scholarly journals Single Cell Analyses Reveal a Non-Canonical EZH2 Activity As a Main Driver of Retinoic Acid Resistance in PLZF/Rara Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2205-2205
Author(s):  
Mathilde Poplineau ◽  
Nadine Platet ◽  
Adrien Mazuel ◽  
Léonard Hérault ◽  
Shuhei Koide ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Single Cell ◽  
Acid Resistance ◽  
Promising Therapeutic Approach ◽  
Main Driver ◽  
M3 Subtype ◽  
Chemical Corporation ◽  
Retinoic Acid Resistance

Abstract Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA. We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature ("Rep" cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance. These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. Disclosures Iwama: Nissan Chemical Corporation: Research Funding.

scholarly journals Single-cell multi-omics analyses reveal EZH2 as a main driver of retinoic acid resistance in PLZF-RARA leukemia.

2022 ◽  
Author(s):  
Mathilde Poplineau ◽  
Nadine Platet ◽  
Adrien Mazuel ◽  
Leonard Herault ◽  
Shuhei Koide ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Single Cell ◽  
Acid Resistance ◽  
Leukemia Cells ◽  
Main Driver ◽  
Leukemia Relapse ◽  
Retinoic Acid Resistance ◽  
Functional Analyses ◽  
Canonical Activity ◽  
Resistant Cells

Cancer relapse is caused by a subset of malignant cells that are resistant to treatment. To characterize resistant cells and their vulnerabilities, we studied the retinoic acid (RA)-resistant PLZF-RARA acute promyelocytic leukemia (APL) using single-cell multi-omics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells and identified a subset of cells resistant to RA that depend on a fine-tuned transcriptional network targeting the epigenetic regulator Enhancer of Zeste Homolog 2 (EZH2). Epigenomic and functional analyses validated EZH2 selective dependency of PLZF-RARA leukemia and its driver role in RA resistance. Targeting pan-EZH2 activities (canonical/non-canonical) was necessary to eliminate leukemia relapse initiating cells, which underlies a dependency of resistant cells on an EZH2 non-canonical activity and the necessity to degrade EZH2 to overcome resistance. Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach.

Constitutive Degradation of PML/RAR Through the Proteasome Pathway Mediates Retinoic Acid Resistance

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1477-1481 ◽  
Author(s):  
Mirco Fanelli ◽  
Saverio Minucci ◽  
Vania Gelmetti ◽  
Clara Nervi ◽  
Carlo Gambacorti-Passerini ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Proteasome Inhibitors ◽  
Acid Resistance ◽  
Parental Cell ◽  
Parental Cell Line ◽  
In Vitro Degradation ◽  
Nb4 Cells ◽  
Proteasome Pathway ◽  
Retinoic Acid Resistance

Abstract PML/RAR is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RAR, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RAR protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RAR is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RAR protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RAR, but not RAR, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RAR protein may lead to RA resistance and that PML/RAR expression is crucial to convey RA sensitivity to APL cells.

scholarly journals Downregulation of CRABP2 Inhibit the Tumorigenesis of Hepatocellular Carcinoma In Vivo and In Vitro

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Qingmin Chen ◽  
Ludong Tan ◽  
Zhe Jin ◽  
Yahui Liu ◽  
Ze Zhang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Tumor Stage ◽  
Migration And Invasion ◽  
Hcc Cells

Cellular retinoic acid-binding protein 2 (CRABP2) binds retinoic acid (RA) in the cytoplasm and transports it into the nucleus, allowing for the regulation of specific downstream signal pathway. Abnormal expression of CRABP2 has been detected in the development of several tumors. However, the role of CRABP2 in hepatocellular carcinoma (HCC) has never been revealed. The current study aimed to investigate the role of CRABP2 in HCC and illuminate the potential molecular mechanisms. The expression of CRABP2 in HCC tissues and cell lines was detected by western blotting and immunohistochemistry assays. Our results demonstrated that the expression levels of CRABP2 in HCC tissues were elevated with the tumor stage development, and it was also elevated in HCC cell lines. To evaluate the function of CRABP2, shRNA-knockdown strategy was used in HCC cells. Cell proliferation, metastasis, and apoptosis were analyzed by CCK-8, EdU staining, transwell, and flow cytometry assays, respectively. Based on our results, knockdown of CRABP2 by shRNA resulted in the inhibition of tumor proliferation, migration, and invasion in vitro, followed by increased tumor apoptosis-related protein expression and decreased ERK/VEGF pathway-related proteins expression. CRABP2 silencing in HCC cells also resulted in the failure to develop tumors in vivo. These results provide important insights into the role of CRABP2 in the development and development of HCC. Based on our findings, CRABP2 may be used as a novel diagnostic biomarker, and regulation of CRABP2 in HCC may provide a potential molecular target for the therapy of HCC.

scholarly journals In vivo analysis of the role of aberrant histone deacetylase recruitment and RARα blockade in the pathogenesis of acute promyelocytic leukemia

2006 ◽  
Vol 203 (4) ◽  
pp. 821-828 ◽  
Author(s):  
Hiromichi Matsushita ◽  
Pier Paolo Scaglioni ◽  
Mantu Bhaumik ◽  
Eduardo M. Rego ◽  
Lu Fan Cai ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Histone Deacetylases ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1–RARα fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML−/− and p53−/− compound mutants lends support to a model by which the RARα and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

scholarly journals Molecular targeting of retinoic acid metabolism in neuroblastoma: the role of the CYP26 inhibitor R116010 in vitro and in vivo

2007 ◽  
Vol 96 (11) ◽  
pp. 1675-1683 ◽  
Author(s):  
J L Armstrong ◽  
G A Taylor ◽  
H D Thomas ◽  
A V Boddy ◽  
C P F Redfern ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acid Metabolism ◽  
Molecular Targeting

Retinoic acid resistance in NB4 APL cells is associated with lack of interferon α synthesis Stat1 and p48 induction

Oncogene ◽  
1999 ◽  
Vol 18 (27) ◽  
pp. 3944-3953 ◽  
Author(s):  
Luis Pelicano ◽  
Caren Brumpt ◽  
Paula M Pitha ◽  
Mounira K Chelbi-Alix
Keyword(s):  
Retinoic Acid ◽  
Acid Resistance ◽  
Interferon Α ◽  
Retinoic Acid Resistance

scholarly journals All-trans Retinoic Acid Counteracts Diarrhea and Inhibition of Downregulated in Adenoma Expression in Gut Inflammation

2019 ◽  
Vol 26 (4) ◽  
pp. 534-545 ◽  
Author(s):  
Shubha Priyamvada ◽  
Arivarasu N Anbazhagan ◽  
Anoop Kumar ◽  
Ishita Chatterjee ◽  
Alip Borthakur ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Intestinal Epithelial ◽  
Gut Inflammation ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Function Expression ◽  
Ifn Γ

Abstract Background Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated. Aims Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo. Methods Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon. Results All-trans retinoic acid (10 μM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1β and CXCL1 and a decrease in DRA mRNA and protein levels in the colon. Conclusion Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea.

Single-cell analyses of nitrergic neurons in simple nervous systems

1999 ◽  
Vol 202 (4) ◽  
pp. 333-341 ◽  
Author(s):  
L.L. Moroz ◽  
R. Gillette ◽  
J.V. Sweedler
Keyword(s):  
Single Cell ◽  
Cell Model ◽  
Single Cells ◽  
Chemical Species ◽  
No Oxidation ◽  
Cell Level ◽  
Pharmacological Manipulations ◽  
Physiological And Biochemical

Understanding the role of the gaseous messenger nitric oxide (NO) in the nervous system is complicated by the heterogeneity of its nerve cells; analyses carried out at the single cell level are therefore important, if not critical. Some invertebrate preparations, most especially those from the gastropod molluscs, provide large, hardy and identified neurons that are useful both for the development of analytical methodologies and for cellular analyses of NO metabolism and its actions. Recent modifications of capillary electrophoresis (CE) allow the use of a small fraction of an individual neuron to perform direct, quantitative and simultaneous assays of the major metabolites of the NO-citrulline cycle and associated biochemical pathways. These chemical species include the products of NO oxidation (NO2-/NO3-), l-arginine, l-citrulline, l-ornithine, l-argininosuccinate, as well as selected NO synthase inhibitors and cofactors such as NADPH, biopterin, FMN and FAD. Diverse cotransmitters can also be identified in the same nitrergic neuron. The sensitivity of CE methods is in the femtomole to attomole range, depending on the species analysed and on the specific detector used. CE analysis can be combined with prior in vivo electrophysiological and pharmacological manipulations and measurements to yield multiple physiological and biochemical values from single cells. The methodologies and instrumentation developed and tested using the convenient molluscan cell model can be adapted to the smaller and more delicate neurons of other invertebrates and chordates.

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