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Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. Significance: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397

Differentiation arrest along the myeloid lineage is a defining feature of acute myeloid leukemia (AML), but its molecular determinants remain poorly defined. Pharmacological removal of the differentiation block leads to leukemia eradication in acute promyelocytic leukemia (APL) patients but has not yet been successfully translated to non-APL AMLs. Here, by investigating the function of hypoxia-inducible transcription factors HIF1α and HIF2α, we identify HIF2α as a new regulator of the AML differentiation block. Mechanistically, HIF2α cooperates with EZH2, the catalytic subunit of polycomb repressive complex 2, to promote deposition of the repressive H3K27me3 histone mark at the regulatory regions of myeloid differentiation genes. Thus, inhibiting HIF2α releases an epigenetic break to leukemic blasts maturation and cooperates with the pro-differentiation agent all-trans retinoic acid to trigger AML differentiation. We propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and forcing leukemia debulking.

Cellular senescence, first described in vitro in 1961, has become a focus for biotech companies that target it to ameliorate a variety of human conditions. Eminently characterized by a permanent proliferation arrest, cellular senescence occurs in response to endogenous and exogenous stresses, including telomere dysfunction, oncogene activation and persistent DNA damage. Cellular senescence can also be a controlled programme occurring in diverse biological processes, including embryonic development. Senescent cell extrinsic activities, broadly related to the activation of a senescence-associated secretory phenotype, amplify the impact of cell-intrinsic proliferative arrest and contribute to impaired tissue regeneration, chronic age-associated diseases and organismal ageing. This Review discusses the mechanisms and modulators of cellular senescence establishment and induction of a senescence-associated secretory phenotype, and provides an overview of cellular senescence as an emerging opportunity to intervene through senolytic and senomorphic therapies in ageing and ageing-associated diseases. Cellular senescence is characterized by a permanent proliferation arrest and the establishment of a senescence-associated secretory phenotype. This Review discusses the mechanisms of cellular senescence and induction of a senescence-associated secretory phenotype, recent insights into how senescence contributes to ageing, and the potential of senolytic and senomorphic therapies in ageing and associated diseases.

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

Mesenchymal stromal cells (MSCs) have been employed in vitro to support hematopoietic stem and progenitor cell (HSPC) expansion and in vivo to promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome. ispartof: MOLECULAR THERAPY vol:31 issue:1 pages:230-248 ispartof: location:United States status: published

Common variable immunodeficiency (CVID) is the most frequent primary antibody deficiency whereby follicular helper T (Tfh) cells fail to establish productive responses with B cells in germinal centers. Here, we analyzed the frequency, phenotype, transcriptome and function of circulating Tfh (cTfh) cells in CVID patients displaying autoimmunity as an additional phenotype. A group of patients showed a high frequency of cTfh1 cells and a prominent expression of PD-1 and ICOS, as well as a cTfh mRNA signature consistent with highly activated, but exhausted, senescent and apoptotic cells. Plasmatic CXCL13 levels were elevated in this group and positively correlated with cTfh1 cell frequency and PD-1 levels. Monoallelic variants in RTEL1, a telomere length- and DNA repair-related gene, were identified in four patients belonging to this group. Their blood lymphocytes showed shortened telomeres, while their cTfh were more prone to apoptosis. These data point toward a novel pathogenetic mechanism in CVID, whereby alterations in DNA repair and telomere elongation might predispose to antibody deficiency. A Th1, highly activated but exhausted and apoptotic cTfh phenotype was associated with this form of CVID. This article is protected by copyright. All rights reserved.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools. We thank S. Gregori and G. Amodio for help with neutrophil isolation and culture experiments; F. Di Salvo, F. Porzio and M. Tassara for patient recruitment and data management; the Center for Omics Sciences, the Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory, Centro Risorse Biologiche at Ospedale San Raffaele; and the Centro Universitario di Statistica per le Scienze Biomediche at Vita-Salute San Raffaele University. Figures were created with Adobe Illustrator and BioRender.com. V.B. and F.V.M. conducted this study as partial fulfillment of a PhD in Molecular Medicine (Basic and Applied Immunology and Oncology program) at Vita-Salute San Raffaele University. R.D.M. is a New York Stem Cell Foundation – Robertson Investigator. M.A.C. and P.S. are supported by grants from the Italian Association for Cancer Research (AIRC) (IG 20339) and the Italian Ministry of University and Research (PRIN 20177J4E75_004). A.A. is supported by the Italian Telethon Foundation (SR-Tiget grant award B02). E.M. and N.C. are supported by fellowships from Fondazione Umberto Veronesi. This study was supported by grants from the Italian Telethon Foundation (SR-Tiget grant award F04 to R.O.) and the Italian Ministry of Health (GR-201602362156 to R.O. and S.C.). Research in the R.O. laboratory is supported by the European Research Council (starting grant 759532, X-TAM) and by AIRC (MFAG 20247 and AIRC 5×1000 special program 22737) Sí

Acute myeloid leukemia (AML) is characterized by epigenetic silencing of differentiation genes and accumulation of aberrant myeloid cells arrested at different stages of myeloid development. The combination of epigenetic and differentiation therapies represents an attractive opportunity for AML patients by promoting chromatin remodeling at differentiation genes and induction of terminal maturation and leukemia debulking. The aim of our work is to define the function of the hypoxia inducible transcription factor HIF2alpha in the pathogenesis of AML. By its genetic inactivation and pharmacological inhibition in AML cell lines and patient-derived xenograft models, we found that HIF2alpha acts as a novel regulator of the AML differentiation block and promotes leukemia progression. Gene expression profiling of HIF2alpha-dependent transcriptome revealed that HIF2alpha mainly promotes the expression of genes involved in transcriptional modulation and epigenetic modifications, whilst inhibiting genes of myeloid maturation and activation. Mechanistically, inhibition of HIF2alpha results in decreased global levels of the heterochromatin marker H3K27me3, which is specifically deposited by the catalytic subunit EZH2 of the Polycomb repressive complex 2. Intriguingly, we found that HIF2alpha and EZH2 cooperate at favoring EZH2-mediated deposition of H3K27me3 at reverse hypoxia responsive elements in the regulatory regions of myeloid maturation genes. Additionally, we demonstrate that HIF2alpha is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we provide new mechanistic insights into the role of HIF2alpha in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. Importantly, small molecule inhibitors of HIF2alpha have been recently generated and are tested for solid cancers. Therefore, HIF2alpha inhibition may open new therapeutic avenues for AML patients and add therapeutic value to ATRA-based therapies by interrupting HIF2alpha upregulation and promoting an epigenetic state permissive to maturation of leukemic blasts and leukemia exhaustion. Citation Format: Daniela Magliulo, Carolina Caserta, Serena Belluschi, Cristina Fracassi, Kety Giannetti, Raffaella Pini, Ettore Zapparoli, Stefano Beretta, Eleonora Draghi, Marco J. Morelli, Raffaella Di Micco, Bernhard Gentner, Luca Vago, Rosa Bernardi. The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B027.

Background: Evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, up to 40% of AML relapses display complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods: Starting from primary AML samples pairwise collected from five patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches and in the different patients were integrated by Multi-Omics Factor Analysis (MOFA) and Gene Set Enrichment Analysis (GSEA). Finally, we tested the immunological effects of epigenetic drugs on AML cells and on their recognition by T cells in ex-vivo short-term cultures and in PDXs. Results: We verified that PDXs faithfully recapitulate immune-related differences between AML diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules. Differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility, and largely unrelated to the DNA methylation profile. In particular, in all five patients analyzed, we documented genomewide chromatin compaction at time of relapse, that was particularly evident for HLA class II genes and their master regulator CIITA, and not detected in relapses after sole chemotherapy. Integration of all the high-throughput technologies by MOFA, and of results from different patients by GSEA, pointed to the Polycomb Repressive Complex 2 (PRC2) as the main candidate mediator of HLA class II silencing.Pharmacological inhibition of PRC2 subunits rescued HLA class II expression in AML relapses ex vivo and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Conclusions: Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML post-transplantation relapse. Citation Format: Valentina Gambacorta, Stefano Beretta, Martina Ciccimarra, Laura Zito, Kety Giannetti, Angela Andrisani, Daniela Gnani, Lucia Zanotti, Giacomo Oliveira, Matteo G. Carrabba, Davide Cittaro, Ivan Merelli, Fabio Ciceri, Raffaella Di Micco, Luca Vago. Integrated multiomic profiling identifies the epigenetic regulator PRC2 as a therapeutic target to counteract leukemia immune escape and relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB563.

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms., BRAF-MAPK activating mutations are reported in histiocytoses—hematological neoplasms with widespread pro-inflammatory myeloid cells. Here, the authors show that an activating mutant BRAF in haematopoietic stem and progenitor cells causes an oncogene-induced senescence response leading to myeloid restricted haematopoiesis, inflammation and histiocytosis.

Summary Globoid cell leukodystrophy (GLD) is a rare neurodegenerative lysosomal storage disease caused by an inherited deficiency of β-galactocerebrosidase (GALC). GLD pathogenesis and therapeutic correction have been poorly studied in patient neural cells. Here, we investigated the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression in induced pluripotent stem cell (iPSC)-derived neural progenitors and neuronal/glial progeny obtained from two GLD patients. GLD neural progeny displayed progressive psychosine storage, oligodendroglial and neuronal defects, unbalanced lipid composition, and early activation of cellular senescence, depending on the disease-causing mutation. The partial rescue of the neural differentiation program upon GALC reconstitution and psychosine clearance suggests multiple mechanisms contributing to neural pathology in GLD. Also, the pathological phenotype associated to supraphysiological GALC levels highlights the need of regulated GALC expression for proper human neural commitment/differentiation. These data have important implications for establishing safe therapeutic strategies to enhance disease correction of GLD., Highlights • GLD hiPSC-derived neural progenitors show defective neuronal/glial differentiation • GLD neural progeny displays psychosine storage and modest lysosomal impairment • Lipidome unbalance and cellular senescence contribute to the GLD phenotype • Restoring GALC activity partially rescues the GLD phenotypic defects, In this article, Mangiameli et al. study the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression in GLD hiPSC-derived neural progenitors and their neuronal/glial progeny. The study uncovers unforeseen mutation- and cell type-specific early pathogenic events (i.e. lipid unbalance and activation of a senescence program) that may contribute to GLD neuropathology along with psychosine storage, with important therapeutic implications.

Cellular senescence constitutes a stable growth arrest characterized by DNA damage response (DDR) activation and by the senescence-associated secretory phenotype (SASP). SASP, through its paracrine effects, stimulates the immune system for senescence eradication. Similarly, chemotherapy-treated cancers activate an interferon-mediated response to induce anti-tumor immunity. Recent studies now uncover a new role for the innate DNA sensing pathway in the recognition of cytosolic chromatin in senescence and cancer.

Editorial summary Recent work has reported that the activation of the p53 pathway in primary cells hampers the efficiency of genome editing by CRISPR/Cas9 via induction of apoptosis or cell cycle arrest. These findings highlight the importance of wholly comprehending cellular responses to programmable nucleases in therapeutically relevant cell types to fulfill the promise of genome editing for therapeutic applications for human diseases.

Background It is becoming increasingly recognized that evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, alterations in the antigen processing and presentation machinery represent one of the most effective strategies enacted by tumor cells to avoid recognition from T cells. Whereas it is now well recognized that genomic loss of HLA is frequently at the basis of post-transplantation relapse, it was only recently reported that up to 40% of AML relapses display transcriptional downregulation and complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods Starting from primary AML samples pairwise collected from patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were analyzed for surface expression of selected immune-related markers (HLA class I and II, PD-L1, B7-H3), and characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), histone modifications associated with active promoters (H3K4me3) or regulatory elements (H3K27ac) (by ChIP-seq) and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches were integrated by Multi-Omics Factor Analysis (MOFA), followed by Gene Set Enrichment Analysis (GSEA). Finally, the immunological effects of epigenetic drugs and recombinant immune-modulatory cytokines on primary and PDX-derived AML samples were tested in ex-vivo short-term cultures on a layer of mesenchymal stromal cells. Results We verified that PDXs faithfully recapitulate immune-related differences between diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules (Figure 1A). Integration of all the high-throughput technologies by MOFA evidenced that the differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility and histone marks, and largely unrelated to the DNA methylation profile (Figure 1B). We documented that the gene sets that emerged upon integrating epigenetic analyses by MOFA matched our previously published immune-related relapse signature (Toffalori et al, Nat Med, 2019) but also and most intriguingly lists of genes known to be targeted by EZH2, the enzymatic subunit of the PRC2 chromatin repressor complex (Figure 1C). These computational analyses were supported by the evidence of a relapse-specific closed chromatin status of HLA class II genes and their regulators (Figure 1D). To revert these epigenetic changes, we inhibited EZH2 with tazemetostat (EPZ-6438), an epigenetic drug currently being tested in early-phase clinical trials for lymphomas, in two AML relapses with non-genomic loss of HLA class II expression. EZH2 inhibition reduced the levels of the repressor mark H3K27me3 (Figure 1E), increased the surface expression of HLA class II molecules on leukemia cells (Figure 1F) and ultimately improved leukemia recognition by CD4+ T cells (Figure 1G). Notably, these effects were even more pronounced when EZH2 inhibition was combined with IFN-g treatment (Figure 1F,G), suggesting synergism between this epigenetic compound and cytokines released by immune cells upon target recognition. Conclusions Our results provide mechanistic insights into epigenetic regulation of HLA class II downregulation in leukemia and a strong therapeutic rationale to test EZH2 inhibition as an innovative strategy for the treatment of AML post-transplantation relapses. Figure 1 Disclosures Vago: GenDx: Research Funding; Moderna Therapeutics: Research Funding.

Age-associated changes in hematopoietic stem and progenitor cells (HSPCs) have been carefully documented in mouse models but poorly characterized in primates and humans. To investigate clinically relevant aspects of hematopoietic aging, we compared the clonal output of thousands of genetically barcoded HSPCs in aged vs young macaques after autologous transplantation. Aged macaques showed delayed emergence of output from multipotent (MP) clones, with persistence of lineage-biased clones for many months after engraftment. In contrast to murine aging models reporting persistence of myeloid-biased HSPCs, aged macaques demonstrated persistent output from both B-cell and myeloid-biased clones. Clonal expansions of MP, myeloid-biased, and B-biased clones occurred in aged macaques, providing a potential model for human clonal hematopoiesis of indeterminate prognosis. These results suggest that long-term MP HSPC output is impaired in aged macaques, resulting in differences in the kinetics and lineage reconstitution patterns between young and aged primates in an autologous transplantation setting.

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a trans-membrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

Summary: Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation. : Di Micco et al. now dissect the mechanisms by which BRD4 regulates embryonic stem cell (ESC) identity by binding to super-enhancers of core pluripotency genes and recruiting active transcription complexes. BRD4 inhibition results in defective elongation of super-enhancer-associated gene transcripts, loss of ESC self-renewal/pluripotency, and commitment to the neuroectodermal lineage.

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However, responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here, we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction, cell-cycle progression, and transcriptional responses in HSPC subpopulations, with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation, constraining proliferation, yield, and engraftment of edited HSPCs. However, functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs. Precise gene editing has the potential to treat immune and hematological diseases. Genovese, Naldini, Di Micco, and colleagues now show that gene-editing procedures are well tolerated by hematopoietic stem cells and provide molecular evidence of the feasibility of seamless gene editing, strengthening translation of such approaches to humans.

Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) niche and serve as a reservoir for mature blood cells throughout life. Aging in the BM is characterized by low‐grade chronic inflammation that could contribute to the reduced functionality of aged HSPC. Mesenchymal stromal cells (MSC) in the BM support HSPC self‐renewal. However, changes in MSC function with age and the crosstalk between MSC and HSPC remain understudied. Here, we conducted an extensive characterization of senescence features in BM‐derived MSC from young and aged healthy donors. Aged MSC displayed an enlarged senescent‐like morphology, a delayed clonogenic potential and reduced proliferation ability when compared to younger counterparts. Of note, the observed proliferation delay was associated with increased levels of SA‐β‐galactosidase (SA‐β‐Gal) and lipofuscin in aged MSC at early passages and a modest but consistent accumulation of physical DNA damage and DNA damage response (DDR) activation. Consistent with the establishment of a senescence‐like state in aged MSC, we detected an increase in pro‐inflammatory senescence‐associated secretory phenotype (SASP) factors, both at the transcript and protein levels. Conversely, the immunomodulatory properties of aged MSC were significantly reduced. Importantly, exposure of young HSPC to factors secreted by aged MSC induced pro‐inflammatory genes in HSPC and impaired HSPC clonogenic potential in a SASP‐dependent manner. Altogether, our results reveal that BM‐derived MSC from aged healthy donors display features of senescence and that, during aging, MSC‐associated secretomes contribute to activate an inflammatory transcriptional program in HSPC that may ultimately impair their functionality.

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.

Primary mouse embryonic fibroblasts (MEFs) are a popular tool for molecular and cell biology studies. However, when MEFs are grown in vitro under standard tissue culture conditions, they proliferate only for a limited number of population doublings (PD) and eventually undergo cellular senescence. Presently, the molecular mechanisms halting cell cycle progression and establishing cellular senescence under these conditions are unclear. Here, we show that a robust DNA damage response (DDR) is activated when MEFs undergo replicative cellular senescence. Senescent cells accumulate senescence-associated DDR foci (SDFs) containing the activated form of ATM, its phosphorylated substrates and gammaH2AX. In senescent MEFs, DDR markers do not preferentially accumulate at telomeres, the end of linear chromosomes. It has been observed that proliferation of MEFs is extended if they are cultured at low oxygen tension (3% O(2)). We observed that under these conditions, DDR is not observed and senescence is not established. Importantly, inactivation of ATM in senescent MEFs allows escape from senescence and progression through the S-phase. Therefore, MEFs undergoing cellular senescence arrest their proliferation due to the activation of a DNA damage checkpoint mediated by ATM kinase. Finally, we observed that spontaneously immortalized proliferating MEFs display markers of an activated DDR, indicating the presence of chromosomal DNA damage in these established cell lines.

The age-related increase in cancer incidence has been taken as a proof of the mutation accumulation theory of cancer in humans. Recent data suggest that longliving stem cells are the most likely target for oncogenic mutations. Nevertheless, a plenty of data point out for a major role of stromal cell in cancer growth. These data suggest that the study of the interaction between aged stromal cells and cancer initiating cells (i.e. cancer stem cells) may be of primary importance to better understand cancer biology.In this investigation we employed human mammospheres (MS) from normal and tumor tissues as a model for normal and cancer stem/progenitor cells of the mammary gland. We here report that, compared to fibroblasts from young individuals, fibroblasts from aged people and nonagenarians show an enhanced capacity to induce invasive behaviour of normal and tumor MS. Similarly, we show that in vitro senescent fibroblasts induce invasiveness of MS at a higher extent than non senescent cells. We also provide details about the molecular pathway involved in such a phenomenon, by showing that the capacity of aged/senescent fibroblasts to enhance the invasive behaviour of stem/progenitor cells is pivoted by p66Shc gene. These data support the notion that in vivo/in vitro aged stromal cells create a pro-tumorigenic environment for stem/progenitor cells of the mammary gland.

The generation of DNA lesions and the resulting activation of DNA damage response (DDR) pathways are both affected by the chromatin status at the site of damaged DNA. In turn, DDR activation affects the chromatin at both the damaged site and across the whole genome. Cellular senescence and cancer are associated with the engagement of the DDR pathways and with profound chromatin changes. In this Opinion article, we discuss the interplay between chromatin and DDR factors in the context of cellular senescence that is induced by oncogenes and in cancer.

Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescence-associated heterochromatic foci (SAHF). Here we show that in human fibroblasts resistant to premature p16(INK4a) induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogene-induced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogene-expressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor (HDACi) causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.

Oncogene activation in normal cells induces a permanent proliferative arrest known as cellular senescence. This phenomenon restrains the expansion of cells that bear an activated oncogene and acts as a powerful tumor-suppressive process. Although the full molecular mechanisms are still being elucidated, it has been observed recently that some oncogenes alter the DNA-replication process and cause DNA-damage accumulation. DNA-damage checkpoint-response activation together with the increased appearance of heterochromatin formation that leads to transcriptional silencing of proliferative genes are, presently, the two main mechanisms known that establish and maintain oncogene-induced senescence. Here, we discuss the most recent advancements in understanding the molecular and cellular mechanisms that control cellular senescence caused by oncogene activation and their impact on cancer studies.

Early tumorigenesis is associated with the engagement of the DNA-damage checkpoint response (DDR). Cell proliferation and transformation induced by oncogene activation are restrained by cellular senescence. It is unclear whether DDR activation and oncogene-induced senescence (OIS) are causally linked. Here we show that senescence, triggered by the expression of an activated oncogene (H-RasV12) in normal human cells, is a consequence of the activation of a robust DDR. Experimental inactivation of DDR abrogates OIS and promotes cell transformation. DDR and OIS are established after a hyper-replicative phase occurring immediately after oncogene expression. Senescent cells arrest with partly replicated DNA and with DNA replication origins having fired multiple times. In vivo DNA labelling and molecular DNA combing reveal that oncogene activation leads to augmented numbers of active replicons and to alterations in DNA replication fork progression. We also show that oncogene expression does not trigger a DDR in the absence of DNA replication. Last, we show that oncogene activation is associated with DDR activation in a mouse model in vivo. We propose that OIS results from the enforcement of a DDR triggered by oncogene-induced DNA hyper-replication.

The incidence of melanoma is increasing faster than that of any other cancer, and predicted to double every 10-20 years. Surgery can be curative in Stage I, II, or III disease, but 75% of patients with deep primary lesions develop extensive recurrence or distant metastases and have dismal prognosis. In fact, there is no curative treatment for stage IV melanoma. Although novel targeted therapies, such as BRAF inhibitors and anti-CTLA4 antibodies are showing promising results in melanoma clinical trials, resistance to these agents and patient relapse rapidly ensue. Therapeutic resistance has been commonly attributed to functional redundancy between intimately hardwired cellular pathways responsible for tumor cell maintenance and survival. In order to avoid redundancy, we propose to directly inhibit the transcription of multiple genes required for the establishment or maintenance of tumors. First, we analyzed the expression of Bromodomain (BrD)-containing proteins, a family of epigenetic readers that bind acetylated lysine. BrDs are present in histone acetyl transferases (i.e. CBP/p300, PCAF, GCN5) and transcriptional regulators (i.e. BET family members: BRD2, 3, and 4). mRNA expression arrays showed several BrD-containing genes as upregulated in primary and metastatic melanoma cell lines compared to normal melanocytes. Analysis of available expression profiles also revealed higher levels of BRD2 and BRD4 in melanoma tissues relative to nevi or normal skin (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2185. doi:1538-7445.AM2012-2185

SummaryTo metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression.