Response to histone deacetylase inhibition of novel PML/RARα mutants detected in retinoic acid–resistant APL cells

Blood ◽  
2002 ◽  
Vol 100 (7) ◽  
pp. 2586-2596 ◽  
Author(s):  
Sylvie Côté ◽  
Angelika Rosenauer ◽  
Andrea Bianchini ◽  
Karen Seiter ◽  
Jonathan Vandewiele ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Fusion Gene ◽  
Combined Treatment ◽  
Resistant Cell Line ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Histone Deacetylase Inhibition

Resistance to all-trans retinoic acid (ATRA) remains a clinical problem in the treatment of acute promyelocytic leukemia (APL) and provides a model for the development of novel therapies. Molecular alterations in the ligand-binding domain (LBD) of the PML/RARα fusion gene that characterizes APL constitute one mechanism of acquired resistance to ATRA. We identified missense mutations in PML/RARα from an additional ATRA-resistant patient at relapse and in a novel ATRA-resistant cell line, NB4-MRA1. These cause altered binding to ligand and transcriptional coregulators, leading to a dominant-negative block of transcription. These mutations are in regions of the LBD that appear to be mutational hot spots occurring repeatedly in ATRA-resistant APL patient cells. We evaluated whether histone deacetylase (HDAC) inhibition could overcome the effects of these mutations on ATRA-induced gene expression. Cotreatment with ATRA and TSA restoredRARβ gene expression in NB4-MRA1 cells, whose PML/RARα mutation is in helix 12 of the LBD, but not in an APL cell line harboring the patient-derived PML/RARα mutation, which was between helix 5 and 6. Furthermore, ATRA combined with TSA increases histone 4 acetylation on the RARβ promoter only in NB4-MRA1 cells. Consistent with these results, the combined treatment induces differentiation of NB4-MRA1 only. Thus, the ability of an HDAC inhibitor to restore ATRA sensitivity in resistant cells may depend on their specific molecular defects. The variety of PML/RARαmutations arising in ATRA-resistant patients begins to explain how APL patients in relapse may differ in response to transcription therapy with HDAC inhibitors.

scholarly journals A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4282-4289 ◽  
Author(s):  
Wenlin Shao ◽  
Laura Benedetti ◽  
William W. Lamph ◽  
Clara Nervi ◽  
Wilson H. Miller
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Ligand Binding ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Regulation Of Gene Expression ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

Abstract The unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.

scholarly journals Multilevel Molecular Profiling to Dissect Resistance to Tyrosine Kinase Inhibitors in TEL/ABL Positive Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3637-3637
Author(s):  
Olga Zimmermannova ◽  
Veronika Kanderova ◽  
Daniela Kuzilkova ◽  
Fridtjof Lund-Johansen ◽  
Eliska Doktorova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Molecular Profiling ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Sensitive Cell Line ◽  
Abl Kinase ◽  
Tki Resistance

Abstract Acute lymphoblastic leukemias (ALLs) bearing TEL/ABL (ETV6/ABL1) fusion gene are generally rare but can be identified within the Ph-like (BCR/ABL-like) ALLs - a poor risk ALL subtype characterized by the presence of various molecular lesions activating kinase signaling. Identification of these druggable lesions brought the possibility to improve the outcome of Ph-like ALL by incorporation of targeted therapeuticals – e.g. tyrosine kinase inhibitors (TKI) – into the current treatment strategies. In vitro studies proved the efficiency of TKI to inhibit TEL/ABL kinase activity justifying TKI use in clinical practice. However, there is a lack of knowledge on potential development of TKI resistance and its mechanism in TEL/ABL-positive ALL. To address this issue we aimed to establish TKI resistant TEL/ABL-positive cell line and study the mechanism of acquired resistance. After a long-term exposure to gradually growing doses of imatinib mesylate we induced a stable pan-TKI resistance not resulting from the most common mechanisms described in BCR/ABL ALL such as ABL kinase domain mutations, fusion gene amplification or enhanced fusion gene expression/activity. To elucidate the molecular basis of the resistance we performed multilevel molecular profiling. Using high density whole genome SNP array we identified a single acquired copy number aberration in the resistant cell line - a 60 kb intragenic deletion in KDM6A gene encoding lysine-specific histone demethylase. However, this deletion did not result in expression of the predicted aberrant protein. Using whole exome sequencing we identified 25 non-synonymous single nucleotide variants (SNVs) within gene coding regions to be gained by the resistant cell line. Only half of these variants (12/25) affected genes transcribed in the studied cell line and were thus subjected to further verification by Sanger sequencing. Finally, 5 of the 12 SNVs were confirmed to be stably present and expressed in the resistant cell line. All the affected genes – AEBP1, GNB1, KMT2C, RREB1, STAM2 – are involved in important biological processes and have not been associated with TKI resistance yet. Gene expression profiling of multiple replicates of parental sensitive and induced resistant cell line identified a gene expression pattern associated with TKI resistance consisting of more than 500 genes with significantly changed expression. Further analysis of this pattern revealed broad changes in many unrelated biological pathways and processes, hence providing a limited potential to specifically focus the study direction. We further employed two independent methods to study the resistant cell line proteome. Using 2-dimensional electrophoresis of the whole cell protein lysates we detected a single differentially expressed protein spot present in the resistant compared to parental sensitive cell line. Strikingly, mass spectrometry identified this protein as a transducin beta chain, product of the aforementioned GNB1 gene, one of the five beta subunits of human G-proteins playing an important role as signal transmitters. Using Size-Exclusion Chromatography-Microsphere-based Affinity Proteomic array (SEC-MAP; Wu, MCP 2009) we analyzed the total expression of more than 300 proteins by flow cytometry. We have found 39 proteins to be differentially expressed in resistant compared to parental sensitive cell line. Interestingly, several distinctive changes indicate enhanced B-cell receptor signaling in the resistant cell line. This mechanism was already implicated as a potential strategy of resistant cells for evading TKI induced apoptosis in BCR/ABL-positive leukemia (Klein, Cell Cycle 2004). In conclusion we have successfully used several high-throughput technologies to study genomic, transcriptomic and proteomic changes associated with acquired TKI resistance in TEL/ABL-positive ALL model and identified dysregulation of G-protein and BCR-signaling as its potential causes. Support: IGA MZ NT/13170-4; GAUK 694414; GAUK 596912; UNCE 204012; RVO-FNM64203; BIOCEV –(CZ.1.05/1.1.00/02.0109) from the ERDF Disclosures No relevant conflicts of interest to declare.

scholarly journals Genome-Wide STAT3 Binding Analysis after Histone Deacetylase Inhibition Reveals Novel Target Genes in Dendritic Cells

2016 ◽  
Vol 9 (2) ◽  
pp. 126-144 ◽  
Author(s):  
Yaping Sun ◽  
Matthew Iyer ◽  
Richard McEachin ◽  
Meng Zhao ◽  
Yi-Mi Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Histone Deacetylase ◽  
Antigen Processing ◽  
Target Genes ◽  
Hdac Inhibition ◽  
Histone Deacetylase Inhibition ◽  
Immune Effector ◽  
Chip Sequencing ◽  
Genome Wide

STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP sequencing coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of noncanonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of proinflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition.

Targeted removal of PML-RARα protein is required prior to inhibition of histone deacetylase for overcoming all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1008-1013 ◽  
Author(s):  
Yongkui Jing ◽  
Lijuan Xia ◽  
Samuel Waxman
Keyword(s):  
Retinoic Acid ◽  
Histone Deacetylase ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Gene Expressions ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nitroblue Tetrazolium Reduction ◽  
A Cell

Abstract All-trans retinoic acid (tRA)–induced differentiation in NB4 cells, a cell line derived from an acute promyelocytic leukemia patient with t(15;17) translocation, is markedly facilitated by sodium butyrate (NaB), a histone deacetylase inhibitor (HDACI), or by hexamethylene bisacetamide (HMBA), a non–HDACI tRA-differentiation inducer, as determined by nitroblue tetrazolium reduction. The tRA-induced expression of RIG-G, Bfl-1/A1, and p21waf1 and, to a lesser extent, of CCAAT/enhancer binding protein–ε (C/EBPε) are also enhanced by such combined treatments. Both responses are associated with a facilitated diminution of the leukemogenic PML-RARα protein and retained ΔPML-RARα, a cleavage product. Treatment with tRA in tRA differentiation–resistant NB4 subclones R4 and MR-2 does not result in PML-RARα diminution and the tested gene expressions. Moreover, the addition of HMBA or NaB with tRA results in only minimal increase of differentiation in the tRA differentiation–resistant subclones. The increases in acetylated histone H3 (AcH3) and AcH4 in NaB-treated NB4, R4, and MR-2 cells are similar and do not correlate with the extent of differentiation induction when NaB and HMBA are given in combination with tRA. Arsenic trioxide (As2O3) treatment results in the total degradation of PML-RARα without increasing AcH3 or AcH4 or inducing differentiation in R4 cells. As2O3 in combination with tRA induces gene (Bfl-1/A1 and C/EBPε) expression and partial differentiation. Both NaB and HMBA addition to As2O3-plus-tRA–treated R4 cells further enhances differentiation. These results suggest that elimination of the dominant negative PML-RARα protein is required prior to inhibition of histone deacetylase to fully overcome tRA-differentiation resistance in APL cells.

scholarly journals Synergistic and additive effect of retinoic acid in circumventing resistance to p53 restoration

2018 ◽  
Vol 115 (9) ◽  
pp. 2198-2203 ◽  
Author(s):  
Connie A. Larsson ◽  
Sydney M. Moyer ◽  
Bin Liu ◽  
Keith A. Michel ◽  
Vinod Pant ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Therapeutic Response ◽  
Retinoic Acid Receptor ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Synthetic Retinoid ◽  
P53 Reactivation

TP53 mutations occur in ∼50% of all human tumors, with increased frequency in aggressive cancers that are notoriously difficult to treat. Additionally, p53 missense mutations are remarkably predictive of refractoriness to chemo/radiotherapy in various malignancies. These observations have led to the development of mutant p53-targeting agents that restore p53 function. An important unknown is which p53-mutant tumors will respond to p53 reactivation-based therapies. Here, we found a heterogeneous impact on therapeutic response to p53 restoration, suggesting that it will unlikely be effective as a monotherapy. Through gene expression profiling of p53R172H-mutant lymphomas, we identified retinoic acid receptor gamma (RARγ) as an actionable target and demonstrated that pharmacological activation of RARγ with a synthetic retinoid sensitizes resistant p53-mutant lymphomas to p53 restoration, while additively improving outcome and survival in inherently sensitive tumors.

scholarly journals The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2826-2832 ◽  
Author(s):  
JV Raelson ◽  
C Nervi ◽  
A Rosenauer ◽  
L Benedetti ◽  
Y Monczak ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Myeloid Differentiation ◽  
Wild Type ◽  
Retinoid Receptor ◽  
Western Blots

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.

Novel Retinoic Acid Metabolism Blocking Agents (RAMBAs) Induce Differentiation in ATRA Resistant Cell Line: Potential New Theraputics for Acute Promyleocytic Leukemia (APL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 746-746
Author(s):  
Kavita B. Kalra ◽  
Xiangfei Cheng ◽  
Marion Womak ◽  
Christopher Gocke ◽  
Jyoti B. Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Breast Cancer Cell Lines ◽  
Hematopoietic Stem

Abstract All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem2004,47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.

Sign in / Sign up

Export Citation Format

Share Document