Differential Regulation of c-Myc/Lin28 Discriminates Subclasses of Rearranged MLL Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 163-163
Author(s):  
Lili Chen ◽  
Yuqing Sun ◽  
Jingya Wang ◽  
Hui Jiang ◽  
Andrew G. Muntean
Keyword(s):  
Growth Rate ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Rna Binding ◽  
Transformed Cells ◽  
Mouse Bone Marrow ◽  
Fusion Partner ◽  
Genome Wide ◽  
Genome Wide Expression

Abstract Rearrangements of the 11q23 locus account for ~70% of infant ALL and ~50% of infant AML1 and about 10% of leukemia overall. The prognosis for 11q23 patients is generally poor, however, outcomes vary depending on the fusion partner2. Rearrangements fuse the N-terminus of MLL with one of >70 different partner genes that includes both nuclear and cytoplasmic proteins. Despite the different intracellular localization of these partner proteins, to date, all studied MLL fusion proteins (MLL-FPs) localize to chromatin in the nucleus and drive aberrant transcriptional activation. Recent seminal work by a number of groups has revealed that several of the most common nuclear translocation partner genes (including AF9, ENL, AFF1 (AF4), AFF4 (AF5q31), AF10, AF17 and ELL) assemble into a transcriptional activation complex that includes p-TEFb and/or the histone H3K79 methyltransferase DOT1l. Translocation of MLL with members of this complex results in deregulated transcriptional activation of target genes. Conversely, oligomerization motifs are necessary for transformation following 11q23 translocation with a cytoplasmic partner. However, these mechanisms fail to explain different survival outcomes observed in patients. Further, the transcriptional programs induced in these diverse 11q23 leukemias are currently not well understood. In this study, we examined the genome wide expression profiles in leukemic cells transformed by several MLL-FPs representative of nuclear translocations [t(9;11) (MLL-AF9), t(10;11) (MLL-AF10) and t(11;19) (MLL-ENL)] or cytoplasmic translocations [t(1;11) (MLL-AF1p), t(6;11) (MLL-AF6) and t(11;17) (MLL-Gas7)]. Leukemia cell lines established from mouse bone marrow cells expressing these MLL-FPs proliferated at different rates and mice transplanted with the transformed cells develop leukemia with different latencies remarkably consistent with differences observed in patients harboring different MLL translocations. To elucidate differences in the gene programs induced by different MLL-FPs we performed genome wide expression profiling by RNA-sequencing. These data demonstrated that while the MLL-AF9 and MLL-ENL fusion proteins induce very similar gene programs, the cytoplasmic fusion proteins (MLL-AF6, MLL-AF1p and MLL-GAS7) all possess unique gene signatures. We then performed a pathway analysis comparing nuclear fusion proteins and cytoplasmic fusion proteins and discovered the Myc transcription factor program as one of the top distinguishing features. Myc overexpression significantly increased the growth rate of slow-growing cells that also had low intrinsic Myc, while the growth rate change of more highly proliferative cells was minimal. While all leukemic cell lines were sensitive to the BET inhibitor JQ1 which regulates c-Myc expression, greater sensitivity was observed in those with low c-Myc expression demonstrating the universal importance of this gene program. The Myc target and micro RNA binding protein Lin28B is also differentially expressed between nuclear and cytoplasmic fusions. Negative regulation of miR-150 by Lin28B was observed in all MLL-FP cell lines, which is necessarily downregulated in 11q23 leukemias. We then investigated another Lin28B microRNA target let-7. Interestingly, let-7g expression was significantly increased in MLL-FP transformed cells associated with the longest disease latency. These data demonstrate that differential activation of the c-Myc/Lin28 program accounts for changes in let-7g expression and is associated with MLL-FP disease latency. These data also suggest that patients harboring different 11q23 rearrangements will respond differentially to therapeutic targeting of c-Myc expression dependent on fusion partner. References: 1. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nature reviews Cancer. 2007;7(11):823-833. 2. Balgobind BV, Raimondi SC, Harbott J, et al. Novel prognostic subgroups in childhood 11q23/MLL-rearranged acute myeloid leukemia: results of an international retrospective study. Blood. 2009;114(12):2489-2496. Disclosures No relevant conflicts of interest to declare.

Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hsiangyu Hu ◽  
Nirmalya Saha ◽  
Yuting Yang ◽  
Sierrah Marie Grigsby ◽  
Rolf Marschalek ◽  
...  
Keyword(s):  
Cell Growth ◽  
Acute Leukemia ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Fusion Partner ◽  
Wild Type

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

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

scholarly journals Generation of An Endogenous FGFR2–BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells

2020 ◽  
Vol 21 (7) ◽  
pp. 2460
Author(s):  
Andreas Reicher ◽  
Antoneicka L Harris ◽  
Felix Prinz ◽  
Tobias Kiesslich ◽  
Miaoyan Wei ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rna Binding ◽  
Human Cancer ◽  
Chromosomal Rearrangements ◽  
Growth Factor Receptor ◽  
Fusion Product ◽  
Fusion Partner ◽  
Gene Fusions ◽  
Endogenous Gene ◽  
Fgfr2 Gene

Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10–15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In this technical report, we describe the generation of an endogenous FGFR2–Bicaudal family RNA binding protein 1 (BICC1) fusion in multiple independent cholangiocarcinoma and immortalized liver cell lines using CRISPR. BICC1 is the most common FGFR2 fusion partner in CCA, and the fusion arises as a consequence of a 58-megabase-sized inversion on chromosome 10. We replicated this inversion to generate a fusion product that is identical to that seen in many human CCA. Our results demonstrate the feasibility of generating large megabase-scale inversions that faithfully reproduce human cancer aberrations.

MLL Fusion Protein Driven AML Is Selectively Inhibited by Targeted Disruption of the MLL-PAFc Interaction

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 56-56 ◽  
Author(s):  
Andrew G. Muntean ◽  
Eric M Granowicz ◽  
Jay L. Hess
Keyword(s):  
Fusion Protein ◽  
Colony Formation ◽  
Fusion Proteins ◽  
Target Genes ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Dominant Negative ◽  
Conditional Knockout ◽  
Transformed Cells ◽  
Mouse Bone Marrow

Abstract Abstract 56 Balanced chromosomal translocations of the MLL gene located on chromosome 11q23 result in the expression of a chimeric fusion proteins with enhanced transcriptional activity. The HOX genes and their co-factors, such as MEIS1 and PBX2, are critical downstream targets of MLL fusion proteins and essential for transformation. Previously we showed MLL fusion proteins are critically dependent on a direct interaction with the RNA Pol II Associated Factor complex (PAFc). PAFc is a protein complex important for the initiation, elongation and termination of transcription. It is also necessary for histone H2B K120 mono-ubiquitination through the direct recruitment of the BRE1/RAD6 E3 ubiquitin ligase complex. MLL fusion proteins make two direct contacts with the PAF1 and CTR9 subunits of the PAFc that are crucial for MLL fusion protein mediated transformation. Deletion of regions of MLL that interact with PAFc abrogates AML in mouse bone marrow transplantation assays. Here we tested the general requirement for PAFc in AML using a conditional knockout mouse model of one component of PAFc, Cdc73. These studies show that PAFc is necessary for growth of both E2A-HLF and MLL-AF9 transformed cells. Excision of Cdc73 leads to decreased expression of the MLL target genes Hoxa9 and Meis1, decreased colony formation and decreased proliferation of leukemic blasts and ultimately apoptosis. We then performed chromatin immunoprecipitation assays to assess the binding of PAFc and MLL to target loci with and without Cdc73. Excision of Cdc73 leads to a rapid decrease in association of PAFc as well as MLL fusion proteins and wild type MLL at target loci confirming that proper targeting of MLL fusion proteins requires PAFc. A decrease in H3K4me3 and H2Bub is also observed and consistent with a role of PAFc in the deposition of these epigenetic marks. We then sought to disrupt the MLL-PAFc interaction through expression of a small 40 amino acid fragment of MLL that interacts with the PAF1 subunit of PAFc. As the MLL-PAFc interaction involves interactions between MLL and both CTR9 and PAF1, it was unknown whether targeting one interaction site would be sufficient to disrupt transformation. Indeed, expression of the short fragment encompassing the pre-CxxC region of MLL acts as a dominant negative and disrupts the MLL-PAFc interaction, significantly decreasing Hox gene expression, colony formation and cell proliferation of MLL-AF9 transformed cells. Importantly, expression of the MLL fragment selectively inhibited MLL fusion mediated leukemic transformation and cell growth while the growth and proliferation of E2A-HLF cells is unaffected. Together these data show that targeting the MLL-PAFc interaction with a small MLL fragment can act as a dominant negative and selectively inhibit the growth of AML cells transformed with MLL fusion proteins. These data also suggest the MLL-PAF1 interaction surface is a promising region for therapeutic targeting. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genome-wide expression profiling of lymphoblastoid cell lines distinguishes different forms of autism and reveals shared pathways †

2007 ◽  
Vol 16 (14) ◽  
pp. 1682-1698 ◽  
Author(s):  
Yuhei Nishimura ◽  
Christa L. Martin ◽  
Araceli Vazquez-Lopez ◽  
Sarah J. Spence ◽  
Ana Isabel Alvarez-Retuerto ◽  
...  
Keyword(s):  
Cell Lines ◽  
Expression Profiling ◽  
Lymphoblastoid Cell Lines ◽  
Genome Wide ◽  
Genome Wide Expression

Assessment of DOT1L as a Therapeutic Target In Acute Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3291-3291
Author(s):  
Stephanie Y Jo ◽  
Eric M Granowicz ◽  
Jay L. Hess
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Fusion Proteins ◽  
Tamoxifen Treatment ◽  
Mouse Bone Marrow ◽  
Knock Out ◽  
H3k79 Methylation

Abstract Abstract 3291 Histone modifying enzymes are crucial regulators of hematopoiesis that are commonly disrupted in acute leukemia. DOT1L has emerged as a particularly important methyltransferase in leukemias with Mixed Lineage Leukemia (MLL) rearrangements. Leukemogenic MLL fusion proteins transform primarily through upregulation of A-cluster HOX genes, including HOXA9 and the HOX cofactor MEIS1. Many of the most common MLL translocation partners including the AF4 family members, AF9, ENL, and AF10, form the Elongation Assisting Proteins (EAP) complex that includes DOT1L. DOT1L is the only known histone methyltransferase that methylates histone H3 on lysine 79 (H3K79). Increasing evidence suggests this histone modification, which is generally associated with transcriptional activation, is essential for MLL fusion protein mediated oncogenicity. Chromatin immunoprecipitation (ChIP) on MLL fusion protein containing cell lines shows higher levels of H3K79 methylation across the HOXA9 and MEIS1 loci compared to non-MLL fusion protein containing cell lines. Similarly, patient samples with MLL fusion proteins show elevated H3K79 methylation. Finally, knockdown of DOT1L has been shown to inhibit growth of MLL rearranged cell lines. These findings suggest that DOT1L may be an effective therapeutic target, however further development of DOT1L inhibitors will be dependent on assessing the efficacy of DOT1L disruption in a wider range of leukemic cells as well as determining the potential toxicity and effect on normal hematopoiesis. Given that constitutive Dot1l knock out is early embryonic lethal, we established conditional Dot1l knockout mouse from gene trap sperm obtained from the Knock Out Mouse Project (KOMP). Dot1l targeted animals were crossed to CreER animals so that Dot1l knock out could be induced with 4-OHT or tamoxifen. To determine the effects of Dot1l deletion in transformation ability, mouse were injected with 5-flurouracil and bone marrow cells were transduced with retrovirus expressing oncogenes in the presence or absence of 4-OHT and growth in methocult media was examined. These experiments showed that growth of cells transformed by MLL-AF9 is completely abolished by Dot1l deletion while transformations by HOXA9/MEIS1 (downstream targets of MLL fusion proteins) and E2A-HLF (which expresses very low levels of HOXA9/MEIS1 and transform through other mechanisms) were unaffected. We also examined the toxicity of Dot1l deletion in vivo by treating mice with tamoxifen and monitoring survival and assessing hematopoiesis. Immunohistochemical studies show that loss of Dot1l is associated with widespread loss of lysine 79 di- and tri-methylation in organs including liver, spleen, bone marrow, testis, muscle and gastrointestinal track with some residual methylation retained in the central nervous system. Immunophenotypic analysis of mouse bone marrow 3–4 weeks after tamoxifen treatment revealed reduction in HSCs, GMPs, MEPs, and CLPs with Dot1l excision. Bone marrow transplantations and cell cycle experiments are currently underway to further characterize the hematopoietic defects in Dot1l deficient animals. Additional experiments will be needed to determine if Dot1l activity is required in other leukemias with high level HOX expression. Together these results suggest that Dot1l is a promising therapeutic target as it is specifically required for transformation by MLL fusion leukemia, however bone marrow suppression occurs with Dot1l inhibition. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks

PLoS ONE ◽  
2015 ◽  
Vol 10 (8) ◽  
pp. e0135958 ◽  
Author(s):  
Prasanna Vidyasekar ◽  
Pavithra Shyamsunder ◽  
Rajpranap Arun ◽  
Rajalakshmi Santhakumar ◽  
Nand Kishore Kapadia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cancer Cell ◽  
Expression Profiling ◽  
Gene Networks ◽  
Cancer Cell Lines ◽  
Microrna Gene ◽  
Genome Wide ◽  
Genome Wide Expression
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