggesting that targeting this interaction may be an effective therapeutic strategy for leukemias with MLL rearrangements.