Myc-Mediated Lymphomagenesis Is Driven by DNA Methylation Changes Induced by DNMT3B7 Expression and Dnmt3b Heterozygosity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 225-225
Author(s):  
Aparna Vasanthakumar ◽  
Janet B Lepore ◽  
Matthew H Zegarek ◽  
Masha Kocherginsky ◽  
Mahi Singh ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mouse Model ◽  
Cancer Cells ◽  
Conflicts Of Interest ◽  
Dna Methyltransferases ◽  
Dominant Negative ◽  
Full Length ◽  
Dna Hypermethylation ◽  
Conserved Sequence ◽  
E Box

Abstract Abstract 225 Cancer cells are characterized by abnormal DNA methylation, including overall genomic hypomethylation with concurrent region-specific hypo- and hyper-methylation, causing aberrant activation of some genes and the silencing of others. Three DNA methyltransferase (DNMT) enzymes catalyze DNA methylation in eukaryotic cells, DNMT1, DNMT3A, and DNMT3B. We discovered previously that cancer cells exhibit aberrant splicing of the DNMT3B gene, which produces transcripts containing premature stop codons that encode truncated proteins lacking the catalytic domain. When we bred transgenic mice expressing DNMT3B7, one of the aberrantly spliced DNMT3B isoforms found most commonly in cancer cells, with the Eμ-Myc mice, a mouse model for B cell lymphomas, we observed an acceleration of mediastinal lymphomagenesis along with changes in the expression of several genes involved in oncogenesis. The acceleration in tumorigenesis was associated with global DNA hypermethylation, and further analyses showed that these changes in DNA methylation were heterogeneous in tumors derived from Eμ-Myc/DNMT3B7 mice, a phenomenon reminiscent of human tumors. We hypothesized that DNMT3B7 altered DNA methylation by functioning as a dominant negative isoform of full-length endogenous mouse Dnmt3b, and therefore tested a second mouse model that has defects in DNA methylation. The introduction of Dnmt3b heterozygosity (Dnmt3b+/−) into the Eμ-Myc background accelerated mediastinal lymphomagenesis to an even greater extent, with more than 90% of the Eμ-Myc/Dnmt3b+/− mice developing mediastinal lymphomas within the first 120 days. This was also associated with an increase in global DNA methylation as measured by liquid chromatography-mass spectrometry, to a larger extent than in the Eμ-Myc/DNMT3B7 mice. Interestingly, the tumors from Eμ-Myc mice themselves showed global hypermethylation when compared to non-transformed cells from Eμ-Myc mice, suggesting that the transformation of cells that express Myc is a key aspect in the induction of global DNA hypermethylation. These observations led us to the hypothesis that Myc-mediated tumorigenesis is particularly sensitive to changes in DNA methylation. Brenner et al. demonstrated that Myc binds to Dnmt3a/b and recruits the methyltransferases to promoter regions of Myc targets, leading to DNA hypermethylation in these regions. We have also found previously that DNNMT3B7 binds with full-length DNMT3B, by co-immunoprecipitation studies. We hypothesize that either in the presence of DNMT3B7 or with Dnmt3b heterozygosity, Myc-Dnmt3a/b binding at promoters is enhanced, which leads to hypermethylation and repression of gene expression. Using Mycbp, a gene that was repressed in Eμ-Myc/DNMT3B7 tumors, we demonstrated that its promoter region was hypermethylated in both Eμ-Myc/DNMT3B7 and Eμ-Myc/Dnmt3b+/− tumors. The E-box, a conserved sequence located ∼100bp upstream of the transcription start site that Myc binds specifically, was hypomethylated in the Eμ-Myc/DNMT3B7 tumors, suggesting that there was an enrichment of Myc binding at this region. Chromatin immunoprecipitation analyses confirmed increased binding of Myc at the E-box of Mycbp in the Eμ-Myc/DNMT3B7 tumors. Furthermore, we also demonstrated that Myc expression induced all the three DNA methyltransferases, suggesting that Myc-mediated lymphomagenesis may occur using a feedback loop which enhances expression of the DNA methyltransferases to regulate particular genes involved in tumorigenesis. This study offers an insight into the mechanism behind Myc-mediated tumorigenesis and provides evidence for the central role played by changes in DNA methylation patterns in this process. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential effects of two HDAC inhibitors with distinct concomitant DNA hypermethylation or hypomethylation in breast cancer cells

2019 ◽  
Author(s):  
Arunasree M. Kalle ◽  
Zhibin Wang
Keyword(s):  
Breast Cancer ◽  
Dna Methylation ◽  
Cancer Cells ◽  
X Chromosome ◽  
Breast Cancer Cells ◽  
Trichostatin A ◽  
Methylation Status ◽  
Dna Methyltransferases ◽  
Dependent Manner ◽  
Dna Hypermethylation

AbstractDNA methylation and histone acetylation are the two important epigenetic phenomena that control the status of X-chromosome inactivation (XCI), a process of dosage compensation in mammals resulting in active X chromosome (Xa) and inactive X chromosome (Xi) in females. While DNA methyltransferases (DNMTs) are known to maintain the DNA hypermethylation of Xi, it remains to be determined how one or a few of 18 known histone deacetylases (HDACs) contribute(s) to Xi maintenance. Herein we found that HDAC1/2/4/6 were overexpressed in breast cancer cells, MDA-MB-231, with Xa/Xa status compared to normal breast epithelial cells, MCF10A, with Xa/Xi status. Inhibition of these overexpressed HDACs with two different drugs, sodium butyrate (SB) and Trichostatin A (TSA), caused surprisingly distinct effects on global DNA methylation: hypermethylation and hypomethylation, respectively, as well as distinct effects on a repressing histone mark H3K27me3 for heterochromatin and an active mark H3K56ac for DNA damage. Surveying three DNMTs through immunoblot analyses for insights revealed the up- or down-regulation of DNMT3A upon drug treatments in a concentration-dependent manner. These results correlated with the decreased XIST and increased TSIX expression in MDA-MB 231 as a possible mechanism of Xi loss and were reversed with SB treatment. Further RNA-seq analysis indicated differential gene expression correlating with the promoter methylation status of a few genes. Collectively, our results demonstrate a crosstalk between HDACs and DNMTs and the novel involvement of HDACs in skewed Xi in breast cancer.

scholarly journals A novel role for BRCA1 in regulating breast cancer cell spreading and motility

2011 ◽  
Vol 192 (3) ◽  
pp. 497-512 ◽  
Author(s):  
Elisabeth D. Coene ◽  
Catarina Gadelha ◽  
Nicholas White ◽  
Ashraf Malhas ◽  
Benjamin Thomas ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cancer Cells ◽  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Cell Spreading ◽  
Dominant Negative ◽  
Full Length ◽  
Suppressor Activity ◽  
Ubiquitin Ligase Activity ◽  
Mcf 7

BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain–interacting proteins. Ezrin–radixin–moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA11634–1863 acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA11634–1863 in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.

Upregulation of eIF4E in Nucleophosmin 1 (NPM1) Haploinsufficient Cells Alters CCAAT Enhancer Binding Protein Alpha (C/EBPα) Activity: Implications for MDS and AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2432-2432
Author(s):  
Nirmalee Abayasekara ◽  
Michelle Levine ◽  
Niccolo Bolli ◽  
Hong Sun ◽  
Matthew Silver ◽  
...  
Keyword(s):  
Gene Expression ◽  
Translation Initiation ◽  
Translational Control ◽  
Conflicts Of Interest ◽  
Mrna Translation ◽  
Dominant Negative ◽  
Full Length ◽  
Initiation Factor ◽  
Specific Gene ◽  
Specific Gene Expression

Abstract Abstract 2432 NPM1, is a highly conserved, ubiquitous nucleolar phosphoprotein that belongs to the nucleoplasmin family of nuclear chaperones. NPM1−/− mice die at mid-gestation (E11.5) from anemia, underscoring the gene's role in embryonic development. NPM1 is one of the most frequently mutated genes in AML. Mutations in NPM1 are found in 50% of normal karyotype AML patients, and mutant NPM1 (NPMc+) is aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Furthermore, NPM1 maps to a region on chromosome 5q that is the target of deletions in both de novo and therapy-associated human MDS. NPM1 thus acts as a haploinsufficient tumor suppressor in the hematological compartment, although the mechanism of its contribution to dysmyelopoiesis remains unknown. NPM-1+/− mice develop a hematological syndrome similar to that observed in human MDS, and develop AML over time. The NPM1 deficient model therefore provides a platform to interrogate the molecular basis of MDS. We identified nucleophosmin (NPM1) in a screen for protein binding partners of C/EBPα. C/EBPα is a single exon gene, but is expressed as two isoforms that arise by alternate translation start sites to yield a full length C/EBPα p42 and a truncated dominant negative C/EBPα p30 isoform. Translational control of isoform expression is orchestrated by a conserved upstream open reading frame (uORF) in the 5' untranslated region (5'UTR) and modulated by the translation initiation factors eIF4E and eIF2. We generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. These lines are IL-3-dependent and inducible toward neutrophil maturation with GM-CSF and/ or all- trans retinoic acid (ATRA). Neutrophils derived from MNPM1+/− cells display defective neutrophil-specific gene expression, including a cassette of C/EBPα-dependent genes. These observations led us to postulate that myeloid abnormalities in NPM1 deficiency reflect an aberrant NPM1-C/EBPα axis. We show that NPM1 haploinsufficiency upregulates eIF4E (eukaryotic initiation factor 4E) (but not eIF2), which binds the mRNA-Cap (m7-GTP) as part of the mRNA translation initiation complex, eIF4F. Increased eIF4E is observed in about 30% of all malignancies. Initial increased eIF4E levels in MNPM+/− cells likely reflect transcriptional activation by the oncoprotein c-Myc, protein levels of which are also elevated in MNPM1+/− cells. We propose that increased eIF4E then induces increased C/EBPαp30 translation. C/EBPαp30 is a dominant negative inhibitor of full length C/EBPαp42 activity and disrupts normal neutrophil development. Furthermore, we demonstrate that C/EBPαp30 but not C/EBPαp42, activates the eIF4E promoter. We propose a positive feedback loop, wherein increased C/EBPαp30 induced by eIF4E further increases the expression of eIF4E. Our data suggest that NPM1 deficiency modulates neutrophil-specific gene expression by altering C/EBPα. We propose an aberrant feed-forward mechanism that increases levels of both eIF4E and C/EBPαp30 and likely contributes to MDS associated with NPM1 deficiency. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CHK Methylation Is Elevated in Colon Cancer Cells and Contributes to the Oncogenic Properties

2021 ◽  
Vol 9 ◽  
Author(s):  
Shudong Zhu ◽  
Yan Zhu ◽  
Qiuwen Wang ◽  
Yi Zhang ◽  
Xialing Guo
Keyword(s):  
Dna Methylation ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Human Colon ◽  
Dna Methyltransferases ◽  
Colon Cancer Cells ◽  
Normal Colon ◽  
Human Colon Cancer ◽  
Colon Cells ◽  
Matrigel Invasion

Src is an important oncogene that plays key roles in multiple signal transduction pathways. Csk-homologous kinase (CHK) is a kinase whose molecular roles are largely uncharacterized. We previously reported expression of CHK in normal human colon cells, and decreased levels of CHK protein in colon cancer cells leads to the activation of Src (Zhu et al., 2008). However, how CHK protein expression is downregulated in colon cancer cells has been unknown. We report herein that CHK mRNA was decreased in colon cancer cells as compared to normal colon cells, and similarly in human tissues of normal colon and colon cancer. Increased levels of DNA methylation at promotor CpG islands of CHK gene were observed in colon cancer cells and human colon cancer tissues as compared to their normal healthy counterparts. Increased levels of DNA methyltransferases (DNMTs) were also observed in colon cancer cells and tissues. DNA methylation and decreased expression of CHK mRNA were inhibited by DNMT inhibitor 5-Aza-CdR. Cell proliferation, colony growth, wound healing, and Matrigel invasion were all decreased in the presence of 5-Aza-CdR. These results suggest that increased levels of DNA methylation, possibly induced by enhanced levels of DNMT, leads to decreased expression of CHK mRNA and CHK protein, promoting increased oncogenic properties in colon cancer cells.

scholarly journals De Novo Nethyltransferases, DNMT3a and DNMT3b Are Underexpressed in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4818-4818 ◽  
Author(s):  
Pavla Latalova ◽  
Jiri Minarik ◽  
Katerina Smesny Trtkova
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cells ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Dna Methyltransferases ◽  
Rt Pcr ◽  
Cpg Dinucleotides

Abstract Background and aims: Presently, there is growing evidence that along with the important role of genetic abnormalities, epigenetic aberrations are relevant factors in multiple myeloma (MM). As was recently found, genome-wide analysis of DNA methylation reveals epigenetic alterations in plasma cells from patients with MM and individuals with monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by predominant hypomethylation. Transformation into MM is accompanied by progressive hypermethylation with maximum methylation seen in relapsed disease. DNA methyltransferases (DNMTs) catalyze DNA methylation through transfer of methyl group to cytosine of the CpG dinucleotides, resulting in 5-methylcytostine. DNMT1 maintains patterns of methylated cytosine residues in human genome. DNMT3A and DNMT3B are de novo DNA methyltransferases, whose role is to maintain new methylation pattern that forms due to formation of the cancer. Methods: 30 bone-marrow aspirates from individuals with MGUS or MM patients before the treatment initiation were used. The cDNA was synthesized using 100 ng of total RNA in a 20 µl reaction volume (Roche, Diagnostics, Basel, Switzerland). Quantification of DNMT1, DNMT3a and DNMT3b levels by TaqMan® probes (Life Technologies, Grand Island, NY) with Xceed qPCR Master Mix (IAB, BioTech-Europe, Czech Republic) was performed. For normalization, the GAPDH was used. Results: Although MM is characterized by widespread alterations in DNA methylation, we observed that DNMT3a and DNMT3b de novo methyltransferases were underexpressed in both, MGUS individuals and MM patients when compared to DNMT1 expression level (Figure 1). The transcribed genes have increased levels of 5-hydroxymethylcytosine, then the DNMTs activities might compensate for active hydroxymethylation - demethylation. Conclusions: Our results confirm that the expression of de novo DNA methyltransferases is deregulated in MM cell lines. The presented analysis is first of its kind that was performed on human myeloma cell lines, especially with the focus on the residual expression of Dnmt3a. With support of the grant NT14393. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of a Pseudomonas aeruginosa PAO1 DNA Methyltransferase, Its Targets, and Physiological Roles

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Sebastian Doberenz ◽  
Denitsa Eckweiler ◽  
Olga Reichert ◽  
Vanessa Jensen ◽  
Boyke Bunk ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Pseudomonas Aeruginosa ◽  
Galleria Mellonella ◽  
Opportunistic Pathogen ◽  
Dna Methyltransferases ◽  
Infection Model ◽  
Sequence Motif ◽  
Regulatory Rna ◽  
Conserved Sequence ◽  
Small Regulatory Rna

ABSTRACT DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N6-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affected P. aeruginosa pathogenicity in a Galleria mellonella infection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits in P. aeruginosa. Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats. IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria. IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria.

scholarly journals Switch to full-length of XAF1 mRNA expression in prostate cancer cells by the DNA methylation inhibitor

2006 ◽  
Vol 118 (10) ◽  
pp. 2485-2489 ◽  
Author(s):  
Xiaolei Fang ◽  
Zhaoxu Liu ◽  
Yidong Fan ◽  
Chengyun Zheng ◽  
Sten Nilson ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Methylation ◽  
Mrna Expression ◽  
Cancer Cells ◽  
Full Length ◽  
Prostate Cancer Cells ◽  
Methylation Inhibitor ◽  
Dna Methylation Inhibitor

scholarly journals piRNA-823 Is Involved in Cancer Stem Cell Regulation Through Altering DNA Methylation in Association With Luminal Breast Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Xin Ding ◽  
Ya Li ◽  
Jinhui Lü ◽  
Qian Zhao ◽  
Yuefan Guo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Methylation ◽  
Stem Cell ◽  
Cancer Cells ◽  
Cancer Biology ◽  
Breast Cancer Cells ◽  
Dna Methyltransferases ◽  
Luminal Breast Cancer ◽  
Stem Cell Markers ◽  
Luminal Subtype

Cancer stem cells (CSCs) are believed to be the main source of cancer relapse and metastasis. PIWI-interacting small non-coding RNAs (piRNAs) have been recently recognized to be relevant to cancer biology. Whether and how piRNAs regulate human CSCs remain unknown. Herein, upregulation of piR-823 was identified in tested luminal breast cancer cells, especially in the luminal subtype of breast CSCs. Enforced expression or targeted knockdown of piR-823 demonstrated its oncogenic function in regulating cell proliferation and colony formation in MCF-7 and T-47D breast cancer cells. In addition, piR-823 induced ALDH (+) breast CSC subpopulation promoted the expression of stem cell markers including OCT4, SOX2, KLF4, NANOG, and hTERT, and increased mammosphere formation. Tail vein injection of magnetic nanoparticles carrying anti-piR-823 into the mammary gland of tumor-burdened mice significantly inhibited tumor growth in vivo. DNA methyltransferases (DNMTs) including DNMT1, DNMT3A, and DNMT3B were demonstrated to be the downstream genes of piR-823, which regulate gene expression by maintaining DNA methylation. piR-823 increased the expression of DNMTs, promoted DNA methylation of gene adenomatous polyposis coli (APC), thereby activating Wnt signaling and inducing cancer cell stemness in the luminal subtype of breast cancer cells. The current study not only revealed a novel mechanism through which piRNAs contribute to tumorigenesis in breast cancer by regulating CSCs, but also provided a therapeutic strategy using non-coding genomes in the suppression of human breast cancer.

scholarly journals Development of an Immunotherapeutic Monoclonal Antibody Recognizing DKK1-HLA-A2 Complex to Treat Human Hematologic Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5551-5551
Author(s):  
Jianfei Qian ◽  
Qiang Wang ◽  
Lintao Liu ◽  
Enguang Bi ◽  
Xingzhe Ma ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Scid Mouse ◽  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
Tumoricidal Activity ◽  
Normal Tissues ◽  
Scid Mouse Model

Immunotherapy is a promising option for cancer treatment. Our previous studies demonstrated that DKK1 is widely expressed by various tumor cells including multiple myeloma (MM) and other hematological malignancies but not normal tissues, and DKK1 peptide (such as P20 and P66v, which bind with HLA-A2 molecule) specific cytotoxic T cells specifically kill myeloma and other cancer cells that express DKK1 and HLA-A2, but not HLA-A2+ normal cells, indicating that DKK1+ tumor cells naturally express these peptides, in the context of HLA-A2 molecules, on their surface. To develop cancer therapeutic antibodies, DKK1 peptide P20-HLA-A2 monomer was synthesized and used to immunize mice. Hybridomas secreting monoclonal antibodies (mAbs) recognizing soluble and cell surface-expressed DKKl P20-HLA-A2 complex were obtained and analyzed. The mAbs bind specifically with DKK1-expressing, HLAA2+ cancer cells but not DKK1-expressing, HLA-A2- cancer cells or DKK1- HLA-A2+ normal blood cells. The mAbs exhibited potent in vitro tumoricidal activity on HLA-A2+DKK1+ U266 multiple myeloma cells, HLA-A2+DKK1+ PC-3 prostate cancer cells and T2 cells loaded with DKK1-P20 peptides. Our results also showed that the anti-DKK1-HLA-A2 mAbs effectively lysed cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which were correlated with and dependent on the surface expression of DKK1-HLA-A2 complex on cancer cells. Furthermore, anti-DKK1/HLA-A2 mAbs were also active and therapeutic in vivo. In MM xenografted SCID mouse model, the mAbs were able to eradicate U266 MM cells and more than 60% of mAb-treated mice survived for 3 months, while control mice all died within 2 months. Toxicity and safety assay in MM xenografted A2-SCID mouse model showed that the mAbs had no significant negative effects on different normal tissues. Therefore, these results support clinical development of anti- DKK1-HLA-A2 mAbs as therapeutic agents to treat hematological malignancies and possibly solid tumors that express surface DKK1-HLA-A2. Disclosures No relevant conflicts of interest to declare.

Direct Suppression of Fas-Mediated Apoptosis by PMLRARa through Forming An Apoptotic Inhibitory Complex with c-FLIP In Acute Promyelocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3148-3148
Author(s):  
Rong-Hua Tao ◽  
Zuzana Berkova ◽  
Jillian F Wise ◽  
Daniluk Urszula ◽  
Li Bai ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Acute Promyelocytic Leukemia ◽  
Fas Ligand ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Death Receptor ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Nb4 Cells ◽  
Fas Signaling

Abstract Abstract 3148 Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while its dominant negative form, promyelocytic leukemia–retinoic acid receptor α (PMLRARα) fusion protein, activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937/PR9 cells and APL primary cells isolated from patients. The PMLRARα-Fas binding was mapped to the PML B-box domain of PMLRARα and death domain of Fas. Flow cytometry analysis of propidium iodide- and Annexin V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody CH-11 indicated that PMLRARα blocks Fas-mediated apoptosis at early and late stages. In line with this finding, knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Detailed analysis showed that expression of PMLRARα prevents procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11) and thus, also prevents cleavage/activation of procaspase-8. Further analysis indicated that PMLRARα recruits caspase-8 inhibitor c-FLIPL/S to Fas to suppress Fas signaling. A significantly higher number of mice transfected with PMLRARα-expressing plasmid than mice transfected with empty vector survived the treatment with the mouse agonistic anti-Fas antibody Jo2 (11 of 12 vs. 0 of 12; P < 0.001). Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with vector-transfected mice. These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which, as we have shown, can occur by regulation via direct interaction. The newly-discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptossis. By neutralizing the effect of death receptor inhibitors such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Disclosures: No relevant conflicts of interest to declare.

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