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 ◽  
...  

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.

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3200-3208 ◽  
Author(s):  
Sylvie Côté ◽  
Dacheng Zhou ◽  
Andrea Bianchini ◽  
Clara Nervi ◽  
Robert E. Gallagher ◽  
...  

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.


Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4282-4289 ◽  
Author(s):  
Wenlin Shao ◽  
Laura Benedetti ◽  
William W. Lamph ◽  
Clara Nervi ◽  
Wilson H. Miller

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.


Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2671-2682 ◽  
Author(s):  
A Rosenauer ◽  
JV Raelson ◽  
C Nervi ◽  
P Eydoux ◽  
A DeBlasio ◽  
...  

All-trans retinoic acid (tRA), a naturally occurring ligand of the nuclear retinoic acid receptors (RARs), induces differentiation of leukemic cells and clinical complete remission in patients with acute promyelocytic leukemia (APL). This differentiation effect can also be seen in vitro in both fresh leukemic cells and in the unique permanent APL cell line, NB4. However, APL cells become resistant to RA-induced differentiation both in vitro and in patients. Although pharmacodynamic mechanisms of resistance have been reported, there is growing evidence that resistance both in patients, as well as in vitro, can be mediated by changes in the sensitivity of leukemic cells to retinoids. To investigate possible mechanisms of retinoid resistance, we established subclones of NB4 that are stably resistant to both tRA and 9-cisRA. Unlike the previously reported NB4.306 retinoid-resistant cells, these subclones expressed PML/RAR-alpha RNA and protein, but demonstrated altered ligand binding patterns of PML/RAR-alpha and differed in retinoid-induced gene expression. They were significantly less able to stimulate transcription of an RARE driven CAT-reporter gene on induction by tRA and showed altered DNA binding activity on a RARE. These data suggest that NB4 cells selected for resistance to retinoids demonstrate abnormal ligand binding to PML/RAR-alpha that lead to altered transcriptional activation by retinoids.


2006 ◽  
Vol 203 (4) ◽  
pp. 821-828 ◽  
Author(s):  
Hiromichi Matsushita ◽  
Pier Paolo Scaglioni ◽  
Mantu Bhaumik ◽  
Eduardo M. Rego ◽  
Lu Fan Cai ◽  
...  

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.


Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2683-2690 ◽  
Author(s):  
Robert L. Redner ◽  
J. Don Chen ◽  
Elizabeth A. Rush ◽  
Hui Li ◽  
Sheri L. Pollock

The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RAR). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RAR sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RAR. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RAR in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RAR. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RAR. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RAR to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.


Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2233-2239 ◽  
Author(s):  
Wenlin Shao ◽  
Angelika Rosenauer ◽  
Koren Mann ◽  
Chao-Pei Betty Chang ◽  
Christophe Rachez ◽  
...  

Retinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARα fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.


Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2683-2690 ◽  
Author(s):  
Robert L. Redner ◽  
J. Don Chen ◽  
Elizabeth A. Rush ◽  
Hui Li ◽  
Sheri L. Pollock

Abstract The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RAR). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RAR sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RAR. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RAR in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RAR. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RAR. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RAR to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.


Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2233-2239 ◽  
Author(s):  
Wenlin Shao ◽  
Angelika Rosenauer ◽  
Koren Mann ◽  
Chao-Pei Betty Chang ◽  
Christophe Rachez ◽  
...  

Abstract Retinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARα fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.


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 ◽  
...  

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.


Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Da-Cheng Zhou ◽  
Soon H. Kim ◽  
Wei Ding ◽  
Cynthia Schultz ◽  
Raymond P. Warrell ◽  
...  

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.


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