The SQSTM1-NUP214 Fusion Protein Cooperates with Crm1 to Activate Hoxa Genes and Drives Leukemogenesis in Mice

Background: The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the NUP phenylalanine-glycine (FG) repeat motifs that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we studied the SQSTM1-NUP214 fusion. Methods: We synthesized a SQSTM1-NUP214 fusion protein which retains only a short C-terminal portion of NUP214 containing FG motifs that mediate interaction with Crm1, and then introduced point mutations targeting these FG motifs (SQSTM1-NUP214FGmut). We compared the activity of these two fusion proteins using co-immunoprecipitation with CRM1, methylcellulose colony assays, murine transplantation, RT-qPCR, and chromatin immunoprecipitation. Results: We found that the ability of the SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Further, mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was greatly diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice (6/6), whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia (0/7). These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impairing the interaction of SQSTM1-NUP214 with Crm1 reduced binding of the fusion protein to Hoxa and Meis1 loci. Conclusions: These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes. Disclosures No relevant conflicts of interest to declare.

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The Nup98-HoxA9 Leukemogenic Fusion Protein Blocks Xpo1 and Tap-Mediated Nuclear Export

Blood ◽

10.1182/blood.v118.21.5228.5228 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5228-5228

Author(s):

Yildirim Dogan ◽

Sutapa Sahay ◽

David C Dorn ◽

Anna Franceschino ◽

Michael Xu ◽

...

Keyword(s):

Nuclear Export ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Shuttling ◽

Nuclear Trafficking ◽

Myeloid Leukemias ◽

Nuclear Export Receptor ◽

Export Signal

Abstract Abstract 5228 In leukemia Nup98 fusion genes are found as a product of the reciprocal translocation between Nucleoporin 98 (nup98) and homeobox-cluster genes. Nup98-HoxA9 (NHA9), the product of the t(7;11) translocation, is detected in acute myeloid leukemias (AMLs) and as a secondary abnormality in blast crisis of chronic myeloid leukemia (CML). The role of NHA9 in leukemogenesis is complex and incompletely understood. Here, we show an abrogation of nucleocytoplasmic shuttling of the nuclear export receptor Xpo1 and Tap in NHA9-expressing cells by using retroviral nuclear trafficking as a model. Lentiviral Rev, the prototype for nuclear export signal (NES)-containing proteins is shuttled through the nucleopore by Xpo1. NHA9 sequestered Xpo1 from the nuclear rim into nuclear aggregates resulting in deficient Xpo1-dependent nuclear exit of Rev and its mRNA substrates. Tap is involved in mRNA nucleocytoplasmic shuttling and is also responsible for the nuclear export of D-type retrovirus CTE-mRNAs. By using Tap/CTE-mRNA nuclear export as a model we also found that Tap colocalized in NHA9 nuclear aggregates leading to impaired Tap-mediated nuclear exit of CTE-mRNA substrates. Leukemogenicity of Nup98 fusion proteins may be accounted for in part by defects in Tap and Xpo1-mediated export of their substrates. Disclosures: No relevant conflicts of interest to declare.

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Divergent Regulatory Pathways Control A and S Motility in Myxococcus xanthus through FrzE, a CheA-CheY Fusion Protein

Journal of Bacteriology ◽

10.1128/jb.187.5.1716-1723.2005 ◽

2005 ◽

Vol 187 (5) ◽

pp. 1716-1723 ◽

Cited By ~ 11

Author(s):

Yinuo Li ◽

Víctor H. Bustamante ◽

Renate Lux ◽

David Zusman ◽

Wenyuan Shi

Keyword(s):

Signal Transduction ◽

Fusion Protein ◽

Myxococcus Xanthus ◽

Point Mutations ◽

Cell Movement ◽

Signal Transduction Pathway ◽

Enteric Bacteria ◽

Gliding Motility ◽

Regulatory Pathways

ABSTRACT Myxococcus xanthus moves on solid surfaces by using two gliding motility systems, A motility for individual-cell movement and S motility for coordinated group movements. The frz genes encode chemotaxis homologues that control the cellular reversal frequency of both motility systems. One of the components of the core Frz signal transduction pathway, FrzE, is homologous to both CheA and CheY from the enteric bacteria and is therefore a novel CheA-CheY fusion protein. In this study, we investigated the role of this fusion protein, in particular, the CheY domain (FrzECheY). FrzECheY retains all of the highly conserved residues of the CheY superfamily of response regulators, including Asp709, analogous to phosphoaccepting Asp57 of Escherichia coli CheY. While in-frame deletion of the entire frzE gene caused both motility systems to show a hyporeversal phenotype, in-frame deletion of the FrzECheY domain resulted in divergent phenotypes for the two motility systems: hyperreversals of the A-motility system and hyporeversals of the S-motility system. To further investigate the role of FrzECheY in A and S motility, point mutations were constructed such that the putative phosphoaccepting residue, Asp709, was changed from D to A (and was therefore never subject to phosphorylation) or E (possibly mimicking constitutive phosphorylation). The D709A mutant showed hyperreversals for both motilities, while the D709E mutant showed hyperreversals for A motility and hyporeversal for S motility. These results show that the FrzECheY domain plays a critical signaling role in coordinating A and S motility. On the basis of the phenotypic analyses of the frzE mutants generated in this study, a model is proposed for the divergent signal transduction through FrzE in controlling and coordinating A and S motility in M. xanthus.

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SIX1 Is a Novel Effector Gene in CALM-AF10 Leukemia

Blood ◽

10.1182/blood-2019-130178 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5035-5035

Author(s):

Waitman K. Aumann ◽

Catherine P. Lavau ◽

Amanda Harrington ◽

Amanda E. Conway ◽

Daniel S. Wechsler

Keyword(s):

Nuclear Export ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Critical Role ◽

Leukemia Cell ◽

The Self ◽

Hek293 Cells ◽

Hematopoietic Progenitors ◽

Self Renewal ◽

Hoxa Genes

Background: The CALM-AF10 translocation is detected in ~10% of T-cell acute lymphoblastic leukemias (T-ALLs), and in some acute myeloid leukemias (AMLs). CALM-AF10 leukemias are characterized by high expression of proleukemic HOXA genes, which serve a critical role in hematopoiesis. We hypothesized that identification of novel CALM-AF10 effector genes may yield new therapeutic targets in this difficult to treat leukemia. We took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal (NES) in CALM. Using next generation sequencing, we determined that, SIX1, similar to HOXA genes, is increased in CALM-AF10 leukemias and decreased in response to CRM1 inhibition with Leptomycin B (LMB). Design/Methods: RT-qPCR and Chromatin Immunoprecipitation were performed using both bone marrow progenitors and murine embryonic fibroblasts (MEFs) transduced with CALM-AF10 or an empty vector, with and without LMB. The ability of SIX1 to enhance self-renewal of hematopoietic progenitors was examined by measuring the colony-forming ability of transduced fetal liver hematopoietic progenitor cells. CRISPR-Cas9 was used to silence SIX1 in Human Embryonic Kidney 293 (HEK293) cells. Results: RT-qPCR confirmed overexpression of SIX1 in both CALM-AF10 transduced MEFs and CALM-AF10 leukemias, with decreased SIX1 expression observed in the presence of LMB. ChIP analysis showed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver cells was sufficient to increase the self-renewal potential of colony-forming progenitors. SIX1 was successfully knocked out in HEK293 cells without a significant effect on HEK293 proliferation. Conclusions: The SIX1 homeobox gene is highly expressed during development and its expression is silenced post-embryogenesis. Increased SIX1 expression has been reported in numerous solid tumors. We have determined that SIX1 is upregulated in CALM-AF10 leukemias, and increases the self-renewal potential of hematopoietic progenitors. Using CRISPR-Cas9 to silence SIX1, we have demonstrated that SIX1 is not essential for cell survival, and that its inhibition may impair CALM-AF10 leukemia cell proliferation. Thus, SIX1 may play a pathogenic role in leukemogenesis and is a potential therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

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The Interplay Between Microrna-223 and E2F1 Regulates Cell Cycle Control During Granulopoiesis.

Blood ◽

10.1182/blood.v114.22.255.255 ◽

2009 ◽

Vol 114 (22) ◽

pp. 255-255

Author(s):

John Anto Pulikkan ◽

Viola Dengler ◽

Philomina Sona Peramangalam ◽

Abdul A. Peer Zada ◽

Carsten Müller tidow ◽

...

Keyword(s):

Cell Cycle ◽

Chromatin Immunoprecipitation ◽

Cell Cycle Progression ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Granulocytic Differentiation ◽

Cycle Progression ◽

Protein Levels ◽

Cell Cycle Inhibition

Abstract Abstract 255 Transcription factor CCAAT enhancer binding protein α (C/EBPα) functions as a master regulator of granulocyte development by co-ordinating cell cycle inhibition and differentiation. Recent findings demonstrate that deregulation of C/EBPα is a critical step in the development of acute myeloid leukemia (AML). Inhibition of E2F1, the key regulator of cell cycle progression by C/EBPα is essential for granulopoiesis and disruption of this function of C/EBPα leads to leukemia. The mechanism with which C/EBPα inhibits E2F1 in granulopoiesis is poorly understood. Recent advances in our understanding about microRNAs suggest that these molecules have profound impact in gene expression programmes. Also, deregulation of microRNAs has been shown as a hall mark of many cancers including leukemia. microRNA-223 (miR-223) is upregulated by C/EBPα during granulopoiesis. The pivotal role of miR-223 in granulopoiesis is shown by the finding that mice deficient for miR-223 display defects in granulopoiesis. In this study, we explored the role of miR-223 in the cell cycle inhibition function of C/EBPα. Computational analysis by using programmes such as Target Scan suggests that E2F1 is a putative target of miR-223. Luciferase assays using 3'UTR of E2F1 suggest E2F1 is a potential target of miR-223. Western blot analysis using bone marrow cells isolated from miR-223 null mice shows accumulation of E2F1 protein levels. Interestingly, E2F1 protein levels were downregulated during miR-223 overexpression in myeloid cells. Analysis of miR-223 by quantitative Real-Time RT-PCR in AML patient samples shows that miR-223 is downregulated in different subtypes of AML. Proliferation assays, cell cycle analysis and BrdU assays show that miR-223 functions as an inhibitor of myeloid cell cycle progression. Several studies have reported the ability of E2F1 to block granulocytic differentiation. We next analysed whether E2F1 is inhibiting myeloid differentiation through miR-223. Promoter assays show that E2F1 inhibits the miR-223 promoter activity. By using Chromatin immunoprecipitation assays, we found that E2F1 binds to miR-223 promoter in leukemia derived cell lines and this binding is reversed during granulocytic differentiation. We also observed that E2F1 is bound to the miR-223 promoter in blast cells isolated from AML patients as analysed by chromatin immunoprecipitation assays. In addition, we show that overexpression of E2F1 leads to down regulation of miR-223 levels in myeloid cells. All these data suggest that E2F1 functions as a transcriptional repressor of the miR-223 gene. Taken together, our data suggest that granulopoiesis is regulated by the interplay between miR-223 and E2F1 and deregulation of this interplay may lead to the development of AML. Overexpression of miR-223 could be a potential strategy in the treatment of AML patients in which E2F1 inhibition by C/EBPα is deregulated. Disclosures: No relevant conflicts of interest to declare.

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Structural Basis for MLL Cxxc Domain Protection against CpG DNA Methylation and the Essential Role of This Function in MLL-AF9 Leukemia.

Blood ◽

10.1182/blood.v114.22.763.763 ◽

2009 ◽

Vol 114 (22) ◽

pp. 763-763

Author(s):

Laurie Risner ◽

Tomasz Cierpicki ◽

Jolanta Grembecka ◽

Stephen M Lukasik ◽

Relja Popovic ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Solution Structure ◽

Conflicts Of Interest ◽

Point Mutations ◽

Chromosomal Translocations ◽

Structural Basis ◽

Cpg Dna ◽

First Time

Abstract Abstract 763 MLL is the target of chromosomal translocations which cause leukemias with poor prognosis. All leukemogenic MLL fusion proteins retain the CXXC domain which binds to nonmethylated CpG DNA. We present the solution structure of the MLL CXXC domain in complex with DNA, showing for the first time how the CXXC domain distinguishes nonmethylated from methylated CpG DNA. Based on the structure, we designed point mutations which disrupt DNA binding. Introduction of these mutations into MLL-AF9 results in increased DNA methylation of specific CpG nucleotides in Hoxa9, increased H3K9 methylation, decreased expression of Hoxa9 locus transcripts, loss of immortalization potential, and inability to induce leukemia in mice. These results establish that DNA binding by the CXXC domain and protection against DNA methylation is essential for MLL fusion leukemia. They also validate this interaction as a potential target for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

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Alcam Mediated Interaction Regulates Normal Hematopoietic Stem Cell Function and Cbfβ-SMMHC Induced Leukemogenesis

Blood ◽

10.1182/blood.v120.21.4091.4091 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4091-4091

Author(s):

Robin Jeannet ◽

Qi Cai ◽

Hongjun Liu ◽

Hieu Vu ◽

Ya-Huei Kuo

Keyword(s):

Fusion Protein ◽

Cell Function ◽

Conflicts Of Interest ◽

Immunoglobulin Superfamily ◽

Leukemic Transformation ◽

Self Renewal ◽

Hematopoietic Stem ◽

A Cell

Abstract Abstract 4091 The inv(16) acute myeloid leukemia (AML)-associated CBFβ-SMMHC fusion protein impairs hematopoietic differentiation and predisposes to leukemic transformation. Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. We found that Alcam expression marks long-term repopulating HSCs (LT-HSC), multipotent progenitors (MPP), a subset of granulocyte-macrophage progenitors (GMP), and that Alcam expression is lost or reduced in subsets of pre-leukemic and leukemic progenitors expressing the Cbfβ-SMMHC fusion protein. We characterized the role of Alcam in HSC differentiation and self-renewal using an Alcam-null (Alcam−/−) mouse model (Weiner et al. 2004 Mol Cell Neurosci 27:1, 59–69). We show that Alcam is highly expressed in LT-HSCs where its level progressively increases with age. Young adult Alcam−/− mice had normal homeostatic hematopoiesis, and normal numbers of phenotypic HSCs. However, Alcam−/− HSCs had reduced long-term replating capacity in vitro and reduced long-term engraftment potential upon transplantation. We show that Alcam−/− BM contain a markedly lower frequency of long-term repopulating cells than wild type (WT). Further, the long-term repopulating potential and engraftment efficiency of Alcam−/− LT-HSCs was greatly compromised despite a progressive increase in phenotypic LT-HSC numbers during long-term serial transplantation. In addition, an age-associated increase in phenotypic LT-HSC cellularity was observed in Alcam−/− mice. This increase was predominately within the CD150hi fraction, and was accompanied by significantly reduced leukocyte output. Moreover, Alcam−/− LT-HSCs display premature elevation of Selp expression, a hallmark of HSC aging. To understand the role of Alcam in leukemic transformation, we generated conditional Cbfb-MYH11 knock-in (Cbfb56M/+/Mx1-Cre), Alcam-deficient (Alcam−/−) mice. Interestingly, we found that loss of Alcam drastically delayed or reduced leukemia incidence. Transplantation of Alcam−/−/Cbfb56M/+/Mx1-Cre pre-leukemic bone marrow cells into WT recipients also led to delayed and reduced incidence of leukemia development. These results suggest that Alcam contributes to leukemia transformation in a cell-intrinsic manner. Collectively, our study reveals that Alcam regulates the functional integrity and self-renewal of LT-HSCs, and contributes to leukemia initiation induced by CBFβ-SMMHC. Disclosures: No relevant conflicts of interest to declare.

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CD24-Siglec-G Interaction Plays an Important in Reducing Experimental Graft-Versus-Host Disease (GVHD)

Blood ◽

10.1182/blood.v120.21.453.453 ◽

2012 ◽

Vol 120 (21) ◽

pp. 453-453

Author(s):

Tomomi Toubai ◽

Rebecca Evers ◽

Yaping Sun ◽

Isao Tawara ◽

Chen Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Fusion Protein ◽

Immune Activation ◽

Conflicts Of Interest ◽

Critical Role ◽

Negative Regulator ◽

Clinical Severity ◽

Allogeneic Bmt

Abstract Abstract 453 The role of host antigen presenting cells (APCs) on negatively regulating GVHD is not well understood. Members of the sialic acid binding Ig–like lectin-G (Siglec-G) is an immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in its intracellular domain that negatively regulates immune activation induced by non-infectious damage associated molecules (DAMPs). Following conditioning for allogeneic BMT, several DAMPs are released which stimulate host APCs and enhance GVHD. But the role of negative regulators of DAMP associated immune activation, such as Siglecs, in regulating allo-reactivity is not known. We therefore utilized well defined clinically relevant murine models of allogeneic BMT to test the hypothesis that deficiency of a negative regulator of responses to DAMPs in the hosts, namely Siglec-G, will increase GVHD. B6 wild type (WT) and Siglec-G−/− animals were lethally irradiated (13Gy) and transplanted on day 0 with 5×106 bone marrow and 3×106 splenic CD90+T cells from either syngeneic WT-B6 or MHC mismatched BALB/c donors. The Siglec-G−/− animals showed significantly worse survival than the allo-WT animals (p=0.0045). The increased mortality was associated with an increase in GVHD specific clinical severity (p<0.05), donor T cell expansion (p<0.03), and serum levels of pro-inflammatory cytokines (TNFα, IFN-γ, p<0.05) on day +7 after BMT. We next evaluated whether this was because of Siglec-G deficiency only on the radiosensitive host APCs. To this end we generated [B6àB6Ly5.2] and [Siglec-G−/−àB6Ly5.2] BM chimeras and utilized them as recipients following lethal radiation. They were injected with 5×106 BM and 3×106 CD90+ T cells from either syngeneic WT B6 or allogeneic BALB/c donors. The allogeneic [Siglec-G−/−àB6Ly5.2] animals demonstrated significantly worse survival than the [B6àB6Ly5.2] animals (p<0.0001). We determined the converse, i.e. analyzed the role of Siglec-G on radio-resistant host APCs by generating [B6Ly5.2àB6] and [B6Ly5.2àSiglec-G−/−] BM chimeras and utilized them as recipients. [B6Ly5.2àSiglec-G−/−] chimeras demonstrated similar survival as [B6Ly5.2àB6] chimeras. These data collectively demonstrate that Siglec-G expression only on the host radiosensitive APCs is critical for protection from GVHD. To confirm the role of increased DAMPs in causing greater mortality we tested whether the intensity of conditioning affects the serum level of DAMPs (HMGB1, proinflammatory cytokines) and found that significantly greater levels of DAMPs were observed in the mice that received 13Gy than 8 Gy. Furthermore, consistent with the increased levels of DAMPs, Siglec-G−/− animals showed higher GVHD only after 13Gy radiation but not after 8Gy conditioning. Because responses to non-infectious DAMPs are regulated by Siglec-G through its interaction with CD24 we next hypothesized that enhanced CD24-Siglec-G interaction would mitigate GVHD following myeloablative conditioning. We first characterized stimulation of allogeneic T cell responses by Siglec-G−/− APCs. We utilized CD24−/− and WT BALB/c T cells as responders in an MLR with B6 and Siglec-G−/−stimulators. We found that Siglec-G−/− APCs expanded the CD24−/− T cells more than WT-B6 APCs. We next tested in vivo whether enhanced CD24-Siglec-G interaction would mitigate GVHD. We utilized a novel CD24 fusion protein (day-1, 100mg/mouse) and found that it decreased GVHD mortality only in the WT but not in the Siglec-G−/− animals. Together our data demonstrate a critical role for CD24-Siglec-G interactions in regulating GVHD and suggest that administration of the novel CD24 fusion protein may be an innovative strategy to mitigate GVHD. Disclosures: No relevant conflicts of interest to declare.

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The GSK3β-Phosphorylation Sites in the DEK-Portion Are Indispensable for the Leukemogenic Potential of the DEK/CAN Fusion Protein in t(6;9)-Positive Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.3574.3574 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3574-3574

Author(s):

Claudia Oancea ◽

Maria Heinßmann ◽

Nathalie Guillen ◽

Oliver G. Ottmann ◽

Martin Ruthardt

Keyword(s):

Acute Myeloid Leukemia ◽

Fusion Protein ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Glycogen Synthase ◽

Confocal Laser ◽

Phosphorylation Sites ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract The t(6,9)(p23,q34) translocation occurs in 1-5% of adult patients with acute myeloid leukemia (AML). It is associated with a poor prognosis and defines a high risk group of AML in the WHO classification. The t(6;9) is in most of the cases the only cytogenetic aberration at diagnosis. The hallmark of t(6;9)-positive AML is the DEK/CAN fusion protein. DEK/CAN is a leukemogenic oncogene, but little is known about the molecular mechanism of DEK/CAN-induced leukemogenesis. The 165 kDa DEK/CAN phosphoprotein is encoded by a single transcript of 5.5Kb. The DEK portion of the DEK/CAN contains all the major functional domains of DEK mediating DNA-binding and multimerization. DEK increases life span of primary cells in culture by inhibiting cellular senescence and apoptosis. Post-translational modifications of DEK, mainly phosphorylation, influence the activity of DEK; unphosphorylated DEK has a higher affinity for DNA than the phosphorylated form, which in turn has a higher ability for multimerization. The main kinases that phosphorylate DEK are Glycogen synthase kinase 3 β (GSK3β) and Casein kinase 2 (CK2). The respective phosphorylation sites are conserved in the DEK portion of DEK/CAN. However, little is known about the role of phosphorylation for the biological functions of DEK/CAN. Therefore we generated several mutants of DEK and DEK/CAN by point-mutating the putative GSK3β-sites (ΔP1) from S to A and by deleting the CK2 sites in addition to these mutations (ΔP2). The reduction of S/T phosphorylation was confirmed by a ProQ staining and affinity chromatography on lysates of 293T cells expressing DEK, DEK/CAN and the respective ΔP1 or ΔP2 mutants. Further biological and biochemical consequences of these mutations for DEK and DEK/CAN were investigated in murine factor dependent 32D progenitor cells and in primary murine Sca1+/lin- hematopoietic stem cells (HSC), retrovirally or lentivirally transduced with DEK, DEK/CAN and/or their phosphorylation mutants ΔP1 or ΔP2, respectively. Here we report that the loss of the GSK3β- and CK2-phosphorylation sites did not interfere with the subnuclear localization of either DEK or DEK/CAN as revealed either by subnuclear fractionation experiments or by co-localization with native DEK/CAN in confocal laser scan microscopy assays on 32D cells co-expressing DEK/CAN and ΔP1-DEK/CAN or ΔP2-DEK/CAN. In contrast, the destruction of GSK3β-phosphorylation sites not only led to a loss of apoptosis inhibition by DEK and DEK/CAN upon factor withdrawal in 32D cells, but also abolished the increased self renewal potential of DEK/CAN-positive HSC. In fact DEK/CAN-positive HSCs significantly increased colony numbers in colony forming units spleen-day 12 (CFU-S12) assays as compared to empty vector controls, whereas ΔP1-DEK/CAN and ΔP2-DEK/CAN did not have any effect. In summary, our results suggest an important role of the GSK3β-phosphorylation for the DEK/CAN-induced leukemogenesis, which establishes the GSK3β-activity as a molecular target for therapeutic intervention in t(6;9)-positive AML. Disclosures No relevant conflicts of interest to declare.

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The SIX1 homeobox Gene Is a Novel Therapeutic Target in CALM-AF10 Leukemogenesis

Blood ◽

10.1182/blood-2020-140754 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 38-39

Author(s):

Waitman K. Aumann ◽

Catherine P. Lavau ◽

Amanda Harrington ◽

Donald Tope ◽

Amanda E. Conway ◽

...

Keyword(s):

Therapeutic Target ◽

Nuclear Export ◽

Homeobox Gene ◽

Leukemia Cells ◽

Hematopoietic Progenitors ◽

Self Renewal ◽

Downstream Targets ◽

Hoxa Genes ◽

Novel Therapeutic

Background : The CALM-AF10 translocation is found 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor, Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. Similar to HOXA genes, SIX1 is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. Additionally, SIX1 and its cofactor EYA2 have been found to be overexpressed in numerous solid tumors, and inhibitor of the SIX1/EYA2 complex has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective: To evaluate the role of SIX1 in CALM-AF10 leukemias. Design/Methods: RT-qPCR and Chromatin Immunoprecipitation (ChIP) were performed using bone marrow progenitors transduced with CALM-AF10 or an empty vector, with and without LMB. Methylcellulose colony assays assessed the ability of SIX1 to enhance self-renewal of hematopoietic progenitors. An inhibitor of the Six1/Eya2 interaction (compound 8430) was used to evaluate cell proliferation. Downstream targets of Six1 were evaluated using RT-qPCR in CALM-AF10 cells treated with Six1/Eya2 inhibitor (8430). Results: RT-qPCR confirmed overexpression of SIX1 in CALM-AF10 leukemia cells, and showed decreased SIX1 expression in the presence of LMB. Furthermore, ChIP revealed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver progenitors was sufficient to increase self-renewal potential. The 8430 Six1/Eya2 inhibitor slowed cell growth in CALM-AF10 cells compared to cells treated with DMSO alone. Finally, downstream targets such as Slc2a1, Cdk2, and Cyclina2 were decreased in 8430-treated CALM-AF10 leukemia cells. Conclusions: The SIX1 homeobox gene is highly expressed during embryogenesis, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. Further, we have shown a potential oncogenic role for Six1, as it was able to increase the self-renewal potential of hematopoietic progenitors. The role of Six1 in CALM-AF10 leukemia is further supported by the ability of a SIX1/EYA2 inhibitor to slow the growth of CALM-AF10 leukemia cells and decrease the expression of downstream targets of SIX1. These observations suggest that Six1 plays a pathogenic role in leukemogenesis, and may be a novel therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

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New Insights into PLZF/Rara Mechanism during APL Onset: EZH2 on the Road

Blood ◽

10.1182/blood-2019-125509 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3761-3761

Author(s):

Mathilde Poplineau ◽

Nadine Platet ◽

Lia N'Guyen ◽

Shuhei Koide ◽

Julien Vernerey ◽

...

Keyword(s):

Retinoic Acid ◽

Mouse Model ◽

Fusion Protein ◽

Cell Line ◽

Conflicts Of Interest ◽

Parent Cell ◽

Promising Therapeutic Approach ◽

Transcriptomic Signature ◽

The Impact

INTRODUCTION Inappropriate recruitment of functional Polycomb-Group proteins (PcG) may trigger epigenetic unbalance at very specific genomic loci that substantially contribute to the pathogenesis of Acute Myeloid Leukemia (AML). This concept was first described in Acute Promyelocytic Leukemia (APL) in which PcG proteins were abnormally addressed due to the expression of X-RARA fusion proteins and were involved in the treatment response of the disease. For instance, in the context of APL with t(11;17)(q23;q21) translocation, the resulted oncogenic fusion protein PLZF/RARA leads to abnormal recruitment of PcG at the promoters of genes involved in acid-trans-retinoic acid (ATRA) response (Boukarabila et al.). As a consequence of that and compared to other APL subtypes (e-g: PML/RARA), APL with PLZF/RARA are insensitive to ATRA. In the recent years, a repertoire of cis-regulatory enhancer elements has been dissected to reveal important insight about leukemia onset and define new subsets of the disease with different treatment responses (Bhagwat et al). As we previously reported that PLZF displayed epigenetic specificity on enhancers (Poplineau et al.) we questioned the role of PLZF/RARA on these regulatory regions during APL onset. METHODS We performed in vivo comparative epigenomic profiling (H3K27ac, H3K4me1, H3K27me3 and H3K4me3 ChIPseq) between normal myeloid progenitors (granulocyte-monocyte progenitors purified from wild-type mice) and PLZF/RARA transformed mouse progenitors (late promyelocytes purified from mice developing APL). To question the role of PcG in APL onset, we used retroviral overexpression of PLZF/RARA and transduced Lineage negative cells from a conditional KO EZH2 mouse model. Transformation was tested by replating assay and cells were characterized by FACS and morphology analyses. We also performed EZH2 pharmacological inhibition using GSK126 and UNC1999 on a human cell line expressing the fusion protein PLZF/RARA. We analyzed the impact of this inhibition on their transcriptomic signature (RNAseq) and their proliferative capacity. RESULTS Upon PLZF/RARA expression and APL progression, specific cis-regulatory enhancer elements were targeted by the H3K27me3 PcG repressive mark. This gain in poised enhancer regions, upon PLZF/RARA expression reflected a reoriented PcG activity, from enhancers regulating developmental processes to those regulating stress and immune responses. To demonstrate the importance of this H3K27me3 switch for APL progression, we investigated the effect of EZH2 loss during PLZF/RARA transformation. Using a conditional KO EZH2 mouse model, we demonstrated that PLZF/RARA required EZH2 activity to efficiently transform progenitors since EZH2 loss promoted differentiation that altered the replating capacity of the PLZF/RARA expressing cells. In addition, EZH2 inhibition by GSK126 revealed some interesting benefits since it sensitized PLZF/RARA transformed progenitors to ATRA treatment. Moreover, inhibition of EZH2 with GSK126 or UNC1999 induced a decrease in the proliferation advantage of a human PLZF/RARA-inducible cell line. This was linked to a change of its transcriptomic signature towards an expression pattern closer to the one observed in the parent cell line. CONCLUSION Taken together, our data showed that PLZF/RARA modifies H3K27me3 profiles at enhancer regions and requires EZH2 activity for APL onset. Finally, our results suggest that EZH2 inhibition could be a new promising therapeutic approach for retinoic-acid resistant APL. Disclosures No relevant conflicts of interest to declare.

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