GATA2 Transcription Factor Mediates Myeloid Expansion Coupled with Lymphoid Block by Transcriptional Regulation of Key Target Genes.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2317-2317
Author(s):  
Satish Kumar Nandakumar ◽  
Geoffrey A. Neale ◽  
Derek A. Persons
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Cell Lines ◽  
Liquid Culture ◽  
Insertional Mutagenesis ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Control Group ◽  
Mouse Bone Marrow

Abstract Abstract 2317 Functions of the transcription factor GATA2 in the hematopoietic system were difficult to explore due to the embryonic lethal phenotype on deletion (Tsai et al., Nature 1994) and HSC quiescence on overexpression (Persons et al., Blood 1999). To overcome this problem we developed a Tamoxifen inducible system by fusing the human GATA2 cDNA to the ligand binding domain of the estrogen receptor (GATA2-ERT). Previously we found that when competitive transplant experiments were performed with mouse bone marrow cells, GATA2-ERT transduced cells greatly outcompeted untransduced myeloid cells while at the same time not contributing to the lymphoid lineage. However, no leukemia was observed. Since these effects were observed in the absence of Tamoxifen, we speculated that GATA2-ERT protein leaked into the nucleus to provide low level tonic activity (known to occur in other ERT systems). Immunoblot analysis of BM cells transduced with the GATA2-ERT vector confirmed the presence of the protein in both nuclear and cytoplasmic fractions. In addition, GATA2-ERT transduced cells also out-competed mock transduced cells in liquid culture and could be serially replated in colony forming assays. GATA2-ERT and control GFP transduced cells were diluted with mock cells to achieve around 25% GFP+ cells. After 9 days in culture the percentage of GFP + cells in the GATA2-ERT group was 91%±6 compared to GFP group which remained at 38%±14. GATA2-ERT transduced cells could be cultured indefinitely while after 7 weeks the control GFP transduced cells differentiated to mast cells. These GATA2-ERT cell lines were dependent only on IL-3 and expressed myeloid cell surface markers (53%±1 Gr+, 87%±4 Mac1+ cells n=3). DNA binding mutants of GATA2-ERT completely abolished both serial colony replating ability and growth advantage in liquid culture. This suggested that GATA2-ERT mediates these effects through transcriptional regulation of target genes. To identify the targets of GATA2 responsible for myeloid expansion, gene expression profiling was done on three independently generated GATA2-ERT cell lines and compared to an immortalized myeloid cell line (due to insertional mutagenesis) which does not express GATA2. Genes involved in myeloid neoplasms like Nmyc, HoxA9 and Bcl11a were significantly elevated (HoxA9 –77-fold, Nmyc 60-fold, Bcl11a 36-fold) in the GATA2-ERT lines. To test if GATA2 mediates myeloid expansion through Nmyc, we knocked down Nmyc in GATA2-ERT cells and tested for growth defects. GATA2-ERT cells were transduced with lentiviral vectors harboring shRNA targeting GATA2-ERT (73%±10.2), Nmyc (77%±9.5) and scrambled (75%±7) and cultured for 18 days. As expected, transduced cell numbers dropped to 5%±1 for GATA2-ERT group, confirming GATA2 dependence, while transduced cells decreased to 40%±8 for the Nmyc group with the control group unchanged. This suggests that Nmyc could be one of the key targets of GATA2 responsible for myeloid expansion. Current studies are aimed at determining whether GATA2 directly regulates the Nmyc promoter. Disclosures: No relevant conflicts of interest to declare.

Mechanism of the Elevated UPR in CN Patients but Not in CyN Patients Carrying Same ELANE Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 14-14
Author(s):  
Rainer Nustede ◽  
Inna Kuznetsova ◽  
Karl Welte ◽  
Julia Skokowa
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia

Abstract Abstract 14 Several studies found that in patients with severe congenital neutropenia (CN) harboring mutations in the ELANE gene mutated NE protein induced unfolded protein response (UPR) leading to elevated apoptosis and diminished differentiation of myeloid cells. However, it is unclear, why UPR was not detected in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations, which have been found in CN patients. Several UPR components have been identified in mammalian cells, which include three transducers (IRE1, PERK, and activating transcription factor 6 (ATF-6) as well as one master regulator (BiP/GRP78). BiP is known to be regulated by ATF6. The activation of ATF6 and its target genes (GADD34, CHOP and BiP) in CN patients has not been studied yet. We were able to detect significantly elevated levels of ATF6 and BiP in myeloid cells of CN patients with ELANE mutations, in comparison to CyN patients and to healthy individuals. Therefore, we investigated the mechanism of UPR and activation of ATF6 and ATF6 target genes in CN patients in comparison to CyN patients. We transduced the myeloid cell lines HL60 and NB4 with lentiviral constructs contained either wild type (WT) ELANE cDNA, or mutated (MUT) ELANE cDNA and measured mRNA and protein expression of ATF6 as well as mRNA expression of ATF6 target genes. We compared the effects of three ELANE mutations: C42R, V145-C152del (both mutations presented in CN patients, but not in CyN patients) and S97L (typical for CN and CyN patients) with WT ELANE. We found that in both cell lines only C42R ELANE MUT, but not V145-C152del ELANE MUT or S97L ELANE MUT induced expression of ATF6, GADD34, CHOP and BiP, as compared to control transduced cells. Furthermore, we hypothesize that degradation of mutated NE protein by Secretory Leukocyte Protease Inhibitor (SLPI) might be involved in UPR induction. However, we detected only very low levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN), as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. The lack of the NE inhibitor, SLPI in CN patients may further contribute to elevated UPR triggered by ELANE MUT and normal levels of SLPI in CyN patients might protect from ELANE MUT-induced UPR. Indeed, inhibition of SLPI using SLPI-specific shRNA led to a significantly elevated expression levels of ATF6, GADD34 and BIP, as compared to ctrl shRNA transduced cells. More importantly, co-transduction of NB4 cells with SLPI shRNA in combination with ELANE S97L MUT (which is common for both CN and CyN patients), but not with WT ELANE led to elevated levels of ATF6, GADD34 and BIP. In summary, different ELANE mutations have different effects on UPR as judged by ATF6 activation and the level of ELANE-triggered UPR is regulated by SLPI. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PICH, an ATP-dependent chromatin remodeling protein, transcriptionally co-regulates oxidative stress response.

2021 ◽  
Author(s):  
Anindita Dutta ◽  
Apurba Das ◽  
Deep Bisht ◽  
Vijendra Arya ◽  
Rohini Muthuswami
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Stress Response ◽  
Chromatin Remodeling ◽  
Dna Sequences ◽  
Target Genes ◽  
Antioxidant Response ◽  
Oxidative Stress Response ◽  
Antioxidant Genes

Cells respond to oxidative stress by elevating the levels of antioxidants, signaling, and transcriptional regulation often implemented by chromatin remodeling proteins.  The study presented in this paper shows that the expression of PICH, an ATP-dependent chromatin remodeler, is upregulated during oxidative stress in HeLa cells. We also show that PICH regulates the expression of Nrf2, a transcription factor regulating antioxidant response, both in the absence and presence of oxidative stress. In turn, Nrf2 regulates the expression of PICH in the presence of oxidative stress. Both PICH and Nrf2 together regulate the expression of antioxidant genes and this transcriptional regulation is dependent on the ATPase activity of PICH. In addition, H3K27ac modification also plays a role in activating transcription in the presence of oxidative stress. Co-immunoprecipitation experiments show that PICH and Nrf2 interact with H3K27ac in the presence of oxidative stress. Mechanistically, PICH recognizes ARE sequences present on its target genes and introduces a conformational change to the DNA sequences leading us to hypothesize that PICH regulates transcription by remodeling DNA. PICH ablation leads to reduced expression of Nrf2 and impaired antioxidant response leading to increased ROS content, thus, showing PICH is essential for the cell to respond to oxidative stress.

scholarly journals Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3932-3939 ◽  
Author(s):  
Yang Du ◽  
Nancy A. Jenkins ◽  
Neal G. Copeland
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Drug Targets ◽  
Insertional Mutagenesis ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Serial Passage ◽  
Mouse Bone Marrow ◽  
Cancer Genes ◽  
Differentiation Potential

Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of hematopoietic cells, which normally have only limited self-renewal. Transduction of mouse bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only neo, followed by serial passage in liquid culture containing stem cell factor (SCF) and interleukin-3 (IL-3), produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologs and are validated human myeloid leukemia genes. Integrations are located in intron 1 or 2, where they promote expression of truncated proteins lacking the PRDI-BF1-RIZ1 homologous (PR) domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Genes identified by insertional mutagenesis by their nature could also be involved in immortalization of leukemic stem cells, and thus represent attractive drug targets for treating cancer.

Cooperation of MLL/AF10(OM-LZ) with K-Ras Mutation Induced Myeloid Sarcoma in a Mouse Bone Marrow Transplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1964-1964
Author(s):  
Jen-Fen Fu ◽  
Lee-Yung Shih
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Flow Cytometric Analysis ◽  
Myeloid Sarcoma ◽  
Mouse Bone Marrow ◽  
Myeloproliferative Disease ◽  
Multiple Tumor

Abstract Abstract 1964 Poster Board I-987 We analyzed genetic mutations in a large cohort of AML patients and found that two of the five patients with MLL/AF10 and N-/K-RAS mutations had cutaneous tumors (myeloid sarcomas). To study the cooperative role of MLL/AF10 and N-/K-RAS in the formation of myeloid sarcoma, we established two cell lines by retroviral transduction of MLL/AF10(OM-LZ) and K-RASG12C into GFP-B6 mouse bone marrow cells. Flow cytometric analysis revealed that the cells with MLL/AF10(OM-LZ) and K-RASG12C showed a decreased Mac-1 and CD115 expression when compared with the cells with a single MLL/AF10(OM-LZ) mutation. Microarray and RT-PCR analyses revealed an increased gene expression in Hoxa10 and Meis1, but not Hoxa9. In addition, the phagocytosis related genes, Cybb and Lyz were decreased in the cells harboring MLL/AF10(OM-LZ) and K-RASG12C. These results suggested that cooperation of MLL/AF10(OM-LZ) and K-RASG12C mutations blocked the cells in a more primitive hematopoietic stage. When the two cell lines were intra-peritoneally injected into B6 mice, the mice developed myeloproliferative disease-like myeloid leukemia as that of the mice transplanted with cells carrying a single MLL/AF10(OM-LZ) fusion gene. The median survival time were 33±4.2 and 31.6±5.1 days, respectively, which were shorter than that of the mice transplanted with cells carrying a single MLL/AF10(OM-LZ) fusion gene (49.8±5.0 days). We found that the majority (84%) of mice transplanted with cells harboring both MLL/AF10(OM-LZ) and K-RASG12C mutations formed multiple tumor masses involving gastrointestinal tract, kidney, peritoneum, paraspinal soft tissue, and/or skin. Cytological examination from the imprint smears of tumor masses showed massive infiltrates of leukemia blastic cells. Immunohistochemical stains of the paraffin-fixed histological sections of tumor masses were positive for GFP, confirmed that the tumor cells were generated from the transplanted cell lines. We have established a mouse model which can be used for further study of the myeloid sarcoma formation. Disclosures: No relevant conflicts of interest to declare.

Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2474-2474
Author(s):  
Jesus Duque-Afonso ◽  
Aitomi Essig ◽  
Leticia M Solari ◽  
Tobias Berg ◽  
Heike L. Pahl ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Functional Role ◽  
Target Gene ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 650-650
Author(s):  
Cailin Collins ◽  
Jingya Wang ◽  
Joel Bronstein ◽  
Jay L. Hess
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Target Genes ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modifications ◽  
Conditional Knockout ◽  
Motif Analysis ◽  
Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

RUNX1-ETO-Dependent Transcriptional Repression of RASSF2 Contributes to t(8;21) Leukemia through Evasion of MST1-Driven Apoptosis Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1547-1547
Author(s):  
Samuel A. Stoner ◽  
Elizabeth T. Andrews ◽  
Russell Dekelver ◽  
Stephanie Weng ◽  
Miao-Chia Lo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Stability ◽  
Cell Lines ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Mouse Bone Marrow ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Regulatory Regions ◽  
Primary Mouse

Abstract The t(8;21) chromosomal translocation is among the most frequent recurring cytogenetic abnormalities associated with acute myeloid leukemia (AML), found in 8-12% of de novo AML patients. The t(8;21) results in the stable fusion of the RUNX1 and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (AML1-ETO). RUNX1-ETO is composed of the N-terminal DNA-binding domain of RUNX1 and nearly the entire ETO protein. RUNX1-ETO promotes leukemia development via the recruitment of transcription factor/transcriptional repression complexes (including NCOR, HDACs, p300, etc.) to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation and function, such as CEBPA, SPI1 (PU.1), NFE2, and CSF1R. Despite this knowledge, additional RUNX1-ETO target genes remain poorly characterized, and the complete molecular mechanism through which RUNX1-ETO leads to leukemic transformation remains to be elucidated. We propose that a better understanding of additional RUNX1-ETO target genes will lead to the potential for development of novel therapeutics to treat these patients. One such gene that we initially identified as markedly downregulated in RUNX1-ETO leukemia cells using a mouse model of t(8;21) AML is RASSF2 (Lo et al, Blood, 2012). Assessment of publicly available gene expression data revealed that RASSF2 is specifically downregulated in the bone marrow of t(8;21) AML patients compared to patients of different cytogenetic subtypes or to non-t(8;21) FAB subtype M2 AML patients. Additionally, RT-qPCR analysis confirmed that RASSF2 transcript is downregulated 10-100-fold in the t(8;21) AML cell lines, Kasumi-1 and SKNO-1, compared to non-t(8;21) AML cell lines and normal CD34+ hematopoietic cells. Expression of RUNX1-ETO in a non-t(8;21) AML cell line led to a reduction in RASSF2 mRNA expression, while knockdown of RUNX1-ETO in Kasumi-1 cells resulted in a ~5-fold increase in RASSF2 expression. Assessment of published ChIP-seq data showed that RUNX1-ETO directly binds at two regulatory regions within the RASSF2 genomic locus in t(8;21) AML cell lines and patient samples. Re-expression of RASSF2 at physiological levels in t(8;21) AML cell lines resulted in a modest negative growth phenotype, and greatly sensitized these cells to apoptosis following stimulation with various pro-apoptotic agents. Re-expression of RASSF2 in RUNX1-ETO-transduced primary mouse bone marrow caused these cells to lose their long-term self-renewal ability after 3 weeks in a serial replating/colony formation assay. This loss of self-renewal ability in co-transduced cells was accompanied by a marked increase in apoptosis during each of the first three weeks of replating. Mechanistically, re-expression of full-length RASSF2, but not of a deletion mutant lacking the SARAH heterodimerization domain (RASSF2ΔSARAH), in t(8;21) AML cell lines resulted in increased protein amount of the pro-apoptotic kinase, MST1. This suggests that RASSF2 may be a critical regulator of MST1 protein stability in AML cells. Importantly, modest (2-3-fold) overexpression of MST1 in t(8;21) AML cell lines resulted in a significant increase in apoptosis and caused growth arrest. The effects of RASSF2 or MST1 expression in non-t(8;21) AML cell lines were greatly reduced, suggesting that the cellular context of RUNX-ETO-driven leukemias makes them highly susceptible to MST1-dependent apoptosis. Overall, we have identified the importance of a MST1-driven pro-apoptotic signaling axis in t(8;21) leukemia. RUNX1-ETO-dependent transcriptional repression of RASSF2 may be essential for evasion of this apoptosis signaling during leukemic transformation via reduction of MST1 protein stability. MST1, perhaps better known as the mammalian orthologue of the drosophila Hippo kinase, is a critical tumor suppressor in many solid tumor types; and we believe our studies warrant the continued investigation of this pathway in hematological malignancy. Disclosures No relevant conflicts of interest to declare.

NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2068-2068
Author(s):  
Xi Huang ◽  
Enfan Zhang ◽  
Xing Guo ◽  
Jing Chen ◽  
Xuanru Lin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Acquired Resistance ◽  
Gene Array ◽  
Cell Counting ◽  
Control Group ◽  
Protein Levels ◽  
Significant Difference

Abstract Background: Multiple myeloma (MM) is among the most common hematologic malignancies. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis of MM. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM. Methods: The mRNA and protein levels of NEDD4-1 and its substrates in MM cell lines (H929, LP-1, RPMI8226, OPM-2 and ARP-1) and MM patients were detected by Quantitative Realtime PCR and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8 (CCK8) and flow cytometry. Gene array was used to compare the gene expression profiles of a panel of Bor treated MM cells vs vehicle-treated MM cells. Results: Gene array showed NEDD4-1 was significantly increased in MM cells treated with Bor. MM cells (CD138+ plasma cells of the bone marrow) from refractory/recurrence patients expressed lower NEDD4-1 than primary patient myeloma cells. Also, MM cell lines H929, ARP-1, LP-1 highly expressed NEDD4-1 at mRNA and protein levels. RPMI8226 and OPM-2 were relatively low expressed. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNAs, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05). We also found that PTEN was decreased in the NEDD4-1 KD H929 cell line. Otherwise, phospho-STAT3 (ser727) and oncoprotein c-Myc and Bcl-2 were upregulated. Conclusion: Collectively, our study reveals that inhibition of NEDD4-1 can reduce MM sensitivity to Bor via regulating PTEN, c-Myc and Bcl-2, may be related to JAK/STAT signaling pathway, which suggests that NEDD4-1 probably acts as a novel drug target and therapeutic paradigm in the battle against multiple myeloma. Disclosures No relevant conflicts of interest to declare.

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