MLLT3

Gene Summary

Gene:MLLT3; myeloid/lymphoid or mixed-lineage leukemia; translocated to, 3
Aliases: AF9, YEATS3
Location:9p22
Summary:-
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:protein AF-9
HPRD
Source:NCBIAccessed: 07 August, 2015

Ontology:

What does this gene/protein do?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 07 August 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 07 August, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: MLLT3 (cancer-related)

Roychoudhury J, Clark JP, Gracia-Maldonado G, et al.
MEIS1 regulates an HLF-oxidative stress axis in MLL-fusion gene leukemia.
Blood. 2015; 125(16):2544-52 [PubMed] Article available free on PMC after 16/04/2016 Related Publications
Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.

Aoki Y, Watanabe T, Saito Y, et al.
Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia.
Blood. 2015; 125(6):967-80 [PubMed] Article available free on PMC after 16/04/2016 Related Publications
Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34(+)CD38(+)CD19(+) and CD34(-)CD19(+) cells initiated leukemia, and in MLL-AF9 patients, CD34(-)CD19(+) cells were LICs. In MLL-ENL patients, either CD34(+) or CD34(-) cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34(+)CD38(-)CD19(-)CD33(-) cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34(+)CD38(-)CD19(-)CD33(-) cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4(+) single positive (SP), CD8(+) SP, and CD4(+)CD8(+) double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34(+)CD38(+) and CD34(-) LICs but not in CD34(+)CD38(-)CD19(-)CD33(-) HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.

Zhang Y, Peng L, Hu T, et al.
La-related protein 4B maintains murine MLL-AF9 leukemia stem cell self-renewal by regulating cell cycle progression.
Exp Hematol. 2015; 43(4):309-18.e2 [PubMed] Related Publications
Our recent study identified a nonsense mutation of La-related protein 4B (LARP4B) from whole genome sequencing of a 3-year-old female monozygotic twin pair discordant for MLL-associated acute myeloid leukemia (AML). To study the role of LARP4B in AML, we established a LARP4B-knockdown MLL-AF9 AML mouse model. Using this mouse model, we found that LARP4B knockdown significantly decreased leukemia cells in the peripheral blood, spleen, and bone marrow and prolonged the survival of AML recipient mice. Additional studies showed that LARP4B knockdown reduced leukemia stem cells (LSCs) and impaired the self-renew capacity of LSCs. Cell cycle analysis revealed that LARP4B knockdown arrested more LSCs in the G0 phase. The transcription of the cell cycle inhibitors p16, p19, and p21 and of the lineage-specific transcription factor CCAAT-enhancer-binding protein α was increased in the LARP4B-knockdown LSCs. Thus, our results demonstrate that LARP4B plays an important role in the maintenance of LSCs and suggest that LARP4B may regulate the cell cycle of LSCs via suppressing the expression of the cell cycle inhibitors p16, p19, and p21 and the myeloid specific transcription factor CCAAT-enhancer-binding protein α.

Sánchez-Aguilera A, Arranz L, Martín-Pérez D, et al.
Estrogen signaling selectively induces apoptosis of hematopoietic progenitors and myeloid neoplasms without harming steady-state hematopoiesis.
Cell Stem Cell. 2014; 15(6):791-804 [PubMed] Related Publications
Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2(V617F)-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2(V617F+) HSPCs in a xenograft model, and sensitized MLL-AF9(+) leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs.

Deshpande AJ, Deshpande A, Sinha AU, et al.
AF10 regulates progressive H3K79 methylation and HOX gene expression in diverse AML subtypes.
Cancer Cell. 2014; 26(6):896-908 [PubMed] Article available free on PMC after 08/12/2015 Related Publications
Homeotic (HOX) genes are dysregulated in multiple malignancies, including several AML subtypes. We demonstrate that H3K79 dimethylation (H3K79me2) is converted to monomethylation (H3K79me1) at HOX loci as hematopoietic cells mature, thus coinciding with a decrease in HOX gene expression. We show that H3K79 methyltransferase activity as well as H3K79me1-to-H3K79me2 conversion is regulated by the DOT1L cofactor AF10. AF10 inactivation reverses leukemia-associated epigenetic profiles, precludes abnormal HOXA gene expression, and impairs the transforming ability of MLL-AF9, MLL-AF6, and NUP98-NSD1 fusions-mechanistically distinct HOX-activating oncogenes. Furthermore, NUP98-NSD1-transformed cells are sensitive to small-molecule inhibition of DOT1L. Our findings demonstrate that pharmacological inhibition of the DOT1L/AF10 complex may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression.

Willer A, Jakobsen JS, Ohlsson E, et al.
TGIF1 is a negative regulator of MLL-rearranged acute myeloid leukemia.
Leukemia. 2015; 29(5):1018-31 [PubMed] Related Publications
Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general importance of this family in malignant hematopoiesis, we therefore tested the potential function of TGIF1 in the maintenance of MLL-rearranged AML. Gene expression analysis of MLL-rearranged patient blasts demonstrated reduced TGIF1 levels, and, in accordance, we find that forced expression of TGIF1 in MLL-AF9-transformed cells promoted differentiation and cell cycle exit in vitro, and delayed leukemic onset in vivo. Mechanistically, we show that TGIF1 interferes with a MEIS1-dependent transcriptional program by associating with MEIS1-bound regions in a competitive manner and that the MEIS1:TGIF1 ratio influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.

Sandhöfer N, Metzeler KH, Rothenberg M, et al.
Dual PI3K/mTOR inhibition shows antileukemic activity in MLL-rearranged acute myeloid leukemia.
Leukemia. 2015; 29(4):828-38 [PubMed] Related Publications
In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.

Sachs Z, LaRue RS, Nguyen HT, et al.
NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia.
Blood. 2014; 124(22):3274-83 [PubMed] Article available free on PMC after 20/11/2015 Related Publications
Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

Lokken AA, Achille NJ, Chang MJ, et al.
Importance of a specific amino acid pairing for murine MLL leukemias driven by MLLT1/3 or AFF1/4.
Leuk Res. 2014; 38(11):1309-15 [PubMed] Article available free on PMC after 01/11/2015 Related Publications
Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.

Kato T, Sakata-Yanagimoto M, Nishikii H, et al.
Hes1 suppresses acute myeloid leukemia development through FLT3 repression.
Leukemia. 2015; 29(3):576-85 [PubMed] Related Publications
In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Cermáková K, Tesina P, Demeulemeester J, et al.
Validation and structural characterization of the LEDGF/p75-MLL interface as a new target for the treatment of MLL-dependent leukemia.
Cancer Res. 2014; 74(18):5139-51 [PubMed] Related Publications
Mixed lineage leukemia (MLL) fusion-driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium-derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75-MLL-MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/p75-MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors-HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL-AF9(+) leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9-expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75-HIV-1 integrase interaction. The newly defined protein-protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75-dependent MLL fusion-driven leukemic disorders. Cancer Res; 74(18); 5139-51. ©2014 AACR.

Sasca D, Hähnel PS, Szybinski J, et al.
SIRT1 prevents genotoxic stress-induced p53 activation in acute myeloid leukemia.
Blood. 2014; 124(1):121-33 [PubMed] Related Publications
SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)(+)-cells protein, expression of SIRT1 is regulated by FLT3 kinase activity. In addition, SIRT1 function is modulated via the ATM-DBC1-SIRT1 axis in a FLT3-ITD-dependent manner. In murine leukemia models driven by MLL-AF9 or AML1-ETO coexpressing FLT3-ITD, SIRT1 acts as a safeguard to counteract oncogene-induced stress, and leukemic blasts become dependent on SIRT1 activity. Pharmacologic targeting or RNAi-mediated knockdown of SIRT1 inhibited cell growth and sensitized AML cells to tyrosine kinase inhibitor treatment and chemotherapy. This effect was a result of the restoration of p53 activity. Our data suggest that targeting SIRT1 represents an attractive therapeutic strategy to overcome primary resistance in defined subsets of patients with AML.

Miyachi M, Watanabe E, Watanabe N, et al.
MRD detection of leukemia relapse using HLA typing by FACS in combination with FISH after mismatched allogeneic stem cell transplantation.
Pediatr Transplant. 2014; 18(5):E180-4 [PubMed] Related Publications
Loss of mismatched HLA is a cause of relapse following HLA-mismatched allo-SCT. We directly detected the loss of mismatched HLA alleles in relapsed leukemic cells at a MRD level using HLA typing by multicolor FACS (HLA-Flow) in combination with FISH in the BM of two patients with MLL-AF9-positive AML, at 6 and 10 months after mismatched allo-SCT. HLA-Flow with FISH analysis detected relapsed leukemic cells not expressing a mismatched HLA allele and harboring the MLL rearrangement. Simultaneously, real-time quantitative RT-PCR detected a low copy number of MLL-AF9 transcripts, consistent with MRD detection. HLA-Flow with FISH is a powerful method for detecting molecular relapse after mismatched allo-SCT and provides important information on the HLA expression status of the relapsed leukemic cells to help determine the next intervention.

Sakamoto K, Imamura T, Yano M, et al.
Sensitivity of MLL-rearranged AML cells to all-trans retinoic acid is associated with the level of H3K4me2 in the RARα promoter region.
Blood Cancer J. 2014; 4:e205 [PubMed] Article available free on PMC after 01/11/2015 Related Publications
All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML-RARα (promyelocytic leukemia-retinoic acid receptor α) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RARα, C/EBPα, C/EBPɛ and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RARα gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RARα gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML.

Placke T, Faber K, Nonami A, et al.
Requirement for CDK6 in MLL-rearranged acute myeloid leukemia.
Blood. 2014; 124(1):13-23 [PubMed] Article available free on PMC after 01/11/2015 Related Publications
Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts.

Bäumer N, Krause A, Köhler G, et al.
Proteinase-Activated Receptor 1 (PAR1) regulates leukemic stem cell functions.
PLoS One. 2014; 9(4):e94993 [PubMed] Article available free on PMC after 01/11/2015 Related Publications
External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.

Jude JG, Spencer GJ, Huang X, et al.
A targeted knockdown screen of genes coding for phosphoinositide modulators identifies PIP4K2A as required for acute myeloid leukemia cell proliferation and survival.
Oncogene. 2015; 34(10):1253-62 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
Given the importance of deregulated phosphoinositide (PI) signaling in leukemic hematopoiesis, genes coding for proteins that regulate PI metabolism may have significant and as yet unappreciated roles in leukemia. We performed a targeted knockdown (KD) screen of PI modulator genes in human acute myeloid leukemia (AML) cells and identified candidates required to sustain proliferation or prevent apoptosis. One of these, the lipid kinase phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A) regulates cellular levels of phosphatidylinositol-5-phosphate (PtsIns5P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). We found PIP4K2A to be essential for the clonogenic and leukemia-initiating potential of human AML cells, and for the clonogenic potential of murine MLL-AF9 AML cells. Importantly, PIP4K2A is also required for the clonogenic potential of primary human AML cells. Its KD results in accumulation of the cyclin-dependent kinase inhibitors CDKN1A and CDKN1B, G₁ cell cycle arrest and apoptosis. Both CDKN1A accumulation and apoptosis were partially dependent on activation of the mTOR pathway. Critically, however, PIP4K2A KD in normal hematopoietic stem and progenitor cells, both murine and human, did not adversely impact either clonogenic or multilineage differentiation potential, indicating a selective dependency that we suggest may be the consequence of the regulation of different transcriptional programs in normal versus malignant cells. Thus, PIP4K2A is a novel candidate therapeutic target in myeloid malignancy.

Pastorczak A, Szczepanski T, Trelinska J, et al.
Secondary acute monocytic leukemia positive for 11q23 rearrangement in Nijmegen breakage syndrome.
Pediatr Blood Cancer. 2014; 61(8):1469-71 [PubMed] Related Publications
Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability disorder characterized by a high incidence of pediatric hematologic malignancies. Majority of patients affected are of Slavic origin and share the same founder mutation of 657del5 within the NBN gene encoding protein involved in DNA double-strand breaks (DSB) repair. We report a case of a pediatric patient with NBS, who developed t(9;11)/AF9-MLL-positive AML as a second malignancy after successful treatment of T-NHL. The coexistence of NBN and MLL mutations suggests that the profound dysfunction of NBN may promote alterations of MLL that is mediated by error-prone non-homologous end joining pathway particularly in patients treated with DNA topoisomerase II inhibitors.

Lim JH, Jang S, Park CJ, et al.
FISH analysis of MLL gene rearrangements: detection of the concurrent loss or gain of the 3' signal and its prognostic significance.
Int J Lab Hematol. 2014; 36(5):571-9 [PubMed] Related Publications
INTRODUCTION: The rearrangement of the mixed-lineage leukemia (MLL) gene occurs through translocations and insertions involving a variety of partner chromosome genes. However, there are few studies on aberrant MLL signal patterns such as concurrent 3' MLL deletion.
METHODS: A total of 84 patients with acute leukemia (AL) who had MLL rearrangements detected by florescence in situ hybridization (FISH) were enrolled in the study. The distribution of MLL fusion partner genes was analyzed, and aberrant MLL signals were evaluated.
RESULTS: Seventy-seven (91.7%) patients had MLL rearrangements, involving previously described translocation partner genes (TPGs). Among these TPGs, the frequencies of MLLT3, AFF1, MLLT4, and ELL were 29.8%, 17.9%, 15.5%, and 13.1%, respectively. A high frequency of MLLT4 in our study was due to the high proportion of acute myeloid leukemia cases in pediatric and adult patients. Aberrant MLL signals were found in 18 patients: 11 (61.1%) with 3' MLL signal loss and 7 with 3' MLL signal gain. All cases with 3' MLL signal gain were due to an extra derivative partner chromosome. The median overall survival period of patients with 3' MLL gain was shorter than that in patients without aberrant MLL signal patterns.
CONCLUSION: Aberrant MLL signals were frequently detected by FISH analysis. The 3' MLL gain was associated with poor prognosis in patients with AL. Therefore, it is important to detect aberrant MLL signal patterns using FISH analysis.

Pattabiraman DR, McGirr C, Shakhbazov K, et al.
Interaction of c-Myb with p300 is required for the induction of acute myeloid leukemia (AML) by human AML oncogenes.
Blood. 2014; 123(17):2682-90 [PubMed] Related Publications
The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interaction of c-Myb with the coactivator CBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore the molecular mechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction in myeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia.

Emerenciano M, Barbosa TC, Lopes BA, et al.
ARID5B polymorphism confers an increased risk to acquire specific MLL rearrangements in early childhood leukemia.
BMC Cancer. 2014; 14:127 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
BACKGROUND: Acute leukemia in early age (EAL) is characterized by acquired genetic alterations such as MLL rearrangements (MLL-r). The aim of this case-controlled study was to investigate whether single nucleotide polymorphisms (SNPs) of IKZF1, ARID5B, and CEBPE could be related to the onset of EAL cases (<24 months-old at diagnosis).
METHODS: The SNPs (IKZF1 rs11978267, ARID5B rs10821936 and rs10994982, CEBPE rs2239633) were genotyped in 265 cases [169 acute lymphoblastic leukemia (ALL) and 96 acute myeloid leukaemia (AML)] and 505 controls by Taqman allelic discrimination assay. Logistic regression was used to evaluate the association between SNPs of cases and controls, adjusted on skin color and/or age. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI).
RESULTS: Children with the IKZF1 SNP had an increased risk of developing MLL-germline ALL in white children. The heterozygous/mutant genotype in ARID5B rs10994982 significantly increased the risk for MLL-germline leukemia in white and non-white children (OR 2.60, 95% CI: 1.09-6.18 and OR 3.55, 95% CI: 1.57-8.68, respectively). The heterozygous genotype in ARID5B rs10821936 increased the risk for MLL-r leukemia in both white and non-white (OR 2.06, 95% CI: 1.12-3.79 and OR 2.36, 95% CI: 1.09-5.10, respectively). Furthermore, ARID5B rs10821936 conferred increased risk for MLL-MLLT3 positive cases (OR 7.10, 95% CI:1.54-32.68). Our data do not show evidence that CEBPE rs2239633 confers increased genetic susceptibility to EAL.
CONCLUSIONS: IKZF1 and CEBPE variants seem to play a minor role in genetic susceptibility to EAL, while ARID5B rs10821936 increased the risk of MLL-MLLT3. This result shows that genetic susceptibility could be associated with the differences regarding MLL breakpoints and partner genes.

Fleischmann KK, Pagel P, Schmid I, Roscher AA
RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes.
Mol Cancer. 2014; 13:27 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
BACKGROUND: The translocation t(9;11)(p22;q23) leading to the leukemogenic fusion gene MLL-AF9 is a frequent translocation in infant acute myeloid leukemia (AML). This study aimed to identify genes and molecular processes downstream of MLL-AF9 (alias MLL-MLLT3) which could assist to develop new targeted therapies for such leukemia with unfavorable prognosis.
METHODS: In the AML cell line THP1 which harbors this t(9;11) translocation, endogenous MLL-AF9 was silenced via siRNA while ensuring specificity of the knockdown and its efficiency on functional protein level.
RESULTS: The differential gene expression profile was validated for leukemia-association by gene set enrichment analysis of published gene sets from patient studies and MLL-AF9 overexpression studies and revealed 425 differentially expressed genes. Gene ontology analysis was consistent with a more differentiated state of MLL-AF9 depleted cells, with involvement of a wide range of downstream transcriptional regulators and with defined functional processes such as ribosomal biogenesis, chaperone binding, calcium homeostasis and estrogen response. We prioritized 41 gene products as candidate targets including several novel and potentially druggable effectors of MLL-AF9 (AHR, ATP2B2, DRD5, HIPK2, PARP8, ROR2 and TAS1R3). Applying the antagonist SCH39166 against the dopamine receptor DRD5 resulted in reduced leukemic cell characteristics of THP1 cells.
CONCLUSION: Besides potential new therapeutic targets, the described transcription profile shaped by MLL-AF9 provides an information source into the molecular processes altered in MLL aberrant leukemia.

Sroczynska P, Cruickshank VA, Bukowski JP, et al.
shRNA screening identifies JMJD1C as being required for leukemia maintenance.
Blood. 2014; 123(12):1870-82 [PubMed] Related Publications
Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.

Zhao X, Chen A, Yan X, et al.
Downregulation of RUNX1/CBFβ by MLL fusion proteins enhances hematopoietic stem cell self-renewal.
Blood. 2014; 123(11):1729-38 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9-mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs.

Kamezaki K, Luchsinger LL, Snoeck HW
Differential requirement for wild-type Flt3 in leukemia initiation among mouse models of human leukemia.
Exp Hematol. 2014; 42(3):192-203.e1 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
FLT3 is one of the most frequently mutated genes in acute leukemias. However, the role in leukemogenesis of wild-type (wt) FLT3, which is highly expressed in many hematologic malignancies, is unclear. We show here that in mouse models established by retroviral transduction of leukemic fusion proteins, deletion of Flt3 strongly inhibits MLL-ENL and to lesser extent p210(BCR-ABL)-induced leukemogenesis, but has no effect in MLL-AF9 or AML1-ETO9a models. Flt3 acts at the level of leukemic stem cells (LSCs), as a fraction of LSCs in MLL-ENL, but not in MLL-AF9-induced leukemia, expressed Flt3 in vivo, and Flt3 expression on LSCs was associated with leukemia development in this model. Furthermore, efficiency of MLL-ENL, but not of MLL-AF9-induced leukemia induction was significantly enhanced after transduction of Flt3(+) compared to Flt3(-) wt myeloid progenitors. However, Flt3 is not required for immortalization of bone marrow cells in vitro by MLL-ENL and does not affect colony formation by MLL-ENL LSCs in vitro, suggesting that in vitro models do not reflect the in vivo biology of MLL-ENL leukemia with respect to Flt3 requirement. We conclude that wt Flt3 plays a role in leukemia initiation in vivo, which is, however, not universal.

Kiyokawa N, Iijima K, Tomita O, et al.
Significance of CD66c expression in childhood acute lymphoblastic leukemia.
Leuk Res. 2014; 38(1):42-8 [PubMed] Related Publications
Upon analyzing 696 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, we identified the characteristics of CD66c expression. In addition to the confirmation of strong correlation with BCR-ABL positivity and hyperdiploid, we further observed that CD66c is frequently expressed in CRLF2-positive (11/15, p<0.01 against chimeric gene-negative) as well as hypodiploid cases (3/4), whereas it is never expressed in ETV6-RUNX1, MLL-AF4, MLL-AF9, MLL-ENL, and E2A-PBX1-positive cases. Although the expression of CD66c itself is not directly linked to the prognosis, the accompanying genetic abnormalities are important prognostic factors for BCP-ALL, indicating the importance of CD66c expression in the initial diagnosis of BCP-ALL.

Huang X, Spencer GJ, Lynch JT, et al.
Enhancers of Polycomb EPC1 and EPC2 sustain the oncogenic potential of MLL leukemia stem cells.
Leukemia. 2014; 28(5):1081-91 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
Through a targeted knockdown (KD) screen of chromatin regulatory genes, we identified the EP400 complex components EPC1 and EPC2 as critical oncogenic cofactors in acute myeloid leukemia (AML). EPC1 and EPC2 were required for the clonogenic potential of human AML cells of multiple molecular subtypes. Focusing on MLL-mutated AML as an exemplar, Epc1 or Epc2 KD-induced apoptosis of murine MLL-AF9 AML cells and abolished leukemia stem cell potential. By contrast, normal hematopoietic stem and progenitor cells (HSPC) were spared. Similar selectivity was observed for human primary AML cells versus normal CD34(+) HSPC. In keeping with these distinct functional consequences, Epc1 or Epc2 KD-induced divergent transcriptional consequences in murine MLL-AF9 granulocyte-macrophage progenitor-like (GMP) cells versus normal GMP, with a signature of increased MYC activity in leukemic but not normal cells. This was caused by accumulation of MYC protein and was also observed following KD of other EP400 complex genes. Pharmacological inhibition of MYC:MAX dimerization, or concomitant MYC KD, reduced apoptosis following EPC1 KD, linking the accumulation of MYC to cell death. Therefore, EPC1 and EPC2 are components of a complex that directly or indirectly serves to prevent MYC accumulation and AML cell apoptosis, thus sustaining oncogenic potential.

Krause DS, Fulzele K, Catic A, et al.
Differential regulation of myeloid leukemias by the bone marrow microenvironment.
Nat Med. 2013; 19(11):1513-7 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-β1 (TGF-β1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.

Liu Y, Cheng H, Gao S, et al.
Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells.
Leukemia. 2014; 28(5):1071-80 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

DeKelver RC, Lewin B, Lam K, et al.
Cooperation between RUNX1-ETO9a and novel transcriptional partner KLF6 in upregulation of Alox5 in acute myeloid leukemia.
PLoS Genet. 2013; 9(10):e1003765 [PubMed] Article available free on PMC after 05/09/2015 Related Publications
Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene Alox5, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of Alox5 expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα in vitro. However, Alox5 is not essential for the induction of leukemia by RUNX1-ETO9a in vivo. Finally, we demonstrate that the upregulation of Alox5 by RUNX1-ETO9a occurs via the C₂H₂ zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, KLF6 is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.

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Cite this page: Cotterill SJ. MLLT3 gene, Cancer Genetics Web: http://www.cancer-genetics.org/MLLT3.htm Accessed:

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