Fabien Guidez

Distinct interactions of PML-RARα and PLZF-RARα with co-repressors determine differential responses to RA in APL

Acute promyelocytic leukaemia (APL), associated with chromosomal translocations involving the retinoic acid receptor α gene (RARA) and the PML gene, is sensitive to retinoic acid (RA) treatment, while APL patients harbouring translocations between RARA and the PLZF gene do not respond to RA. We have generated PML-RARA and PLZF-RARA transgenic mice and show here that these fusion proteins play a critical role in leukaemogenesis and in determining responses to RA in APL, because PLZF-RARA transgenic mice develop RA-resistant leukaemia, while PML-RARA mice are responsive to RA treatment. We demonstrate that both PML-RARα and PLZF-RARα fusion proteins can act as transcriptional repressors and are able to interact with nuclear receptor transcriptional co-repressors, such as SMRT. PLZF-RARα, but not PML-RARα, can form, via its PLZF moiety, co-repressor complexes which are insensitive to RA. Histone deacetylase inhibitors such as Trichostatin A (TSA), in combination with RA, can overcome the transcriptional repressor activity of PML-RARα and PLZF-RARα as well as the unresponsiveness of PLZF-RARα-expressing leukaemic cells to RA. Thus, our findings unravel a crucial role for transcriptional silencing in APL pathogenesis and resistance to RA in APL.

Translocations of the RARalpha gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) has been recognized as a distinct clinical entity for over 40 years. Although relatively rare among hematopoietic malignancies (approximately 10% of AML cases), this disease has attracted a particularly good share of attention by becoming the first human cancer in which all-trans-retinoic acid (ATRA), a physiologically active derivative of vitamin A, was able to induce complete remission (CR). ATRA induced remission is not associated with rapid cell death, as in the case of conventional chemotherapy, but with a restoration of the ‘normal’ granulocytic differentiation pathway. With this remarkable medical success story APL has overnight become a paradigm for the differentiation therapy of cancer. A few years later, excitement with APL was further enhanced by the discovery that a cytogenetic marker for this disease, the t(15:17) reciprocal chromosomal translocation, involves a fusion between the retinoic acid receptor alpha (RARα) gene and a previously unknown locus named promyelocytic leukemia (PML). Consequence of this gene rearrangement is expression of the PML–RARα chimeric oncoprotein, which is responsible for the cellular transformation as well as ATRA response that is observed in APL. Since this initial discovery, a number of different translocation partner genes of RARα have been reported in rarer cases of APL, strongly suggesting that disruption of RARα underlies its pathogenesis. This article reviews various rearrangements of the RARα gene that have so far been described in literature, functions of the proteins encoded by the different RARα partner loci, and implications that these may have for the molecular pathogenesis of APL.

Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein

ABSTRACT Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.

Colocalization and heteromerization between the two human oncogene POZ/zinc finger proteins, LAZ3 (BCL6) and PLZF

Most acute promyelocytic leukemia (APL) cases are associated with recurrent translocations between the gene of retinoic receptor alpha and that of PML (t(15;17)) or PLZF (t(11;17)). PML localizes onto discrete intranuclear domains, the PML-nuclear bodies, and displays anti-oncogenic and pro-apoptotic properties. PLZF encodes a transcription factor belonging to the POZ/domain and Krüppel zinc finger (POK) family which interacts directly with PML. PLZF is related to another POK protein, LAZ3(BCL6), which is structurally altered, and presumably misexpressed, in many non-Hodgkin lymphoma (NHL) cases. PLZF and LAZ3 share many functional properties: both inhibit cell growth, concentrate into punctated nuclear subdomains and are sequence-specific transcriptional repressors recruiting a histone deacetylase-repressing complex. Given these similarities, we tested whether both proteins could be targeted by each other. Here, LAZ3 and PLZF are shown to colocalize onto nuclear dots. Moreover, truncated derivatives of one protein, which display a diffuse nuclear localization, are recruited onto nuclear dots by the full-length other. The colocalization and the reciprocal ‘rescue’ is the result of a direct interaction between LAZ3 and PLZF, as indicated by yeast two hybrid assays, in vitro immunoprecipitations, and GST pull down experiments. In contrast to LAZ3 homomerization, LAZ3/PLZF heteromerization in yeast does not solely depend on POZ/POZ contacts but rather also relies on interactions between the two zinc finger regions and ‘cross’ contacts between the zinc finger region and the POZ domain of each partner. Likewise, LAZ3 shows some colocalization with the PLZF partner PML upon stable overexpression of both proteins in CHO cells and interacts with PML in yeast. Finally, endogenous LAZ3 and PLZF are co-induced and partially colocalized in myeloid MDS cells. These data indicate that a physical interaction between LAZ3 and PLZF underlies their simultaneous recruitment onto multiproteic nuclear complexes, presumably involved in transcriptional silencing and whose integrity (for APL) and/or function (for APL and NHL) may be altered in oncogenesis.

Translocations of the RARα gene in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) has been recognized as a distinct clinical entity for over 40 years. Although relatively rare among hematopoietic malignancies (approximately 10% of AML cases), this disease has attracted a particularly good share of attention by becoming the first human cancer in which all-trans-retinoic acid (ATRA), a physiologically active derivative of vitamin A, was able to induce complete remission (CR). ATRA induced remission is not associated with rapid cell death, as in the case of conventional chemotherapy, but with a restoration of the ‘normal’ granulocytic differentiation pathway. With this remarkable medical success story APL has overnight become a paradigm for the differentiation therapy of cancer. A few years later, excitement with APL was further enhanced by the discovery that a cytogenetic marker for this disease, the t(15:17) reciprocal chromosomal translocation, involves a fusion between the retinoic acid receptor alpha (RARα) gene and a previously unknown locus named promyelocytic leukemia (PML). Consequence of this gene rearrangement is expression of the PML–RARα chimeric oncoprotein, which is responsible for the cellular transformation as well as ATRA response that is observed in APL. Since this initial discovery, a number of different translocation partner genes of RARα have been reported in rarer cases of APL, strongly suggesting that disruption of RARα underlies its pathogenesis. This article reviews various rearrangements of the RARα gene that have so far been described in literature, functions of the proteins encoded by the different RARα partner loci, and implications that these may have for the molecular pathogenesis of APL.

RARalpha-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARα by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARα oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARα-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARα+RARα/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARα APL. RARα-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARα-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype.

Acute Promyelocytic Leukemia: A Paradigm for Differentiation Therapy

Acute promyelocytic leukemia(APL) is characterized by the t(15;17) chromosomal translocation leading to the formation of the PML-RARalpha oncoprotein. This leukemia has attracted considerable interest in recent years, being the first in which therapies that specifically target the underlying molecular lesion, i.e., all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), leading to induction of differentiation and apoptosis have been successfully used in clinical practice. The advent of ATRA therapy has transformed APL from being a disease with a poor outlook to one of the most prognostically favorable subsets of acute myeloid leukemia. Further improvements in outcome may be achieved with the use of ATO, which achieves high rates of remission in the relatively small proportion of patients now relapsing following standard first-line therapy with ATRA and anthracycline-based chemotherapy. Moreover, recent studies have suggested that ATO and ATRA, or even ATO alone, used as front-line treatment of PML-RARA- associated APL can induce long-term remissions. This raises the possibility that some patients can be cured using differentiation therapies alone, without the need for chemotherapy, thereby potentially reducing treatment-related toxicity. It is clear that the success of such an approach is critically dependent upon molecular diagnostics and monitoring for minimal residual disease (MRD) to distinguish those patients who can potentially be cured with differentiation therapy from those requiring additional myelosuppressive agents. This represents an exciting new phase in the treatment of acute leukemia, highlighting the potential of molecularly targeted and MRD-directed therapies to achieve an individualized approach to patient management.

Juvenile myelomonocytic leukemia displays mutations in components of the RAS pathway and the PRC2 network

Juvenile myelomonocytic leukemia (JMML) is a rare and severe myelodysplastic and myeloproliferative neoplasm of early childhood initiated by germline or somatic RAS-activating mutations. Genetic profiling and whole-exome sequencing of a large JMML cohort (118 and 30 cases, respectively) uncovered additional genetic abnormalities in 56 cases (47%). Somatic events were rare (0.38 events/Mb/case) and restricted to sporadic (49/78; 63%) or neurofibromatosis type 1 (NF1)-associated (8/8; 100%) JMML cases. Multiple concomitant genetic hits targeting the RAS pathway were identified in 13 of 78 cases (17%), disproving the concept of mutually exclusive RAS pathway mutations and defining new pathways activated in JMML involving phosphoinositide 3-kinase (PI3K) and the mTORC2 complex through RAC2 mutation. Furthermore, this study highlights PRC2 loss (26/78; 33% of sporadic JMML cases) that switches the methylation/acetylation status of lysine 27 of histone H3 in JMML cases with altered RAS and PRC2 pathways. Finally, the association between JMML outcome and mutational profile suggests a dose-dependent effect for RAS pathway activation, distinguishing very aggressive JMML rapidly progressing to acute myeloid leukemia.

Regulation of Hoxb2 by APL-associated PLZF protein

The PLZF gene is translocated in a subset of all-trans-retinoic acid resistant acute promyelocytic leukaemia (APL) cases, encodes a DNA binding transcription factor and is expressed highly in haematopoietic progenitor cells as well-developing central nervous system (CNS). The spatially restricted and temporally dynamic pattern of PLZF expression in the developing CNS suggested that it might play a role in the circuitry regulating hindbrain segmentation. We have now identified a PLZF binding site (PLZF-RE) in an enhancer region of Hoxb2 that itself is required for directing high-level expression in rhombomers 3 and 5 of the developing hindbrain. The wild-type r3/r5 enhancer linked to a heterologous promoter was responsive to regulation by PLZF, and this activity was lost in variants containing a mutated PLZF-RE. Compared with the wild-type protein, the binding of the APL-associated reciprocal RARα–PLZF fusion to PLZF-RE was much stronger, suggesting that the N-terminal PLZF sequences missing from the fusion may play a role in the regulation of DNA binding. Consistent with this, the N-terminal POZ domain was required for cooperative binding of PLZF to a multimerized PLZF-RE. In the context of the r3/r5 enhancer, the PLZF-RE cooperated for PLZF binding with an additional A/T-rich motif positioned downstream of the PLZF-RE. This A/T motif was previously shown to be essential for the regulation of Hoxb2 expression in r3 and r5 in cooperation with another Krüppel-like zinc finger protein Krox 20. The presence of both the PLZF-RE and the A/T-rich motif was required for a maximal effect of PLZF on a heterologous promoter and was essential in vivo to direct the expression of a lacZ reporter in the chick neural tube. Hence, both PLZF and Krox20 cooperate with a common A/T motif in mediating in vivo activity of the Hoxb2 enhancer. Our findings indicate that Hoxb2 is a direct target for regulation by PLZF in the developing CNS and suggest that deregulation of Hox gene expression may contribute to APL pathogenesis.

HDAC4 as a potential therapeutic target in neurodegenerative diseases: a summary of recent achievements

For the past decade protein acetylation has been shown to be a crucial post-transcriptional modification involved in the regulation of protein functions. Histone acetyltransferases (HATs) mediate acetylation of histones which results in the nucleosomal relaxation associated with gene expression. The reverse reaction, histone deacetylation, is mediated by histone deacetylases (HDACs) leading to chromatin condensation followed by transcriptional repression. HDACs are divided into distinct classes: I, IIa, IIb, III, and IV, on the basis of size and sequence homology, as well as formation of distinct repressor complexes. Implications of HDACs in many diseases, such as cancer, heart failure, and neurodegeneration, have identified these molecules as unique and attractive therapeutic targets. The emergence of HDAC4 among the members of class IIa family as a major player in synaptic plasticity raises important questions about its functions in the brain. The characterization of HDAC4 specific substrates and molecular partners in the brain will not only provide a better understanding of HDAC4 biological functions but also might help to develop new therapeutic strategies to target numerous malignancies. In this review we highlight and summarize recent achievements in understanding the biological role of HDAC4 in neurodegenerative processes.