Frequent mutations in the ligand-binding domain of PML-RARα after multiple relapses of acute promyelocytic leukemia: analysis for functional relationship to response to all-transretinoic acid and histone deacetylase inhibitors in vitro and in vivo

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Da-Cheng Zhou ◽  
Soon H. Kim ◽  
Wei Ding ◽  
Cynthia Schultz ◽  
Raymond P. Warrell ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Histone Deacetylase ◽  
Acute Promyelocytic Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Promyelocytic Leukemia ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Missense Mutations

This study identified missense mutations in the ligand binding domain of the oncoprotein PML-RARα in 5 of 8 patients with acute promyelocytic leukemia (APL) with 2 or more relapses and 2 or more previous courses of all-trans retinoic acid (RA)–containing therapy. Four mutations were novel (Lys207Asn, Gly289Arg, Arg294Trp, and Pro407Ser), whereas one had been previously identified (Arg272Gln; normal RARα1 codon assignment). Five patients were treated with repeat RA plus phenylbutyrate (PB), a histone deacetylase inhibitor, and one patient experienced a prolonged clinical remission. Of the 5 RA + PB-treated patients, 4 had PML-RARα mutations. The Gly289Arg mutation in the clinical responder produced the most defective PML-RARα function in the presence of RA with or without sodium butyrate (NaB) or trichostatin A. Relapse APL cells from this patient failed to differentiate in response to RA but partially differentiated in response to NaB alone, which was augmented by RA. In contrast, NaB alone had no differentiation effect on APL cells from another mutant case (Pro407Ser) but enhanced differentiation induced by RA. These results indicate that PML-RARα mutations occurred with high frequency after multiple RA treatment relapses, indicate that the functional potential of PML-RARα was not correlated with clinical response to RA + PB treatment, and suggest that the response to RA + PB therapy in one patient was related to the ability of PB to circumvent the blocked RA-regulated gene response pathway.

Altered ligand binding and transcriptional regulation by mutations in the PML/RARα ligand-binding domain arising in retinoic acid–resistant patients with acute promyelocytic leukemia

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3200-3208 ◽  
Author(s):  
Sylvie Côté ◽  
Dacheng Zhou ◽  
Andrea Bianchini ◽  
Clara Nervi ◽  
Robert E. Gallagher ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Ligand Binding ◽  
Acute Promyelocytic Leukemia ◽  
Transcriptional Activation ◽  
Histone Deacetylase Inhibitors ◽  
Molecular Mechanisms ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Mechanism Of Resistance

Acute promyelocytic leukemia (APL) is characterized by a specific translocation, t(15;17), that fuses the promyelocytic leukemia (PML) gene with the RA receptor RARα. Pharmacologic doses of retinoic acid (RA) induce differentiation in human APL cells and complete clinical remissions. Unfortunately, APL cells develop resistance to RA in vitro and in vivo. Recently, mutations in PML/RARα have been described in APL cells from patients clinically resistant to RA therapy. The mutations cluster in 2 regions that are involved in forming the binding pocket for RA. These mutant PML/RARα proteins have been expressed in vitro, which shows that they cause a diversity of alterations in binding to ligand and to nuclear coregulators of transcription, leading to varying degrees of inhibition of retinoid-induced transcription. This contrasts with the nearly complete dominant negative activity of mutations in PML/RARα previously characterized in cell lines developing RA resistance in vitro. Current data from this study provide additional insight into the molecular mechanisms of resistance to RA and suggest that alterations in the ability of mutants to interact with coregulators can be determinant in the molecular mechanism of resistance to RA. In particular, ligand-induced binding to the coactivator ACTR correlated better with transcriptional activation of RA response elements than the ligand-induced release of the corepressor SMRT. The diversity of effects that are seen in patient-derived mutations may help explain the partial success to date of attempts to overcome this mechanism of resistance in patients by the clinical use of histone deacetylase inhibitors.

Histone Deacetylase Inhibitors and Filgrastim Do Not Synergize with ATRA in the Induction of Changes of Acute Promyelocytic Leukemia Cells Adhesive Properties.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2548-2548
Author(s):  
Gil C. De Santis ◽  
Suzana E. Moreno ◽  
Hamilton L.G. Teixera ◽  
Ana Silvia G. Lima ◽  
Aglair B. Garcia ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Retinoic Acid ◽  
Histone Deacetylase ◽  
Acute Promyelocytic Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Myeloid Cells ◽  
Promyelocytic Leukemia ◽  
Myeloperoxidase Activity ◽  
Nb4 Cells

Abstract All-trans retinoic acid (ATRA) in combination with anthracyclines induces long term complete remission in approximately 80% of patients with acute promyelocytic leukemia (APL). However, ATRA causes the retinoic acid syndrome (RAS) characterized by respiratory distress, pleural effusions, fever and weight gain. RAS is associated with changes in the expression of adhesion molecules (AMs) in the leukemic blasts. Nevertheless, which AMs are essential to RAS development is not clear. In addition, the effect on AMs expression of new therapeutic agents for APL such as histone deacetylase inhibitors (HDACis) or filgrastim is presently unknown. HDACis have been successfully used to treat ATRA-refractory cases and they potentiate ATRA-induced differentiation. The association of ATRA+ filgastrim induced remission in an APL patient harboring the t(11;17)/PLZF/RARα, which is resistant to ATRA. In order to determine the effect of ATRA, filgrastim, HDACis and their associations on cell adhesion, we analyzed the expression of the AMs: CD11a, CD11b, CD18, CD29, CD54, CD62L and CD162 on leukemic cells from 18 patients with APL and in NB4 cells treated ex vivo for 12 hours with DMSO (control), ATRA (1mM), filgrastrim (100ng/mL), trichostatin A (TSA, 0.1 mM), phenyl butirate (PB, 1 mM) (the latter two are bona fine HDACis), ATRA+TSA, ATRA+PB and ATRA+filgrastim (at the same doses). The number of positive cells for each of this markers and their respective fluorescence intensity was determined by flow cytometry. We detected a significant increase in the number of CD54+ and CD18+ cells, associated with an increase in the intensity of expression of CD54, CD11a, CD11b and CD18 in both NB4 and primary cells treated with ATRA alone or associated with PB or G-CSF. No difference was observed between samples treated exclusively with ATRA and those with the associations. We then analyzed if the changes in AMs expression were accompanied by changes in the adhesion to Matrigel or endothelial cells. ATRA and its associations, but not TSA, PB or filgrastim alone, increased significantly cell adhesion in vitro, an effect that was reversed by pre-incubating treated cells with anti-CD54 or anti-CD18 antibodies (Abs), or with dexametasone. ATRA induced cell adhesion was not dependent on myeloid maturation as it could be detected after short (12h) incubations. Finally, we analyzed the effects of ATRA, filgrastim and their association in a mouse model. NB4 cells were treated with ATRA, filgrastim, ATRA+filgrastim and injected IV through the tail vein. After 6h mice, the number of myeloid cells retained in the lungs was evaluated by measuring the myeloperoxidase activity. Compared to control groups (untreated cells or saline), the ATRA and ATRA+filgrastim but not the Filgrastim alone group presented a significant increase in the number of myeloid cells infiltrating the lungs. Similarly to the observed in vitro, pre incubation with anti-CD54, anti-CD18 Abs or with dexametasone reversed the increased cell adhesion in vivo. In conclusion, our results show that treatment with HDACis or filgrastim alone do not affect AM expression or cell adhesion and that there is no significant synergism between these agents and ATRA. In addition, our data suggest that SAR development is dependent on ATRA induced changes in CD54 and CD18 expression.

Altered ligand binding and transcriptional regulation by mutations in the PML/RARα ligand-binding domain arising in retinoic acid–resistant patients with acute promyelocytic leukemia

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3200-3208 ◽  
Author(s):  
Sylvie Côté ◽  
Dacheng Zhou ◽  
Andrea Bianchini ◽  
Clara Nervi ◽  
Robert E. Gallagher ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Ligand Binding ◽  
Acute Promyelocytic Leukemia ◽  
Transcriptional Activation ◽  
Histone Deacetylase Inhibitors ◽  
Molecular Mechanisms ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Mechanism Of Resistance

Abstract Acute promyelocytic leukemia (APL) is characterized by a specific translocation, t(15;17), that fuses the promyelocytic leukemia (PML) gene with the RA receptor RARα. Pharmacologic doses of retinoic acid (RA) induce differentiation in human APL cells and complete clinical remissions. Unfortunately, APL cells develop resistance to RA in vitro and in vivo. Recently, mutations in PML/RARα have been described in APL cells from patients clinically resistant to RA therapy. The mutations cluster in 2 regions that are involved in forming the binding pocket for RA. These mutant PML/RARα proteins have been expressed in vitro, which shows that they cause a diversity of alterations in binding to ligand and to nuclear coregulators of transcription, leading to varying degrees of inhibition of retinoid-induced transcription. This contrasts with the nearly complete dominant negative activity of mutations in PML/RARα previously characterized in cell lines developing RA resistance in vitro. Current data from this study provide additional insight into the molecular mechanisms of resistance to RA and suggest that alterations in the ability of mutants to interact with coregulators can be determinant in the molecular mechanism of resistance to RA. In particular, ligand-induced binding to the coactivator ACTR correlated better with transcriptional activation of RA response elements than the ligand-induced release of the corepressor SMRT. The diversity of effects that are seen in patient-derived mutations may help explain the partial success to date of attempts to overcome this mechanism of resistance in patients by the clinical use of histone deacetylase 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 Peptide Vaccines and Peptidomimetics of EGFR (HER-1) Ligand Binding Domain Inhibit Cancer Cell Growth In Vitro and In Vivo

2013 ◽  
Vol 191 (1) ◽  
pp. 217-227 ◽  
Author(s):  
Kevin Chu Foy ◽  
Ruthie M. Wygle ◽  
Megan J. Miller ◽  
Jay P. Overholser ◽  
Tanios Bekaii-Saab ◽  
...  
Keyword(s):  
Cell Growth ◽  
Ligand Binding ◽  
Cancer Cell ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Peptide Vaccines ◽  
Cancer Cell Growth

Combination Treatment of p53-Null HL-60 cells with Histone Deacetylase Inhibitors and Chlorambucil Augments Apoptosis and Increases BCL6 and p21 Gene Expression

2019 ◽  
Vol 12 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Faith A.A. Kwa ◽  
Merrole F. Cole-Sinclair ◽  
Miroslav K. Kapuscinski
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Combination Treatment ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Control Cell ◽  
Leukemia Cell Line ◽  
Tetrazolium Salt ◽  
Treatment Type

Background:Treatment of hematological malignancies with conventional DNA-damaging drugs, such as chlorambucil (CLB), commonly results in p53-dependent chemo-resistance. Chromatin modifying agents, such as histone deacetylase inhibitors (HDACIs), sodium butyrate (NaBu) and trichostatin A (TSA), may reverse chemo-resistance by modulating the activity of chromatin remodeling enzymes and/or genes that control cell proliferation, differentiation and survival.Objective:This study examined the potential use of HDACIs and CLB combination therapies in an in vitro chemo-resistant leukemia model.Methods:The p53-null promyelocytic leukemia cell line, HL60, was used as an in vitro model of chemo-resistant leukemia. Drug cytotoxicity was determined by tetrazolium salt-based colorimetric assays and Annexin V/propidium iodide staining (flow cytometry). The level of mRNA expression of the chromatin modifying genes was measured by quantitative real-time PCR.Results:Micromolar concentrations of CLB combined with either NaBu or TSA triggered synergistic cytotoxic effects in HL-60 cells (p < 0.001). The effects of the combination treatments resulted in upregulated p21 gene expression (up to 59-fold; p<0.001) that preceded an increase in BCL6 gene expression (up to 20-fold; p < 0.001). Statistically significant but smaller magnitude changes (≤ 2-fold; p <0.05) were noted in the expression of other genes studied regardless of the treatment type.Conclusion:The combination treatment of p53-null HL-60 cells with DNA-damaging agent CLB and HDACIs NaBu and TSA triggered additive to synergistic effects on apoptosis and upregulated BCL6 and p21 expression. These findings reveal BCL6 and p21 as potential targets of chemo-resistance for the development of anti-leukemic drugs.

Different Histone Deacetylase Inhibitors Affect Distinct Cellular Targets within the Hierarchy of Hematopoietic Stem / Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1721-1721
Author(s):  
Hiroto Araki ◽  
Ronald Hoffman ◽  
Nadim Mahmud
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase ◽  
Hdac Inhibitor ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Recently several laboratories have examined the in vitro effects of chromatin modifying agents on hematopoiesis. We have previously reported that the sequential addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) to cultures of human cord blood (CB) CD34+ cells containing SCF, thrombopoietin and FLT-3 ligand (cytokines) resulted in a 10-fold expansion of SCID repopulating cells (SRC) (Araki et al. Blood2004: 104:881a). Recently others have shown that another HDAC inhibitor, valproic acid (VA) resulted in an expansion of CB CD34+ cells and murine hematopoietic stem / progenitor cells (HSPC) (DeFelice et al. Cancer Res.2005:65:1505, Beg et al. Cancer Res.2005:65:2537). In our current studies we have compared the efficacy of VA, TSA or 5azaD as single agents or in combination to promote the expansion of CB HSPC in vitro. The frequency and fold expansion of colony forming cells (CFC), cobblestone area-forming cells (CAFC) as well as SRC generated from CB CD34+ cells after 9 days of culture were examined. The addition of cytokines alone result in a 1.5-fold expansion of CD34+CD90+ cells. By contrast the addition of cytokines with VA led to a 65-fold expansion of the numbers of CD34+CD90+ cells as compared to a 1.3-fold, 5.6-fold, 4.2-fold or 12.5-fold expansion of CD34+CD90+ cells in cultures receiving cytokines with 5azaD, TSA or 5azaD/VA or 5azaD/TSA respectively. In vitro biological assays (CFC, CAFC) were performed to determine the correlation between CD34+CD90+ cell expansion and function. Cultures receiving cytokines alone or cytokines with VA had the greatest degree of expansion of CFC (14.4 and 18.6-fold respectively). By contrast cultures receiving cytokines alone contained only 70% of the numbers of CAFC as did the primary CB CD34+ cells. Cultures receiving VA or 5azaD/TSA had the greatest degree of expansion of CAFC numbers (9.6-fold and 11.5-fold respectively). The marrow repopulating potential of these various expanded cell populations were then assayed by transplanting them into NOD/SCID mice. CD34+ cells from cultures receiving cytokines alone or cytokines with 5azaD/VA were devoid of human hematopoietic cell chimerism. By contrast, all NOD/SCID mice receiving grafts from cultures treated with cytokines with 5azaD/TSA had evidence of human multilineage hematopoietic engraftment (7.5% ± 3.7%). Cells from cultures treated with cytokines with VA are capable of engraftment in 2 out of 6 mice with a barely detectable level of human cell chimerism (0.11%, 0.14%). We then assessed using western blot analysis whether the chromatin modifying agents might alter HSC function by upregulating HOXB4 protein levels. HOXB4 protein was detectable in cells cultured in the presence of cytokines with VA, cytokines with 5azaD/VA, cytokines with 5azaD/TSA but only cells treated with cytokines with 5azaD/TSA contained readily assayable SRC. These studies suggest that treatment with different chromatin modifying agents are capable of altering the differentiation program of distinct populations of HSPC. Some treatments (VA, 5azaD/VA) primarily affect CFC and CAFC but not SRC. While 5azaD/TSA targets CAFC and SRC but not CFC. In addition, although HOXB4 may participate in HSC self-renewal, additional genes are likely altered following 5azaD/TSA treatment which are required for the maintenance of SRC potential.

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