Bcr-Abl Represses IRF8 Gene Transcription in a Stat5-Dependent Manner

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3497-3497
Author(s):  
Elizabeth Hjort ◽  
Weiqi Huang ◽  
Elizabeth A. Eklund
Keyword(s):  
Gene Transcription ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Consensus Sequence ◽  
Myeloproliferative Neoplasm ◽  
Proximal Promoter ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Interleukin Receptors

Abstract Abstract 3497 The interferon consensus sequence binding protein is a member of the interferon regulatory factor family of transcription factors (referred to as Icsbp or Irf8). The first described functions for Icsbp involved regulation of phagocyte and B-cell effector genes, including genes encoding components of the phagocyte NADPH-oxidase, Toll-like receptors and interleukin receptors. However, subsequent studies in murine models and human disease indicated that Icsbp also functions as a myeloid leukemia suppressor. For example, decreased Icsbp expression is found in chronic myeloid leukemia (CML) in association with uncontrolled disease, drug resistance and progression to blast crisis (BC). Decreased Icsbp expression is also found in the bone marrow of subjects with some subtypes of acute myeloid leukemia (AML). Consistent with this clinical correlative data, IRF8−/− mice exhibit a myeloproliferative neoplasm that is similar to CML and progresses to BC over time. However, the mechanism for decreased Icsbp expression in leukemia is not known, although preliminary studies indicate that DNA-methylation of the IRF8 locus is not altered. Therefore, in these studies, we investigate the effects of Bcr-abl on IRF8 transcription. This is clinically relevant, because previous studies in our laboratory identified a set of Icsbp-target-genes that contribute to the pathogenesis of CML. We find that Bcr-abl decreases expression of Icsbp mRNA and protein in a kinase dependent manner. Since it is unlikely that Bcr-abl directly binds to the promoter to regulate gene transcription, we hypothesized that Bcr-abl regulates IRF8 through an intermediary transcription factor. In this study, we determine that Stat5 negatively regulates IRF8 transcription through a proximal promoter cis-element. We also find that Stat5 repression activity is necessary for Bcr-abl dependent regulation of IRF8. Bcr-abl is known to phosphorylate and activate Stat5 in CML. In our studies, we find that Stat5 protein (but not mRNA) is also increased in Bcr-abl+ cells. Stat5 is a known substrate for calpain; a serine protease. We previously demonstrated that Icsbp regulates calpain protease activity through repression of the gene encoding Gas2; an endogenous calpain inhibitor. Consistent with this, our current studies demonstrate that Stat5 protein stability is increased in Bcr-abl+ cells in an Icsbp/Gas2/calpain-dependent manner. These results identify novel mechanisms by which Bcr-abl-kinase activity controls a positive feedback loop that leads to decreased Icsbp expression and stabilization of Stat5 protein. These studies suggest that targeting Gas2/calpain might be a novel therapeutic approach to CML. Disclosures: No relevant conflicts of interest to declare.

Fap1 Controls Beta-Catenin Activity in a Gsk3-Beta Dependent Manner in Chronic Myeloid Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2404-2404
Author(s):  
Weiqi Huang ◽  
Ling Bei ◽  
Elizabeth A. Eklund
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Functional Significance ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Consensus Sequence ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Apoptosis Resistance ◽  
Adenomatous Polyposis Coli Protein ◽  
Interaction Domain

Abstract Abstract 2404 Fap1 (Fas-associated phosphatase 1) interacts with the C-terminus of multiple proteins via a PDZ-type protein-protein interaction domain. Interaction of Fap1 with Fas results in Fas-de-phosphorylation and inhibits Fas-induced apoptosis. In previous studies, we determined that PTPN13, the gene encoding Fap1, is a target gene for the interferon consensus sequence binding protein (referred to as Icsbp or Irf8). Icsbp functions as a leukemia suppressor in chronic myeloid leukemia (CML), and we previously demonstrated Icsbp/Fap1-dependent apoptosis resistance in cells expressing the CML-associated oncoprotein, Bcr-abl. Another known interaction partner for Fap1 is the adenomatous polyposis coli protein (Apc). However, the functional significance of this interaction has not been previously explored. In the current stuides, we investigate whether interaction between Fap1 and Apc impacts the pathogenesis of CML. Apc is involved in assembly of a multi-protein complex that includes Apc, Axin, Gsk3-beta and beta-catenin. Association of these proteins results in serine/threonine phosphorylation (pS/T) of beta-catenin by Gsk3-beta. pS/T beta-catenin is released from the complex and degraded by the proteasome. Previous studies demonstrated that pS/T of beta-catenin, and consequent beta-catenin degradation, is impaired in CML, but the mechanism for this impairment is unknown in most cases. We find that Icsbp-dependent increase in Fap1 results in increased interaction between Fap1 and Apc in Bcr-abl+ myeloid progenitor cells. We also find that increased Fap1/Apc interaction is associated with inactivation (de-phosphorylation) of Gsk3-beta and a decrease in inhibitory pS/T of βcatenin. We find that this results in increased beta-catenin protein and activity. We also find that Fap1 co-immuno-precipitates with Apc, Axin, Gsk3-beta and beta-catenin. This result suggests the possibility that Gsk3-beta is a substrate for Fap1 protein tyrosine phosphatase activity. Consistent with this hypothesis, we find that Fap1 is able to dephosphorylate Gsk3-beta in vitro. We tested the functional significance of this observation in experiments using an activated form of Gsk3-beta that cannot be dephosphorylated at a key residue (Y216D-Gsk3-beta). We find that expression of this Gsk3-beta mutant partly reverses the effect of Bcr-abl and Fap1 on beta-catenin protein stability and activity. Increased beta-catenin activity is a characteristic of leukemia stem cells (LSC) in CML, and increasing beta-catenin activity in the bone marrow is associated with poor prognosis in CML. Therefore, these studies identify a novel pathway that regulates events significant to CML pathogenesis. These studies also identify a role for Apc in the pathogenesis of leukemia, and another mechanism for the leukemia suppressor effect of Icsbp. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interferon Consensus Sequence Binding Protein (ICSBP) Decreases β-Catenin Activity in Myeloid Cells by Repressing GAS2 Transcription

2010 ◽  
Vol 30 (19) ◽  
pp. 4575-4594 ◽  
Author(s):  
Weiqi Huang ◽  
Wei Zhou ◽  
Gurveen Saberwal ◽  
Iwona Konieczna ◽  
Elizabeth Horvath ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Target Genes ◽  
Binding Protein ◽  
Blast Crisis ◽  
Consensus Sequence ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Histone Deacetylase 3 ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

ABSTRACT The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and β-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases β-catenin protein and activity in a Gas2- and calpain-dependent manner. Conversely, decreased ICSBP expression increases β-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased β-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in β-catenin activity in Bcr/abl-positive (Bcr/abl+) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences β-catenin activity in myeloid leukemia.

scholarly journals IGF2BP3 As an N6-Methyladenosine Reader Promotes Acute Myeloid Leukemia Progression By Regulating the Stability of Target RNA

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4322-4322
Author(s):  
Nan Zhang ◽  
Jianchuan Deng ◽  
Fuling Zhou
Keyword(s):  
Acute Myeloid Leukemia ◽  
Genetic Risk ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Clinical Samples ◽  
Dependent Manner ◽  
High Genetic Risk ◽  
Acute Myeloid

Abstract Background: N6-methyladenosine (m6A) is the most common post-transcriptional modification of eukaryotic mRNA. Recent evidence suggests that dysregulated m6A-associated proteins and m6A modifications play a pivotal role in the initiation and progression of diseases such as cancer. Here, we identified that IGF2BP3 is specifically overexpressed in acute myeloid leukemia (AML), which constitutes a subtype of this malignancy associated with poor prognosis and high genetic risk. Methods: Bioinformatics analysis of public databases was performed to screen the differentially expressed m6A regulators in AML. Clinical samples were collected to detect the expression of IGF2BP3 in AML by RT-qPCR. The effects of IGF2BP3 on the proliferation, apoptosis and cycle of AML cells were detected by CCK-8 and flow cytometry. RNA-seq was used to identify target genes of IGF2BP3 by integrating analysis with RIP-Seq, iCLIP-Seq and MeRIP-Seq data sets. Results:High expression of IGF2BP3 is closely associated with poor prognosis of AML and is higher in patients with high genetic risk group. IGF2BP3 was the lowest expressed in AML-M3 and the highest expressed in RUNX1 mutant type. IGF2BP3 is required for maintaining AML cell survival in an m6A-dependent manner, and knockdown of IGF2BP3 suppressed dramatically induces apoptosis, reduces proliferation and impaired leukemic capacity AML cells in vitro and in vivo. Mechanistically, IGF2BP3 interacts with RCC2 mRNA and stabilizes the expression of m6A-tagged RNA. Conclusions:We provided compelling evidence to demonstrate that m6A reader IGF2BP3 contributed to tumorigenesis and poor prognosis of AML, which can serve as a target to develop therapeutics for cancer treatment. Disclosures No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

The Flt3 ITD Accelerates An Already Established Myeloid Leukemia and Alters Chemotherapy Response In Vitro and In Vivo in a p53 Dependent Manner.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1719-1719
Author(s):  
Timothy Pardee ◽  
Johannes Zuber ◽  
Scott Lowe
Keyword(s):  
Myeloid Leukemia ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Disease Onset ◽  
High Dose ◽  
Chemotherapy Response ◽  
Dependent Manner ◽  
Response To Chemotherapy

Abstract Abstract 1719 Poster Board I-745 Acute myeloid leukemia (AML) is an aggressive disease with heterogeneous genetics and variable prognosis. The presence of an internal tandem duplication within the FLT3 gene (Flt3 ITD) is a marker for poor prognosis and has been linked to anthracycline resistance in cell lines and primary patient samples in vitro. The effect of this mutation on response to chemotherapy in vivo has not been examined and its effect on response to cytarabine is not known. In this study we use a genetically defined mouse model of AML to examine the effects of the Flt3 ITD on response to cytarabine and the anthracycline doxorubicin in vitro and in vivo. In vitro the Flt3 ITD conferred resistance to doxorubicin and the combination of doxorubicin and cytarabine but sensitivity to cytarabine alone in comparison to the identical leukemia without the Flt3 ITD. In vivo the presence of the Flt3 ITD provided an advantage in leukemic engraftment and accelerated disease onset. This advantage could be partially reversed by treatment of the animals with cytarabine but not by treatment with doxorubicin. Surprisingly, in vivo the Flt3 ITD conferred a marked increase in sensitivity to cytarabine when compared to the parental leukemia without this mutation. In contrast to the parental leukemia, the addition of doxorubicin to cytarabine provided no advantage over cytarabine alone. When the DNA damage response was assessed the presence of the Flt3 ITD resulted in an increase in the levels of p53 following treatment with either doxorubicin or cytarabine. Induction of the p53 target genes p21 and MDM2 was also increased. Surprisingly, the Flt3 ITD had no effect on disease onset or chemotherapy response in vitro or in vivo in the setting of p53 null AML. These data when taken together demonstrate that the Flt3 ITD confers a mixed sensitivity and resistance to standard chemotherapy and provides an engraftment advantage in a manner that depends on an intact p53 allele. This may at least in part explain the rarity of dual p53 null and Flt3 ITD positive AML. Furthermore, these data suggest that patients with Fl3 ITD positive AML may benefit more from treatment with high dose Ara-C then with combinations containing an anthracycline. Disclosures No relevant conflicts of interest to declare.

scholarly journals SirR, a Novel Iron-Dependent Repressor inStaphylococcus epidermidis

1998 ◽  
Vol 66 (9) ◽  
pp. 4123-4129 ◽  
Author(s):  
Philip J. Hill ◽  
Alan Cockayne ◽  
Patrick Landers ◽  
Julie A. Morrissey ◽  
Catriona M. Sims ◽  
...  
Keyword(s):  
Binding Site ◽  
Dna Sequences ◽  
Blot Analysis ◽  
Target Genes ◽  
Consensus Sequence ◽  
Dependent Manner ◽  
Chromosomal Dna ◽  
Iron Starvation ◽  
Mobility Shift ◽  
Western Blots

ABSTRACT In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated lipoproteins are induced in response to iron starvation. To gain insights into the molecular basis of iron-dependent gene regulation in the staphylococci, we sequenced the DNA upstream of the 3-kb S. epidermidis sitABC operon, which Northern blot analysis indicates is transcriptionally regulated by the growth medium iron content. We identified two DNA sequences which are homologous to elements of the Corynebacterium diphtheriae DtxR regulon, which controls, in response to iron stress, for example, production of diphtheria toxin, siderophore, and a heme oxygenase. Upstream of thesitABC operon and divergently transcribed lies a 645-bp open reading frame (ORF), which codes for a polypeptide of approximately 25 kDa with homology to the DtxR family of metal-dependent repressor proteins. This ORF has been designated SirR (staphylococcal iron regulator repressor). Within thesitABC promoter/operator region, we also located a region of dyad symmetry overlapping the transcriptional start ofsitABC which shows high homology to the DtxR operator consensus sequence, suggesting that this region, termed the Sir box, is the SirR-binding site. The SirR protein was overexpressed, purified, and used in DNA mobility shift assays; SirR retarded the migration of a synthetic oligonucleotide based on the Sir box in a metal (Fe2+ or Mn2+)-dependent manner, providing confirmatory evidence that this motif is the SirR-binding site. Furthermore, Southern blot analysis of staphylococcal chromosomal DNA with the synthetic Sir box as a probe confirmed that there are at least five Sir boxes in the S. epidermidis genome and at least three in the genome of S. aureus, suggesting that SirR controls the expression of multiple target genes. Using a monospecific polyclonal antibody raised against SirR to probe Western blots of whole-cell lysates of S. aureus, S. carnosus,S. epidermidis, S. hominis, S. cohnii, S. lugdunensis, and S. haemolyticus, we identified an approximately 25-kDa cross-reactive protein in each of the staphylococcal species examined. Taken together, these data suggest that SirR functions as a divalent metal cation-dependent transcriptional repressor which is widespread among the staphylococci.

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3376-3376
Author(s):  
Romain Gioia ◽  
Cedric Leroy ◽  
Claire Drullion ◽  
Valérie Lagarde ◽  
Serge Roche ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Dependent Manner ◽  
Stable Isotope Labelling ◽  
Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

Ezh2 Plays a Critical Role in the Progression of MLL-AF9-Induced Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 57-57
Author(s):  
Satomi Tanaka ◽  
Goro Sashida ◽  
Satoru Miyagi ◽  
Koutaro Yokote ◽  
Chiaki Nakaseko ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Histone H3 ◽  
Chip Sequencing ◽  
Acute Myeloid

Abstract Abstract 57 The polycomb group proteins function in gene silencing through histone modifications. They have been characterized as a general regulator of stem cells, but also play a critical role in cancer. EZH2 is a catalytic component of the polycomb repressive complex 2 (PRC2) and tri-methylates histone H3 at lysine 27 to transcriptionally repress the target genes. Although EZH2 is over-expressed in various cancers including hematological malignancies, it remains unknown how EZH2 contributes to the initiation and/or progression of acute myeloid leukemia (AML). To understand the role of EZH2 in AML, we transformed granulocyte macrophage progenitors (GMPs) from Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 fusion gene. Then, Ezh2 was deleted by inducing nuclear translocation of Cre by adding tamoxifen to culture. We found that proliferation of Ezh2δ/δ transformed cells was severely compromised upon deletion of Ezh2 (Ezh2δ/δ) in liquid culture. They gave rise to a significantly reduced number of colonies in replating assays. Of note, while Ezh2+/+ cells formed compact colonies composed of immature myeloblasts, Ezh2δ/δ cells formed dispersed colonies composed of differentiated myeloid cells. We next transplanted Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox GMPs transformed by MLL-AF9 into recipient mice. All the recipient mice developed AML by 3 weeks after transplantation. At 3 weeks after transplantation, we depleted Ezh2 by intraperitoneal injection of tamoxifen. Deletion of Ezh2 significantly prolonged the survival of the recipient mice (60 days vs. 76 days, p<0.0001), although all the mice eventually died of leukemia. Nonetheless, as was detected in vitro, Ezh2δ/δ AML cells in BM were apparently differentiated in morphology compared with the control. Ezh2δ/δ AML cells in BM gave rise to 10-fold fewer colonies in methylcellulose medium compared with Ezh2+/+ AML cells, and again showed an obvious tendency of differentiation. These observations imply that Ezh2 is critical for the progression of MLL-AF9 AML and maintains the immature state of AML cells. To elucidate the mechanism how Ezh2 promotes the progression of MLL-AF9-induced AML, we examined the genome-wide distribution of tri-methylation of histone H3 at lysine 27 (H3K27me3) by ChIP-sequencing and microarray-based expression analysis. ChIP-sequencing using Ezh2+/+ and Ezh2δ/δ BM AML cells identified 3525 and 89 genes exhibiting a ≧ 10-fold enrichment in H3K27me3 levels in Ezh2+/+ and Ezh2δ/δ AML cells, respectively, confirming a drastic reduction in the levels of global H3K27me3 in the absence of Ezh2. Microarray analysis using lineage marker (except for Mac1)−Sca-1−c-Kit+FcγRII/IIIhi BM AML cells revealed 252 upregulated and 154 downregulated genes (≧ 2-fold) in Ezh2δ/δ AML cells compared with Ezh2+/+ AML cells. Of interest, the absence of Ezh2 did not affect the transcriptional activation of the major target genes of MLL-AF9, including HoxA9 and Meis1. Because Ezh2 functions as transcriptional repressor, de-repressed genes could be direct targets of Ezh2. Based on these data, we are now engaged in further comprehensive analysis to narrow down the direct target genes of Ezh2 responsible for the progression of AML. Collectively, our findings suggest that Ezh2 is the major enzyme for H3K27me3 in AML and contributes to the progression of AML by regulating transcription a cohort of genes that are supposedly relevant to the self-renewal capacity and perturbed differentiation of AML stem cells. Disclosures: No relevant conflicts of interest to declare.

Mechanistic Deficits Of a Leukemogenic GATA-2 Mutant

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3668-3668
Author(s):  
Koichi Ricardo Katsumura ◽  
Chenxi Yang ◽  
Jing Zhang ◽  
Lingjun Li ◽  
Kirby D Johnson ◽  
...  
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Phosphorylation Site ◽  
Spectrometric Analysis ◽  
Ras Signaling ◽  
Dependent Manner ◽  
Subnuclear Localization ◽  
Transactivation Activity ◽  
N Terminus ◽  
Fold Activation

Abstract Recent studies have demonstrated a role for the master regulator of hematopoiesis GATA-2 in MonoMAC Syndrome, a human immunodeficiency disorder associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Though GATA2 coding region and cis-regulatory element mutations underlie MonoMAC syndrome, many questions remain unanswered regarding how GATA-2 is controlled physiologically and how it is dysregulated in pathological contexts. We dissected how a T354M mutation in the GATA-2 DNA binding zinc finger, which is frequently detected in MonoMAC syndrome and familial MDS/AML, alters GATA-2 activity. The T354M mutation reduced GATA-2 chromatin occupancy, induced GATA-2 hyperphosphorylation, and disrupted GATA-2 subnuclear localization. These molecular phenotypes also characterized an additional familial MDS/AML-associated GATA-2 mutant (Δ355T). T354M hyperphosphorylation and ectopic subnuclear localization were detected in hematopoietic and non-hematopoietic cell lines. We developed a new model system in mouse aortic endothelial (MAE) cells to quantitate GATA-2 activity to regulate endogenous target genes. T354M exhibited significantly reduced activity in this assay (GATA-2: 200-fold activation; T354M: 7.7-fold activation). Mass spectrometric analysis of the phosphorylation states of GATA-2 and T354M revealed that the T354M mutation enhanced phosphorylation at several GATA-2 residues. Analysis of single phosphorylation site mutants indicated that only mutation of S192 (S192A) abolished T354M-induced hyperphosphorylation. The S192A mutation attenuated phosphorylation of sites within wild-type GATA-2 and reduced transactivation activity (50% decrease, p < 0.01). A distinct 60 amino acid (aa) region within the GATA-2 N-terminus was required for T354M hyperphosphorylation and ectopic subnuclear localization. Deletion of this sequence decreased GATA-2 transactivation activity (60 aa deletion: 85% decrease, p < 0.01; 10 aa deletion: 45% decrease, p < 0.05). GATA-1 lacks an analogous subnuclear targeting sequence, and accordingly, a GATA-1(T263M) mutant, which corresponds to the GATA-2(T354M) mutant, localized normally and was not hyperphosphorylated. However, a GATA-1 chimera containing the GATA-2 subnuclear targeting sequence localized to ectopic subnuclear foci in a T263M-dependent manner. The GATA-2 N-terminus endowed GATA-1 with the capacity to induce GATA-2 target genes. By contrast, a GATA-2 chimera containing the GATA-1 N-terminus exhibited normal subnuclear localization. Thus, the leukemogenic T354M mutation utilizes the GATA-2-specific subnuclear targeting sequence to disrupt the normal subnuclear localization pattern, and this disruption is associated with S192-dependent hyperphosphorylation. In addition to its involvement in AML, GATA-2 interfaces with RAS signaling to promote the development of non-small cell lung cancer. We discovered that RAS signaling promotes S192-dependent GATA-2 hyperphosphorylation and ectopic subnuclear localization and propose that GATA-2 is an important component in oncogenic RAS-dependent leukemogenesis, which is being formally tested using innovative mouse models. In summary, dissecting the mechanistic deficits of a leukemogenic GATA-2 mutant revealed unexpected insights into mechanisms underlying physiological GATA-2 function and GATA-2-dependent hematologic pathologies. Disclosures: No relevant conflicts of interest to declare.

Early Different Downstream Target of Glucocorticoid Receptor Contributing to Glucocorticoids Sensitivity in Kasumi-1 Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5132-5132
Author(s):  
Wenbin Gu ◽  
Meng Li ◽  
Liang Liang ◽  
Jian Zhang ◽  
Chongye Guo ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Fusion Protein ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Non Coding Rnas

Abstract The t(8;21) chromosome translocation frequently occurs in acute myeloid leukemia (AML), resulting in an in-frame fusion between the DNA-binding domain of AML1 and almost the entire of ETO gene. The fusion AML1-ETO protein is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells, such as Kasumi-1 and SKNO-1 cells. Glucocorticoids (GC) can induce apoptosis in these cells at low concentrations, whereas most other myeloid leukemia cell lines are resistant to glucocorticoid-induced apoptosis. To experimentally address possible sensitive mechanisms in leukemia cells with AML1-ETO translocation, we generated aGC-resistant Kasumi-1 cell line by induction of 10-6 M dexamethasone (Dex) for three weeks. The IC50 of Dex to cells is increased from 2.5×10-8 M for original GC-sensitive Kasumi-1 cell line ( K-S cell line) to more than 1×10-5 M for induced GC-resistant Kasumi-1 cell line (K-R cell line). Since GC resistance often results from mutations in the glucocorticoid receptor (GR), all the exons of GR gene were sequenced and no mutation was found in K-R cells. Comparing to those in K-S cells, the GR protein level didn't decrease in K-R cells after 2h, 4h, 8h, 12h and 24h exposure to dexamethasone. Given that the difference of direct GR downstream genes between K-S and K-R cells may play a key role in the GC sensitivity, we systematically analyzed the changes of gene expression induced by Dex versus ethanol vehicle for 8h in K-S and K-R cells by high throughput RNA sequencing. The time point of 8h was selected according to the expression peaks of several foregone GR target genes after Dex induction. There were found 32 genes conversely regulated in K-S and K-R cells, including 14 mRNAs and 18 long non-coding RNAs. Pathway analysis indicated that the upregulated genes in K-S cells might promote the AML1-ETO fusion protein degradation by proteasomes, while the component genes of this pathway were downregulated in K-R cells. Further validation and function studies of these mRNAs and long non-coding RNAs are ongoing. Our data suggested that the downstream targets of GR among GC-sensitive and -resistant Kasumi-1 cells were significant different and they may contribute to the GC sensitivity and resistance by degradation or reservation of AML-ETO fusion protein and the regulation of apoptosis in t(8;21) leukemia cell subtype. Disclosures No relevant conflicts of interest to declare.

Results from the first-in-human study of mivebresib (ABBV-075), a pan-inhibitor of bromodomain and extra terminal proteins, in patients with relapsed/refractory acute myeloid leukemia.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 7030-7030 ◽  
Author(s):  
Olatoyosi Odenike ◽  
Johannes E. Wolff ◽  
Gautam Borthakur ◽  
Ibrahim Taha Aldoss ◽  
David Rizzieri ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Febrile Neutropenia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Phase 1 ◽  
Human Study ◽  
Dependent Manner ◽  
Bet Inhibitor ◽  
Acute Myeloid

7030 Background: Bromodomain and extra-terminal (BET) proteins bind to acetyllysines and upregulate oncogenic target genes. Mivebresib (ABBV-075) is a pan-BET inhibitor with antitumor activity in vitro and xenograft models of AML. This 2-part phase 1 study evaluates the safety and pharmacokinetics of mivebresib at monotherapy or combination dosing schedules in patients with solid tumors (part 1) and acute myeloid leukemia (AML; part 2) (NCT02391480). Here, we report preliminary data from part 2 in patients with relapsed/refractory (RR) AML. Methods: Mivebresib monotherapy (MIV-mono), or combined with venetoclax (MIV-VEN), were administered daily to adult patients with AML. The dose-limiting toxicity (DLT) period was 28 d. Results: As of Dec 2018, 41 patients (median age: 69 y [range, 29–84]; 19 patients had > 2 prior therapies) were enrolled: 19 in MIV-mono (5 of whom switched to MIV-combo) and 22 who began treatment in MIV-VEN cohorts. 23 patients had high cytogenetic risk. Median time on treatment was 28 d (range, 8–562). There were no DLTs. All patients experienced a treatment-emergent adverse event (AE), most commonly (≥40% patient incidence), fatigue (56%), dysgeusia (46%), decreased appetite (44%), diarrhoea (42%), nausea (42%), vomiting (42%). 40 patients had grade ≥3 AEs (febrile neutropenia (37%), anemia (34%) and thrombocytopenia (32%). 33 patients had serious AEs, most commonly febrile neutropenia (19%). 25 deaths were reported; 15 patients died of causes unrelated to mivebresib and 10 patients due to AML progression. The median best % bone marrow blast change for 26 evaluable patients was -20% (range, -98% to +300%). Gene expression analysis in pre- and post-treatment peripheral blood samples showed that HEXIM1, DCXR and CD93 genes were reliable PD biomarkers of ABBV-075 which were consistently modulated in a dose-dependent manner. At the cutoff date, median overall survival for all patients was 2.6 m. Conclusions: Mivebresib was well tolerated and showed antileukemic effects in patients with RR AML. Clinical trial information: NCT02391480.

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