Insights Into the Role of ASPP2 (Apoptosis Stimulating Protein of p53-2) in Lymphomagenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1967-1967
Author(s):  
Kerstin M Kampa ◽  
Sandra Mueller ◽  
Michael Bonin ◽  
Marcus M Schittenhelm ◽  
Charles D Lopez
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
T Cell ◽  
Cell Growth ◽  
Tumor Suppressor ◽  
Target Genes ◽  
Tumor Formation ◽  
Transcriptional Networks ◽  
Damage Response

Abstract Abstract 1967 Poster Board I-990 ASPP2 is a member of a family of p53 binding proteins that enhance apoptosis, in part through selective stimulation of p53 transactivation of pro-apoptotic target genes. Low ASPP2 expression is found in many human cancers and has been associated with poor clinical outcome in patients with aggressive lymphoma. Using an ASPP2+/- mouse model, we have previously demonstrated that ASPP2 is a haploinsufficient tumor suppressor and that reduced ASPP2 expression results in attenuated damage-response thresholds (Kampa et al., PNAS 2009). While ASPP2-/- mice are not viable, ASPP2+/- mice have an increased incidence of -irradiation-induced tumors compared to ASPP2+/+ mice.γspontaneous and ASPP2+/- mice develop high-grade thymic T-cell lymphomas after -irradiation. Moreover, primary ASPP2+/- thymocytes have an attenuatedγ -irradiation compared to ASPP2+/+ thymocytes.γapoptotic response after To explore the mechanisms of how attenuated ASPP2 expression could increase thymic lymphomagenesis and attenuate apoptosis, we performed global gene expression profiling on unirradiated, and 5 Gy irradiated ASPP2+/+ and ASPP2+/- thymocytes using an Affymetrix Mouse GeneChip® Array. We found significant differences in gene expression between ASPP2+/+ and ASPP2+/- thymocytes, in both unirradiated and irradiated sets. Using Ingenuity Pathway Analysis software, we found that amongst the highest scoring pathways displaying differences were those associated with cell growth, tumor formation, hematologic malignancies, immune response, cell death and cell cycle regulation. We additionally studied global phosphorylation patterns using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectrometry to determine the posttranscriptional mechanisms mediated by attenuated ASPP2 expression. Analysis of the phosphoproteome of ASPP2+/+ and ASPP2+/- mouse embryonic fibroblasts (with and without irradiation) revealed differences in the phosphorylation status of 108 peptides/proteins including those involved in regulating cell cycle checkpoints, T-cell receptor signaling, cell stress response, DNA repair mechanisms, cell growth, translation and transcription. Differential expression of the identified genes and proteins was verified by PCR and Western Blot. Thus, reduced ASPP2 expression affects global transcriptional as well as post-transcriptional networks intimately involved in the development of hematologic disorders–suggesting that ASPP2 function is by far more complex than solely enhancing the expression of pro-apoptotic p53 target genes. Given that ASPP2 is a bona fide tumor suppressor, reduced ASPP2 levels result in global dysregulation of pathways engaged in tumor suppression networks and the cellular damage response, which may ultimately promote genomic instability and tumor formation. Our findings provide insights into the role of ASPP2 in lymphomagenesis and reveal possible new targets for cancer therapy. Disclosures: No relevant conflicts of interest to declare.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2

2019 ◽  
Vol 105 (3) ◽  
pp. 839-853
Author(s):  
Aglaia Kyrilli ◽  
David Gacquer ◽  
Vincent Detours ◽  
Anne Lefort ◽  
Frédéric Libert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Iodide Uptake ◽  
Transcriptomic Response ◽  
Uptake Inhibition ◽  
Γ Radiation ◽  
Damage Response

Abstract Background The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

Williams-Beuren Syndrome Critical Region-5/Non-T Cell Activation Linker: A Novel Target Gene of AML1/ETO.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2898-2898
Author(s):  
Michael Lübbert ◽  
Michael Stock ◽  
Tobias Berg ◽  
Manfred Fliegauf
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
T Cell Activation ◽  
Critical Region ◽  
Cell Activation ◽  
Target Genes ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
Acute Myeloid

Abstract The chromosomal translocation (8;21) fuses the AML1 gene on chromosome 21 and the ETO gene on chromosome 8 in human acute myeloid leukemias, resulting in expression of the chimeric transcription factor AML1/ETO. AML1/ETO-mediated dysregulation of target genes critical for hematopoietic differentiation and proliferation is thought to contribute to the leukemic phenotype. Several mechanisms, including recruitment of histone deacetylases (HDACs) to AML1 target genes, may be responsible for altered gene expression. We used an ecdysone-inducible expression system in the human monoblastic U-937 cell line to isolate genes that were differentially expressed upon induction of AML1/ETO expression. By representational difference analysis (cDNA-RDA), we identified 26 genes whose expression levels were significantly modulated following AML1/ETO induction for 48 hours. None of these genes has previously been described as a target of AML1, ETO or AML1/ETO. One gene down-regulated by AML1/ETO in vitro, Williams Beuren Syndrome critical region 5 (WBSCR5), was expressed in primary t(8;21) negative AML blasts but not in primary t(8;21) positive AML blasts, strongly implying a role of this gene in the phenotype of t(8;21) positive AML. WBSCR5 is part of the critical region located on chromosome 7q11.23 that is deleted in the Williams Beuren syndrome (OMIM 194050), an autosomal dominant disorder comprising vascular, neurological, behavioral and skeletal abnormalities. WBSCR5 has recently been shown to have a role in the activation and differentiation of B cells (Brdicka et al., J. Exp. Med. 196:1617, 2002) and thus was also termed Non-T cell activation linker.. WBSCR5 as well as seven other regulated genes were further studied using all-trans-retinoic acid (ATRA), an inducer of differentiation of U-937 cells, and Trichostatin A (TSA), an HDAC inhibitor. WBSCR5 and two other out of these eight genes were regulated during ATRA-induced monocytic differentiation of U-937 cells, however none of them antagonistically, upon both ATRA-treatment and AML1/ETO-induction. Since repression of WBSCR5 might be mediated by recruitment of HDACs through the fusion gene, cells were treated with TSA prior to transgene induction. However, the AML1/ETO-associated dysregulation of WBSCR5 gene expression (as well as that of the other seven genes studied) was not mediated by a TSA-sensitive mechanism. The identified genes provide a useful model to study the mechanism by which the AML1/ETO fusion protein exerts its function in transcriptional dysregulation in acute myeloid leukemia. The role of WBSCR5 in malignant hematopoietic cells warrants further study.

scholarly journals Restoration of miR-193a-5p and miR-146 a-5p Expression Induces G1 Arrest in Colorectal Cancer through Targeting of MDM2/p53

2019 ◽  
Vol 10 (1) ◽  
pp. 130-134 ◽  
Author(s):  
Saeed Noorolyai ◽  
Elham Baghbani ◽  
Leili Aghebati Maleki ◽  
Amir Baghbanzadeh Kojabad ◽  
Dariush Shanehbansdi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cell Cycle Progression ◽  
Cyclin B ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Ht 29

Purpose: Colorectal cancer (CRC) remains a universal and lethal cancer owing to metastatic and relapsing disease. Currently, the role of microRNAs has been checked in tumorigeneses. Numerous studies have revealed that between the tumor suppressor miRNAs, the reduced expression of miR-146a-5p and -193a-5p in several cancers including CRC tissues are related with tumor progression and poor prognosis of patients. The purpose of this study is to examine the role of miR-146 a-5p and -193 a-5p in CRC cell cycle progression. Methods: The miR-193a-5p and -146 a-5p mimics were transfected into HT-29 CRC cells via jetPEI transfection reagent and their impact was assessed on p53, cyclin B, and NF-kB gene expression. The inhibitory effect of these miRNAs on cell cycle was assessed by flow cytometry. The consequence of miR-193a-5p and miR-146 a-5p on the protein expression level of Murine double minute 2 (MDM2) was assessed by western blotting. Results: miR193a-5p and -146a-5p regulated the expression of MDM2 protein and p53, cyclin B, and NF-kB gene expression in CRC cells. Treatment of HT-29 cells with miRNA-146a-5p and -193a-5p induced G1 cell cycle arrest. Conclusion: The findings of our study suggest that miR146a-5p and -193a-5p may act as a potential tumor suppressor by their influence on cell cycle progression in CRC cells. Thus, miRNA-146a-5p and -193a-5p restoration may be recommended as a potential therapeutic goal in the treatment of CRC patients.

Apoptosis Stimulating Protein of p53 (ASPP2) Heterozygous Mice Are Lymphoma-Prone and Display Attenuated Apoptosis and Cell Cycle Checkpoints

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2844-2844
Author(s):  
Kerstin Maria Kampa ◽  
Jared Acoba ◽  
Dexi Chen ◽  
Hun-Joo Lee ◽  
Kelly Beemer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Preliminary Analysis ◽  
Ex Vivo ◽  
Global Gene Expression ◽  
Tumor Formation ◽  
Cell Cycle Checkpoints ◽  
Cellular Damage ◽  
High Grade ◽  
Damage Response

Abstract Introduction: p53 is an important tumor suppressor, and loss of p53 pathway function is a common event in human cancer. ASPP2 is a damage-inducible p53 binding protein that enhances apoptosis, at least in part, by selective stimulation of p53 pro-apoptotic target genes. Low ASPP2 expression occurs in many human tumors and as we have previously demonstrated, correlates with poor clinical outcome in patients with B-cell lymphomas. However, the mechanisms by which ASPP2 suppresses tumor formation remain unknown. Methods: To rigorously study ASPP2 in vivo function, we targeted the ASPP2 allele in a mouse model by homologous recombination by replacing exons 10–17 with a neoR gene. Aging mice were followed for spontaneous tumor formation. Additionally, six week old mice were irradiated at different doses and followed for tumor development. To explore the functional consequences of low ASPP2 expression, ASPP2+/− and ASPP2+/+ thymocytes were subjected ex vivo to 5Gy ionizing irradiation and apoptosis was assessed by Annexin-V/PI staining and flow cytometry. We also irradiated ASPP2+/+ and ASPP2+/−MEFs (mouse embryonic fibroblasts) with 5Gy and performed cell cycle analysis. Furthermore, we conducted global gene expression profiling between the unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using an Affymetrix GeneChip® Mouse Gene 1.0 ST Array. Moreover, phosphoproteomic analysis was performed on unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectroscopy. Results: We were unable to generate ASPP2−/− mice due to an early embryonic lethal defect. However, ASPP2+/− mice appear developmentally normal and reproduce. We observed an increased formation of spontaneous tumors in aging ASPP2+/− mice compared to ASPP2 +/+ mice (43% vs. 22%, at 115 weeks, p=0.011). Additionally we were able to show that after ionizing radiation, ASPP2+/− mice develop high-grade lymphomas in a dose-dependent manner at a significantly higher incidence at 50 weeks compared to ASPP2+/+ mice (p = 0.024 and p = 0.045, 6 Gy and 10.5 Gy respectively). Immunophenotyping demonstrated that these were high-grade T-cell lymphomas of thymic origin. Since ASPP2 is damage-inducible and promotes apoptosis, we wished to determine the extent to which reduction in ASPP2 expression attenuated the cellular damage response. We therefore gamma-irradiated ex vivo ASPP2+/+ and ASPP2+/−thymocytes in short-term culture and found a two-fold reduction in apoptosis in ASPP2+/− thymocytes compared to ASPP2+/+ thymocytes. Additionally, after 5 Gy gamma-irradiation, ASPP2+/− MEFs exhibited an attenuated G0/G1 checkpoint compared to ASPP2+/+ MEFs. Preliminary analysis of global gene expression in ASPP2+/+ and ASPP2+/− MEFs shows specific differences in gene expression patterns after damage. Likewise, preliminary analysis of phosphoproteomics between ASPP2+/+ and ASPP2+/− MEFs after damage, demonstrate differences in the phosphophorylation status of 170 proteins. Conclusions: ASPP2 is a haploinsufficient tumor suppressor, and reduction in ASPP2 expression attenuates both cell cycle checkpoints and apoptosis-induction after damage. These results suggest that reduction of ASPP2 levels modulate the cellular damage response, possibly at transcriptional as well as post-transcriptional levels, and provide a functional explanation for the increased tumor incidence in ASPP2+/− mice---since attenuated damage-response thresholds would lead to an impaired ability to eliminate cells that have accumulated tumorigenic mutations. Our study provides important insights into the p53-ASPP2 axis, and opens new avenues for investigation into its role in tumorigenesis and response to therapy.

Genome-Wide Chromatin Immunopreciptiation and Gene Expression Analysis Indicates That MMSET Is a Transcriptional Repressor in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 627-627
Author(s):  
Dong-Joon Min ◽  
Julia Meyer ◽  
Eva Martinez-Garcia ◽  
Josh Lauring ◽  
Jonathan D. Licht
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Signaling Pathway ◽  
Target Genes ◽  
Transcriptional Repressor ◽  
Low Levels

Abstract Multiple myeloma (MM) is a malignancy of plasma cells characterized by frequent chromosomal translocations of the immunoglobulin heavy chain (IgH) locus. The multiple myeloma SET domain (MMSET) gene is a recurrent chromosomal partner in the t(4;14) translocation, and MMSET levels are elevated in these patients relative to other myeloma cases and normal cells. Previously, we showed that MMSET is a histone methyltransferase with specific activity for lysine 20 on histone H4 and acts as a transcriptional repressor when tethered to a model target gene. To reveal the function of MMSET in t(4;14) MM in vivo, we identified MMSET target genes in the KMS11 t(4;14) MM cell line. Chromatin from these cells was subjected to immunoprecipitation with a polyclonal anti-MMSET antibody in biological replicate, amplified by ligation-mediated PCR and hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,659 human promoters. Data analysis using the MaxFour algorithm ranked putative binding sites based upon intensities of 4 consecutive probes. The top 2000 promoters identified from each experiment were combined to yield a list of 1,412 putative MMSET target genes. This list was analyzed using the DAVID program (david.abcc.ncifcrf.gov/). Genes bound by MMSET includes those implicated in antigen processing and presentation (p<8.7×10-4), cell cycle (p<2.2×10-3), the p53 signaling pathway (p<0.03) apoptosis (p<1.6×10-6) and DNA repair (p<3.3×10-4) Among genes bound by MMSET were XBP1, IRF2, and BCL6, all important transcription factors regulating B cell development. Real-time quantitative PCR validated MMSET binding in 6/6 promoters tested so far. To investigate the role of MMSET in transcriptional regulation, we profiled gene expression in KMS11 cells using Illumina arrays to determine the expression of MMSET bound genes. Nearly 50% of genes bound by MMSET had very low levels of expression (≤100, Range on arrays 10–18,000) while only 13% of genes bound by MMSET were expressed at high levels (>1000). This supports the notion that MMSET represses target genes in vivo. Functional annotation of genes bound by MMSET and expressed at low levels showed over-representation of genes implicated in toll-like receptor signaling pathway (p<2.8×10-3), cytokine-signaling (p<3.3×10-3) and JAK2/STAT signaling (p<0.08), transmembrane receptor function (p<4×10-8), and apoptosis (p<0.01), while those bound yet expressed at high levels were implicated in oxidative phosphorylation (p<3.9×10-4) and protein synthesis (p<4.1×10-6). The effects of MMSET on gene expression were further investigated using KMS11KO cells in which the rearranged MMSET allele was ablated by homologous recombination. RNA from KMS11 and KMS11KO cells was profiled by Illumina arrays and genes showing a significant change in gene expression were determined by significance analysis of microarray (SAM) testing with 1% of false discovery rate. Among the 720 genes bound by MMSET and expressed at a level of >100 in the wild-type KMS11 cells, 35 genes were up-regulated and 20 genes were down-regulated (>1.5 fold) in the KMS11KO cell line. Among the 692 genes bound by MMSET and expressed at a level of ≤100, 9 genes were up-regulated in KMS11KO cells. The up-regulated genes (presumably bound and repressed by MMSET) were categorized in cytokine receptor (p<0.02) and JAK2/STAT signaling pathway (p<0.05), nucleosome assembly (p<6×10-4), apoptosis (p<0.01), and cell differentiation (p<0.05). Collectively these data suggest that MMSET may interfere with signal transduction, chromatin modulation and apoptosis pathways involved in the terminal differentiation of the plasma cell. Intriguingly MMSET also bound and was associated with repression of RB1 and RBL2 suggesting a role of MMSET in cell cycle control. Chromatin immunoprecipitation analysis of a MMSET bound gene (ARHGAP25) revealed that MMSET binding was correlated with increased tri-methylation of H4K20, a repression-associated chromatin mark. MMSET binding of this promoter was decreased but still detectable in the KMS11KO cells. Collectively these data suggest that MMSET binds and represses many target genes in vivo. However MMSET could still bind to genes expressed at a high level and MMSET ablation was associated with activation of some MMSET target genes, suggesting that its role in gene regulation may be complex and potentially gene-specific.

The Role Of BIN1 Tumor Suppressor Isoforms In Regulation Of Proliferation, Apoptosis and Tumor Formation Of Human Cutanous T-Cell Lymphoma Cells In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2517-2517
Author(s):  
Sharmin Esmailzadeh ◽  
Youwen Zhou ◽  
Xiaoyan Jiang
Keyword(s):  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Tumor Formation ◽  
Post Injection ◽  
Apoptosis Pathway ◽  
Sézary Cells

Abstract Cutaneous T-cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders that are characterized by homing of malignant T-cells to the surface of skin. There are two main types of CTCL: Mycosis Fungoides (MF) and its leukemic variant Sezary Syndrome (SS), which together represent about 65-70% of all CTCL cases. The precise genetic pathogenesis of these diseases remains largely undetermined. Recently, our research group has demonstrated that AHI-1 (Abelson Helper Integration site-1) oncogene is involved in CTCL. Expression of AHI-1 is increased in human leukemia cell lines, with marked upregulation (up to 40 fold) in CTCL lines (Hut78 and Hut102). Moreover, in FACS-purified CD4+CD7- Sezary cells from patients with Sezary Syndrome, AHI-1 expression is higher at both the RNA and protein levels compared to normal CD4+ cells. Furthermore, stable suppression of endogenous AHI-1 in Hut78 cells using small interfering RNA, normalizes their transforming activity both in vitro and in vivo. Thus, lymphomagenic activity of Hut78 cells is partially dependent on the expression of AHI-1. Interestingly, BIN1 (Bridging integrator 1) was identified through microarray analysis as one of the genes that may be involved in AHI-1-mediated leukemic transformation in CTCL cells. BIN1 is a nucleocytosolic adaptor protein with more than ten isoforms; some isoforms, including the BIN1 isoform (+10, +13), act as tumor suppressors, whereas the BIN1 (+12A) behaves as a cancer-related isoform in solid tumor models. However, the role of BIN1 in regulation of normal hematopoiesis and lymphomagenesis remains unknown. We have recently demonstrated that transcript levels of BIN1 isoforms are significantly lower in patients with MF or SS compared to controls. Four isoforms of BIN1 have been identified in Hut78 and primary CD4+CD7- Sezary cells. To investigate the role of BIN1 in CTCL, the BIN1 isoforms (+10, +13) and BIN1 (+12A) lentiviral constructs were transduced into two CTCL cell lines, Hut78 and HH cells. Overexpression of BIN1 isoforms led to a significant reduction in cell proliferation, as assessed by colony forming cell assays and 3H-Thymidine uptake assays (2-3 fold, p<0.05). Furthermore, a significant increase in spontaneous and specific apoptosis was observed in BIN1-transduced cells, with and without exogenous FAS-ligand (2-3 fold, p<0.05). Interestingly, a significant reduction in protein expression of c-FLIP (inhibitor of the FAS-mediated apoptosis pathway) and upregulation of downstream cleaved caspase-8 and caspase-3 was demonstrated in BIN1-transduced cells, suggesting that BIN1 isoforms induce apoptosis by downregulating the expression of c-FLIP, which leads to activation of the FAS-mediated apoptosis pathway. These findings show anti-proliferative and pro-apoptotic roles for BIN1 isoforms in human CTCL cells. In addition, subcellular fractionation and confocal microscopy further indicated that both the BIN1 (+10, +13) and BIN1 (+12A) isoforms are mainly located in the nucleus in Hut78 and HH cells. Furthermore, to investigate the effects of overexpression of BIN1 isoforms on the ability to induce tumors in vivo, we tested their leukemogenic potential by injecting transduced HH cells into non-obese diabetic/severe-combined immunodeficiency (NOD/SCID) mice. Mice injected subcutaneously with either parental HH or control empty vector cells (2 x 107/per mouse), showed local tumor formation in 6 of 6 mice within 4 days post-injection. The local tumors enlarged progressively and were often 1.5–2 cm in diameter by 3 weeks after injection. In contrast, no local tumors formed in mice given injections of equal numbers of BIN1-transduced HH cells after 14 days, in 12 out of 12 mice. Tumor formation was only observed in BIN1-transduced HH cells after 3 weeks post-injection. However, the local tumors were significantly smaller in BIN1-transduced HH cells compared to controls (∼4-fold). These findings further indicate that the two BIN1 isoforms have tumor suppressor activities in NOD/SCID mice and can significantly delay tumor formation and reduce tumor size in vivo. Disclosures: No relevant conflicts of interest to declare.

Retinoic Acid Receptor Alpha Expression Drives Cell-Cycle Progression and Is Associated with Increased Sensitivity to Retinoids in Peripheral T-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1749-1749
Author(s):  
Rebecca L Boddicker ◽  
Xueju Wang ◽  
Surendra Dasari ◽  
Grzegorz S. Nowakowski ◽  
Konstantinos N Lazaridis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Research Funding ◽  
Retinoic Acid Receptor ◽  
Wild Type ◽  
T Cell Lymphomas

Abstract Background: Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with marked clinical, pathological, and molecular heterogeneity. Outcomes following standard therapy generally are poor; however, few candidate therapeutic targets have been identified for precision medicine approaches. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to natural or synthetic retinoids. Retinoids have been used successfully to treat acute promyelocytic leukemia and some cutaneous T-cell lymphomas (CTCLs). However, the function of RARA and the action of retinoids in PTCL have not been defined. Methods:Based on identification of a PTCL patient with a non-synonymous point mutation, RARA R394Q, identified in the Mayo Clinic Center for Individualized Medicine, we sought to characterize the role of RARA in PTCL cells. To investigate the role of wild-type and mutant RARA, we constructed expression vectors containing either wild-type RARA or RARA R394Q coding sequences, and also used siRNAs targeting RARA to study the role of native RARA expression. Cell lines derived from post-thymic T-cell malignancies were used for in vitro studies, including HuT78 and Mac-1 (both derived from circulating tumor cells from CTCL patients) and Karpas 299 (from an ALK-positive anaplastic large cell lymphoma). Following RARA overexpression or knockdown, we measured cell growth, cell cycle regulation, and sensitivity to synthetic retinoids. In addition, RNA sequencing and pathway analysis were performed to profile the transcriptomic response to retinoids in malignant T cells. Results:In two RARAlow cell lines, Karpas 299 and HuT78, overexpression of wild-type RARA or RARA R394Q significantly increased cell growth (p<0.001), with a greater increase observed from mutant versus wild-type RARA in Karpas 299 (136% of control versus 122%; p=0.04). Accordingly, knockdown of wild-type RARA in the RARAhigh cell line, Mac-1, resulted in a 22% inhibition of cell growth (p=0.0002). This inhibition specifically was associated with G1 cell cycle arrest (120% of control; p=0.004) and decreased protein expression of the G1-S-associated cyclin-dependent kinases, CDK2, CDK4, and CDK6. These kinases were up-regulated by overexpression of RARA in RARAlow HuT78 cells. The relatively RARA-specific retinoid, AM80 (tamibarotene), and the less specific retinoid, all-trans retinoic acid (ATRA), resulted in RARA protein degradation, cell growth inhibition that was both dose-dependent and proportional to baseline RARA expression, G1 arrest, and CDK protein up-regulation. Gene-set enrichment analysis (GSEA) of transcriptome data confirmed that genes down-regulated by AM80 were highly enriched for regulators of cell cycle and particularly G1-S transition. Finally, overexpressing RARA in RARAlow Karpas 299 and HuT78 cell lines significantly increased the ability of AM80 to inhibit CDK2/4/6 expression and cell growth (16% to 23% greater growth inhibition than control; p<0.05). Conclusions:RARA drives cyclin-dependent kinase expression and G1-S transition in malignant T cells, and promotes cell growth. These functions may be enhanced by specific RARA gene mutations. Synthetic retinoids inhibit these functions in a dose-dependent fashion, and are most effective in cells with high RARA expression. These data suggest RARA as a candidate therapeutic target in some PTCL patients. Disclosures Nowakowski: Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

scholarly journals The Histone Demethylase KDM6B Is a Genetic Dependency of NOTCH1-Driven T-ALL

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 782-782
Author(s):  
Nancy Issa ◽  
Hamza Celik ◽  
Hassan Bjeije ◽  
Wangisa M.B. Dunuwille ◽  
Won Kyun Koh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
T Cell ◽  
Genetic Alterations ◽  
Histone Demethylase ◽  
Control Group ◽  
Post Transplant ◽  
T Cell Leukemogenesis ◽  
Over Time

Abstract T-cell acute lymphoblastic leukemia (T-ALL) arises from the accumulation of genomic abnormalities and the malignant proliferation of immature T-cells. Despite recent advancements in understanding the genetic alterations driving T cell leukemogenesis, patients still suffer from recurrent relapses and treatment-related toxicities. Genome sequencing has revealed significant heterogeneity and important insights into the genetic landscape of T-ALL. Mutations in epigenetic modifiers are frequently observed and serve as an attractive target for novel therapeutic approaches. Histone demethylase enzymes play a critical role in the regulation of gene expression programs in T-ALL. KDM6A (UTX) is known to behave as a tumor suppressor in most T-ALL subtypes. However, it's gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed in patients. Here, we show that KDM6B is required for T-ALL initiation. Using genetic mouse models, Sca-1 enriched WBM from Vav-Cre: Kdm6b+/+, Vav-Cre: Kdm6bfl/+, and Vav-Cre: Kdm6bfl/fl adult mice was transduced with a retrovirus expressing Notch1 Intracellular Domain (NICD). NOTCH1 gain-of-function mutations are the most frequent driver events in adult T-ALL, and this model recapitulates many of the human pathologies. Transduced cells were transplanted into irradiated mice. While there was robust engraftment in all groups at four weeks post-transplant, T-ALL cells were not sustained in the genetic absence of Kdm6b. Mice receiving control NICD-GFP+ cells succumbed to T-ALL with median survival of 79 days, whereas the only mice receiving Kdm6b-null NICD-GFP+ cells that developed disease were found to retain one copy of the Kdm6b floxed allele (Fig 1). To investigate the translational potential, we targeted KDM6B for genetic inactivation by CRISPR/Cas9 in primary T-ALL patient cells, followed by xenograft into NSG mice. The effect of KDM6B targeting was quantified over time by monitoring the variant allele fraction (VAF) of the transplanted cells. In most patient samples, KDM6B-targeted cells were significantly outcompeted over time, thus further highlighting the requirement of KDM6B in sustaining T-ALL tumorigenesis. To examine the mechanism by which KDM6B sustain T-ALL cells, gene expression profiling was performed by RNA-seq on mouse T-ALL cells of genetic backgrounds Control, Kdm6b-Het, and Kdm6b-KO. Three gene sets were significantly downregulated in Kdm6b-deficient T-ALL cells compared to the Control group, all of which are involved in cell cycle processes. Additional validation of these findings with cell cycle functional studies is currently ongoing. Additionally, while Kdm6b has been described for its H3K27me3 histone demethylase function, recent studies have shown its involvement in regulating various gene expression programs through demethylase-independent mechanisms. To determine if the role of Kdm6b in T-ALL oncogenesis was related to it demethylase activity, we performed the same NICD retroviral transduction experiment with inclusion of a Kdm6b +/H1388Amouse genotype, a point mutation which renders Kdm6b catalytically dead. Our data shows that Kdm6b +/H1388Aphenocopies T-ALL with Kdm6b homozygous loss-of-function, showing no evidence of disease in the blood beyond 8-weeks post-transplant. We conclude from this that the leukemogenic role of Kdm6b requires it's H3K27me3 demethylase function. In summary, these data reveal Kdm6b as an oncogenic dependency in Notch1-driven T-ALL in both mouse and human systems, and present Kdm6b as a high value therapeutic target in adult T-ALL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals EGFR-ERK induced activation of GRHL1 promotes cell cycle progression by up-regulating cell cycle related genes in lung cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Yiming He ◽  
Mingxi Gan ◽  
Yanan Wang ◽  
Tong Huang ◽  
Jianbin Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Potential Role ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Phosphorylation Site ◽  
Promoter Regions ◽  
Cycle Progression

AbstractGrainyhead-like 1 (GRHL1) is a transcription factor involved in embryonic development. However, little is known about the biological functions of GRHL1 in cancer. In this study, we found that GRHL1 was upregulated in non-small cell lung cancer (NSCLC) and correlated with poor survival of patients. GRHL1 overexpression promoted the proliferation of NSCLC cells and knocking down GRHL1 inhibited the proliferation. RNA sequencing showed that a series of cell cycle-related genes were altered when knocking down GRHL1. We further demonstrated that GRHL1 could regulate the expression of cell cycle-related genes by binding to the promoter regions and increasing the transcription of the target genes. Besides, we also found that EGF stimulation could activate GRHL1 and promoted its nuclear translocation. We identified the key phosphorylation site at Ser76 on GRHL1 that is regulated by the EGFR-ERK axis. Taken together, these findings elucidate a new function of GRHL1 on regulating the cell cycle progression and point out the potential role of GRHL1 as a drug target in NSCLC.

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