PML–RARA-RXR Oligomers Mediate Retinoid and Rexinoid/cAMP Cross-Talk in Acute Promyelocytic Leukemia Cell Differentiation

Abstract

PML–RARA was proposed to initiate acute promyelocytic leukemia (APL) through PML–RARA homodimer–triggered repression. Here, we examined the nature of the PML–RARA protein complex and of its DNA targets in APL cells. Using a selection/amplification approach, we demonstrate that PML–RARA targets consist of two AGGTCA elements in an astonishing variety of orientations and spacings, pointing to highly relaxed structural constrains for DNA binding and identifying a major gain of function of this oncogene. PML–RARA-specific response elements were identified, which all conveyed a major transcriptional response to RA only in APL cells. In these cells, we demonstrate that PML–RARA oligomers are complexed to RXR. Directly probing PML–RARA function in APL cells, we found that the differentiation enhancer cyclic AMP (cAMP) boosted transcriptional activation by RA. cAMP also reversed the normal silencing (subordination) of the transactivating function of RXR when bound to RARA or PML–RARA, demonstrating that the alternate rexinoid/cAMP-triggered APL differentiation pathway also activates PML–RARA targets. Finally, cAMP restored both RA-triggered differentiation and PML–RARA transcriptional activation in mutant RA-resistant APL cells. Collectively, our findings directly demonstrate that APL cell differentiation parallels transcriptional activation through PML–RARA-RXR oligomers and that those are functionally targeted by cAMP, identifying this agent as another oncogene-targeted therapy.

A conceptual framework for the identification of candidate drugs and drug targets in acute promyelocytic leukemia

“…4,6 In APL cells, PML-RARA forms homodimers, multimeric complexes and heterodimers with retinoid X receptor that all bind to retinoic acid responsive element and repress transcription at physiological levels of RA through recruitment of co-repressor complexes containing HDACs, DNA methyltransferases and polycomb histone methyltransferases. 4,[7][8][9][10] At pharmacological levels, however, RA reverses PML-RARA mediated repression of RARA target genes resulting in terminal neutrophil differentiation of APL cells. 4,5 Although these findings highlight the progress in our understanding of how PML-RARA represses transcription at the molecular level, little is currently known regarding the gene-expression signature that emerges as a consequence of PML-RARA and secondary genetic aberrations in human APL.…”

Towards novel paradigms for cancer therapy

“…This chromosomal rearrangement juxtaposes a portion of the PML and RARA genes (other X-RARa oncofusions are similarly leukemogenic; Melnick and Licht, 1999). The aberrant PML-RARa fusion protein displays a plethora of molecular features distinct from RARa, including altered homo-and hetero-oligomerization, different DNA-binding site repertoires, changes in the interaction with key factors like p53 or abnormal recruitment of epigenetically active complexes to RARa targets (Grignani et al, 1998;He et al, 1998;Lin et al, 1998Lin et al, , 2004Di Croce et al, 2002;Bernardi et al, 2004;Insinga et al, 2004;Kamashev et al, 2004;de Stanchina et al, 2004;Zeisig et al, 2007). However, which of these abnormal characteristics, if not all, are responsible for the initiation of leukemogenesis has remained elusive.…”

RXR, un cofacteur essentiel à la transformation dans les leucémies aiguës promyélocytaires

“…Néanmoins, la pré-sence d'AMPc permet la « désubordination » de RXR quand il est associé à RAR ou à PML-RARa [7][8][9]. L'AMPc active la PKA (protéine kinase A) induisant alors une phosphorylation de RARa et/ou des corépresseurs et le décrochage de ces derniers de la sous-unité RAR de l'hétérodimère ( Figure 2B, 2C) ainsi qu'une augmentation de la fixation de RARa à l'ADN [7,10].…”

“…L'oncoprotéine de fusion PML-RARa peut s'homodi- les sites non canoniques, RXRa les rend beaucoup plus fortes que celles de l'homodimère PML-RARa [9]. Le complexe PML-RARa:RXRa se fixant sur une gamme de sites plus large que le dimère RAR:RXR [22], il est alors capable de réguler un ensemble distinct de gènes, dont une grande partie n'est pas contrôlée par les voies classiques de signalisation de l'AR.…”

“…Certains complexes contiennent ainsi quatre domaines de liaison à l'ADN (deux dans RXRa et deux dans RARa) permettant alors d'expliquer la coopération dans la liaison à l'ADN : les domaines non fixés sur les motifs AGGTCA peuvent contribuer à la stabilisation du complexe via des interactions à l'ADN non spécifiques des séquences ( Figure 3A). Plusieurs études ont montré que la liaison de l'hétérocomplexe PML-RARa:RXRa n'a plus de préférence de structure ou d'orientation au niveau des répétitions AGGTCA : la fixation de PML-RARa a ainsi été observée dans des cellules de LAP humaines sur des répétitions directes espacées de 1 à 20 nucléotides mais aussi sur des répéti-tions inversées ou reversées aussi bien in vitro que in vivo [9,21,22]. En modifiant les propriétés de liaison à l'ADN du complexe sur …”