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Cancer cell heterogeneity is a major driver of therapy resistance. To characterize resistant cells and their vulnerabilities, we studied the PLZF-RARA variant of acute promyelocytic leukemia, resistant to retinoic acid (RA), using single-cell multiomics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells. We identified a subset of cells resistant to RA with proliferation, DNA replication, and repair signatures that depend on a fine-tuned E2F transcriptional network targeting the epigenetic regulator enhancer of zeste homolog 2 (EZH2). Epigenomic and functional analyses validated the driver role of EZH2 in RA resistance. Targeting pan-EZH2 activities (canonical/noncanonical) was necessary to eliminate leukemia relapse-initiating cells, which underlies a dependency of resistant cells on an EZH2 noncanonical activity and the necessity to degrade EZH2 to overcome resistance. Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach. Beyond RA resistance and acute promyelocytic leukemia context, our study also demonstrates the power of single-cell multiomics to identify, characterize, and clear therapy-resistant cells.

BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein ‘cytokine inducible SH2-containing protein’ (CISH) in NK cells to evaluate the impact on their functions and antitumor properties.MethodsTo further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions.ResultsIn Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions.ConclusionThis study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA. We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature ("Rep" cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance. These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. Disclosures Iwama: Nissan Chemical Corporation: Research Funding.

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.