FLT3-ITD impedes retinoic acid, but not arsenic, responses in murine acute promyelocytic leukemias

Abstract Acute promyelocytic leukemia (APL) is often associated with activating FLT3 signaling mutations. These are highly related to hyperleukocytosis, a major adverse risk factor with chemotherapy-based regimens. APL is a model for oncogene-targeted therapies: all-trans retinoic acid (ATRA) and arsenic both target and degrade its ProMyelocytic Leukemia/Retinoic Acid Receptor α (PML/RARA) driver. The combined ATRA/arsenic regimen now cures virtually all patients with standard-risk APL. Although FLT3-internal tandem duplication (ITD) was an adverse risk factor for historical ATRA/chemotherapy regimens, the molecular bases for this effect remain unknown. Using mouse APL models, we unexpectedly demonstrate that FLT3-ITD severely blunts ATRA response. Remarkably, although the transcriptional output of initial ATRA response is unaffected, ATRA-induced PML/RARA degradation is blunted, as is PML nuclear body reformation and activation of P53 signaling. Critically, the combination of ATRA and arsenic fully rescues therapeutic response in FLT3-ITD APLs, restoring PML/RARA degradation, PML nuclear body reformation, P53 activation, and APL eradication. Moreover, arsenic targeting of normal PML also contributes to APL response in vivo. These unexpected results explain the less favorable outcome of FLT3-ITD APLs with ATRA-based regimens, and stress the key role of PML nuclear bodies in APL eradication by the ATRA/arsenic combination.

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Role of Promyelocytic Leukemia (Pml) Sumolation in Nuclear Body Formation, 11s Proteasome Recruitment, and as2O3-Induced Pml or Pml/Retinoic Acid Receptor α Degradation

Journal of Experimental Medicine ◽

10.1084/jem.193.12.1361 ◽

2001 ◽

Vol 193 (12) ◽

pp. 1361-1372 ◽

Cited By ~ 331

Author(s):

Valérie Lallemand-Breitenbach ◽

Jun Zhu ◽

Francine Puvion ◽

Marcel Koken ◽

Nicole Honoré ◽

...

Keyword(s):

Retinoic Acid ◽

Nuclear Matrix ◽

Viral Infections ◽

Retinoic Acid Receptor ◽

Nuclear Body ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Acid Receptor ◽

Pml Nuclear Bodies ◽

Retinoic Acid Receptor Α

Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) α expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARα and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARα degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation.

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Synergy against PML-RARa: targeting transcription, proteolysis, differentiation, and self-renewal in acute promyelocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20131121 ◽

2013 ◽

Vol 210 (13) ◽

pp. 2793-2802 ◽

Cited By ~ 76

Author(s):

Guilherme Augusto dos Santos ◽

Lev Kats ◽

Pier Paolo Pandolfi

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Target Genes ◽

Retinoic Acid Receptor ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Pml Nuclear Bodies ◽

Molecular Events

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by a chimeric oncoprotein containing the C terminus of the retinoic acid receptor-a (RARa) fused to an N-terminal partner, most commonly promyelocytic leukemia protein (PML). Mechanistically, PML-RARa acts as a transcriptional repressor of RARa and non-RARa target genes and antagonizes the formation and function of PML nuclear bodies that regulate numerous signaling pathways. The empirical discoveries that PML-RARa–associated APL is sensitive to both all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO), and the subsequent understanding of the mechanisms of action of these drugs, have led to efforts to understand the contribution of molecular events to APL cell differentiation, leukemia-initiating cell (LIC) clearance, and disease eradication in vitro and in vivo. Critically, the mechanistic insights gleaned from these studies have resulted not only in a better understanding of APL itself, but also carry valuable lessons for other malignancies.

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Arsenic-Induced SUMO-Dependent Recruitment of RNF4 into PML Nuclear Bodies

Molecular Biology of the Cell ◽

10.1091/mbc.e10-05-0449 ◽

2010 ◽

Vol 21 (23) ◽

pp. 4227-4239 ◽

Cited By ~ 41

Author(s):

Marie-Claude Geoffroy ◽

Ellis G. Jaffray ◽

Katherine J. Walker ◽

Ronald T. Hay

Keyword(s):

Retinoic Acid Receptor ◽

E3 Ligase ◽

Nuclear Body ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Dependent Manner ◽

Nuclear Trafficking ◽

Sumo Modification ◽

Ubiquitin E3 Ligase ◽

Pml Nuclear Bodies

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.

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Trivalent Antimonials Induce Degradation of the PML-RAR Oncoprotein and Reorganization of the Promyelocytic Leukemia Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells

Blood ◽

10.1182/blood.v92.11.4308.423k36_4308_4316 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4308-4316 ◽

Cited By ~ 5

Author(s):

Stefan Müller ◽

Wilson H. Miller ◽

Anne Dejean

Keyword(s):

Retinoic Acid ◽

Fusion Protein ◽

Acute Promyelocytic Leukemia ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Antimony Trioxide ◽

Nb4 Cells ◽

Pml Nuclear Bodies

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor  (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.

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The Promyelocytic Leukemia Gene Product (PML) Forms Stable Complexes with the Retinoblastoma Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.1084 ◽

1998 ◽

Vol 18 (2) ◽

pp. 1084-1093 ◽

Cited By ~ 111

Author(s):

Myriam Alcalay ◽

Lucia Tomassoni ◽

Emanuela Colombo ◽

Stephan Stoldt ◽

Francesco Grignani ◽

...

Keyword(s):

Retinoic Acid ◽

Nuclear Protein ◽

Retinoic Acid Receptor ◽

Retinoblastoma Protein ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Oncogenic Potential ◽

Nuclear Structures ◽

Retinoic Acid Receptor Α

ABSTRACT PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor α (RARα) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the expression of PML-RARα. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RARα expression. Both PML and PML-RARα form complexes with the nonphosphorylated form of pRB in vivo, and they interact with the pocket region of pRB. The regions of PML and PML-RARα involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RARα, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RARα further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RARα protein may derive from the alteration of PML-regulated transcription.

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Trivalent Antimonials Induce Degradation of the PML-RAR Oncoprotein and Reorganization of the Promyelocytic Leukemia Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells

Blood ◽

10.1182/blood.v92.11.4308 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4308-4316 ◽

Cited By ~ 57

Author(s):

Stefan Müller ◽

Wilson H. Miller ◽

Anne Dejean

Keyword(s):

Retinoic Acid ◽

Fusion Protein ◽

Acute Promyelocytic Leukemia ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Antimony Trioxide ◽

Nb4 Cells ◽

Pml Nuclear Bodies

Abstract Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor  (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.

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Evaluation of semiquantitative reverse transcriptase-polymerase chain reaction assay of PML/retinoic acid receptor .ALPHA. mRNA and in vitro differentiation assay as prognostic prediction in acute promyelocytic leukemia treated with all-trans retinoic acid.

Journal of Nippon Medical School ◽

10.1272/jnms1923.63.259 ◽

1996 ◽

Vol 63 (4) ◽

pp. 259-267

Author(s):

Rika Iwakiri ◽

Koiti Inokuchi ◽

Kazuo Dan ◽

Ichiji Wakabayashi

Keyword(s):

Retinoic Acid ◽

Retinoic Acid Receptor ◽

Promyelocytic Leukemia ◽

In Vitro Differentiation ◽

Chain Reaction ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Polymerase Chain ◽

Prognostic Prediction

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Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies

Journal of Virology ◽

10.1128/jvi.01442-10 ◽

2010 ◽

Vol 84 (23) ◽

pp. 12210-12225 ◽

Cited By ~ 54

Author(s):

Mario A. Pennella ◽

Yue Liu ◽

Jennifer L. Woo ◽

Chongwoo A. Kim ◽

Arnold J. Berk

Keyword(s):

Nuclear Export ◽

P53 Mutation ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Live Cells ◽

High Concentration ◽

Sumoylation Site ◽

Pml Nuclear Bodies ◽

Induced Apoptosis

ABSTRACT Oncogenic transformation by adenovirus E1A and E1B-55K requires E1B-55K inhibition of p53 activity to prevent E1A-induced apoptosis. During viral infection, E1B-55K and E4orf6 substitute for the substrate-binding subunits of the host cell cullin 5 class of ubiquitin ligases, resulting in p53 polyubiquitinylation and proteasomal degradation. Here we show that E1B-55K alone also functions as an E3 SUMO1-p53 ligase. Fluorescence microscopy studies showed that E1B-55K alone, in the absence of other viral proteins, causes p53 to colocalize with E1B-55K in promyelocytic leukemia (PML) nuclear bodies, nuclear domains with a high concentration of sumoylated proteins. Photobleaching experiments with live cells revealed that E1B-55K tethering of p53 in PML nuclear bodies decreases the in vivo nuclear mobility of p53 nearly 2 orders of magnitude. E1B-55K-induced p53 sumoylation contributes to maximal inhibition of p53 function since mutation of the major p53 sumoylation site decreases E1B-55K-induced p53 sumoylation, tethering in PML nuclear bodies, and E1B-55K inhibition of p53 activity. Mutation of the E1B-55K sumoylation site greatly inhibits E1B-55K association with PML nuclear bodies and the p53 nuclear export to cytoplasmic aggresomes observed in E1A-E1B-transformed cells. Purified E1B-55K and p53 form high-molecular-weight complexes potentially through the formation of a network of E1B-55K dimers bound to the N termini of p53 tetramers. In support of this model, a p53 mutation that prevents tetramer formation greatly reduces E1B-55K-induced tethering in PML nuclear bodies and p53 nuclear export. These data indicate that E1B-55K's association with PML nuclear bodies inactivates p53 by first sequestering it in PML nuclear bodies and then greatly facilitating its nuclear export.

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ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression

Blood ◽

10.1182/blood-2005-07-3068 ◽

2006 ◽

Vol 107 (8) ◽

pp. 3330-3338 ◽

Cited By ~ 139

Author(s):

Beatrice U. Mueller ◽

Thomas Pabst ◽

José Fos ◽

Vibor Petkovic ◽

Martin F. Fey ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Myeloid Leukemia ◽

Retinoic Acid Receptor ◽

Promyelocytic Leukemia ◽

All Trans Retinoic Acid ◽

Conditional Expression ◽

Neutrophil Differentiation ◽

Acute Myeloid

Abstract Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor α (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

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