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BackgroundThe epigenetic machinery is frequently altered in acute myeloid leukemia. Focusing on cytogenetically normal (CN) AML, we previously described an abnormal H3K27me3 enrichment covering 70 kb on theHIST1cluster (6.p22) in CN-AML patient blasts. Here, we further investigate the molecular, functional, and prognosis significance of this epigenetic alteration named H3K27me3HIST1inNPM1-mutated (NPM1mut) CN-AML.ResultsWe found that three quarter of theNPM1mut CN-AML patients were H3K27me3HIST1high. H3K27me3HIST1highgroup of patients was associated with a favorable outcome independently of known molecular risk factors. In gene expression profiling, the H3K27me3HIST1highmark was associated with lower expression of the histone genesHIST1H1D,HIST1H2BG,HIST1H2AE, andHIST1H3Fand an upregulation of genes involved in myelomonocytic differentiation. Mass spectrometry analyses confirmed that the linker histone protein H1d, but not the other histone H1 subtypes, was downregulated in the H3K27me3HIST1highgroup of patients. H1d knockdown primed ATRA-mediated differentiation of OCI-AML3 and U937 AML cell lines, as assessed on CD11b/CD11c markers, morphological and gene expression analyses.ConclusionsOur data suggest thatNPM1mut AML prognosis depends on the epigenetic silencing of theHIST1cluster and that, among the H3K27me3 silenced histone genes,HIST1H1Dplays a role in AML blast differentiation.Graphical abstract

International audience; The transcription factor PLZF (promyelocytic leukemia zinc finger protein) acts as an epigenetic regulator balancing self-renewal and differentiation of hematopoietic cells through binding to various chromatin-modifying factors. First described as a transcriptional repressor, PLZF is also associated with active transcription, although the molecular bases underlying the differences are unknown. Here, we reveal that in a hematopoietic cell line, PLZF is predominantly associated with transcribed genes. Additionally, we identify a new association between PLZF and the histone methyltransferase, EZH2 at the genomic level. We find that co-occupancy of PLZF and EZH2 on chromatin at PLZF target genes is not associated with SUZ12 or trimethylated lysine 27 of histone H3 (H3K27me3) but with the active histone mark H3K4me3 and active transcription. Removal of EZH2 leads to an increase of PLZF binding and increased gene expression. Our results suggest a new role of EZH2 in restricting PLZF positive transcriptional activity independently of its canonical PRC2 activity.

INTRODUCTION Inappropriate recruitment of functional Polycomb-Group proteins (PcG) may trigger epigenetic unbalance at very specific genomic loci that substantially contribute to the pathogenesis of Acute Myeloid Leukemia (AML). This concept was first described in Acute Promyelocytic Leukemia (APL) in which PcG proteins were abnormally addressed due to the expression of X-RARA fusion proteins and were involved in the treatment response of the disease. For instance, in the context of APL with t(11;17)(q23;q21) translocation, the resulted oncogenic fusion protein PLZF/RARA leads to abnormal recruitment of PcG at the promoters of genes involved in acid-trans-retinoic acid (ATRA) response (Boukarabila et al.). As a consequence of that and compared to other APL subtypes (e-g: PML/RARA), APL with PLZF/RARA are insensitive to ATRA. In the recent years, a repertoire of cis-regulatory enhancer elements has been dissected to reveal important insight about leukemia onset and define new subsets of the disease with different treatment responses (Bhagwat et al). As we previously reported that PLZF displayed epigenetic specificity on enhancers (Poplineau et al.) we questioned the role of PLZF/RARA on these regulatory regions during APL onset. METHODS We performed in vivo comparative epigenomic profiling (H3K27ac, H3K4me1, H3K27me3 and H3K4me3 ChIPseq) between normal myeloid progenitors (granulocyte-monocyte progenitors purified from wild-type mice) and PLZF/RARA transformed mouse progenitors (late promyelocytes purified from mice developing APL). To question the role of PcG in APL onset, we used retroviral overexpression of PLZF/RARA and transduced Lineage negative cells from a conditional KO EZH2 mouse model. Transformation was tested by replating assay and cells were characterized by FACS and morphology analyses. We also performed EZH2 pharmacological inhibition using GSK126 and UNC1999 on a human cell line expressing the fusion protein PLZF/RARA. We analyzed the impact of this inhibition on their transcriptomic signature (RNAseq) and their proliferative capacity. RESULTS Upon PLZF/RARA expression and APL progression, specific cis-regulatory enhancer elements were targeted by the H3K27me3 PcG repressive mark. This gain in poised enhancer regions, upon PLZF/RARA expression reflected a reoriented PcG activity, from enhancers regulating developmental processes to those regulating stress and immune responses. To demonstrate the importance of this H3K27me3 switch for APL progression, we investigated the effect of EZH2 loss during PLZF/RARA transformation. Using a conditional KO EZH2 mouse model, we demonstrated that PLZF/RARA required EZH2 activity to efficiently transform progenitors since EZH2 loss promoted differentiation that altered the replating capacity of the PLZF/RARA expressing cells. In addition, EZH2 inhibition by GSK126 revealed some interesting benefits since it sensitized PLZF/RARA transformed progenitors to ATRA treatment. Moreover, inhibition of EZH2 with GSK126 or UNC1999 induced a decrease in the proliferation advantage of a human PLZF/RARA-inducible cell line. This was linked to a change of its transcriptomic signature towards an expression pattern closer to the one observed in the parent cell line. CONCLUSION Taken together, our data showed that PLZF/RARA modifies H3K27me3 profiles at enhancer regions and requires EZH2 activity for APL onset. Finally, our results suggest that EZH2 inhibition could be a new promising therapeutic approach for retinoic-acid resistant APL. Disclosures No relevant conflicts of interest to declare.

Ciliary neurotrophic factor (CNTF) has been shown to be expressed after brain lesions and in particular after demyelination. Here, we addressed the role of this cytokine in the regulation of neural progenitor migration in the adult rodent brain. Using an acute model of demyelination, we show that CNTF is strongly re-expressed after lesion and is involved in the postlesional mobilization of endogenous progenitors that participate in the myelin regenerative process. We show that CNTF controls the migration of subventricular zone (SVZ)-derived neural progenitors toward the demyelinated corpus callosum. Furthermore, an ectopic source of CNTF in adult healthy brains changes SVZ-derived neural progenitors' migratory behavior that migrate toward the source by activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway. Using variousin vitroassays (Boyden chambers, explants, and video time-lapse imaging), we demonstrate that CNTF controls the directed migration of SVZ-derived progenitors and oligodendrocyte precursors. Altogether, these results demonstrate that in addition to its neuroprotective activity and its role in progenitor survival and maturation, CNTF acts as a chemoattractant and participates in the recruitment of endogenous progenitors during myelin repair.

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.