Heme oxygenase-1 (HO-1, encoded by Hmox1 ) is a cytoprotective enzyme with well-established roles in defending against oxidative stress. Global Hmox1 deficiency in mice accelerates hematopoietic stem cell (HSC) exhaustion and aging, effects previously attributed primarily to loss of HO-1 activity within the bone marrow (BM) niche. However, the cell-intrinsic contribution of HO-1 to HSC regulation has remained unclear. Here, we show that global Hmox1 deficiency results in accumulation of an expanded but largely quiescent HSC pool characterized by compromised genome maintenance, altered apoptotic signaling, and defective cell-cycle checkpoint control. We further demonstrate that HO-1 protein is expressed in HSCs and exhibits a predominantly nuclear, non-canonical localization. Using Hoxb5 -CreERT2-mediated conditional deletion of Hmox1 in HSCs, we uncover an intrinsic requirement for HO-1 in controlling early hematopoietic differentiation. HSC-specific loss of HO-1 skews stem cell output toward short-term progenitors and increases colony-forming capacity. Transcriptomic profiling of Hmox1 fl/fl ; Hoxb5 -CreERT2 HSCs revealed broad dysregulation of pathways involved in translation and RNA metabolism, together with aberrant expression of key transcription factors controlling hematopoietic differentiation. Collectively, these findings identify a non-canonical, cell-intrinsic role for HO-1 in regulating HSC homeostasis, differentiation, and aging.

Multiple myeloma (MM) is a malignancy of immunoglobulin-secreting plasma cells that represents 10% of all hematological cancers and remains essentially incurable, despite recent advances. Long non-coding RNAs (lncRNAs) are an important class of regulatory molecules that have been strongly implicated in the aetiology of MM. Colorectal Neoplasia Differentially Expressed (CRNDE) is one lncRNA that is upregulated in tumor plasma cells of MM patients and contributes to disease progression and outcome. In order to characterise the molecular mechanisms of CRNDE action in MM, here we have carried out a high-throughput screen to identify proteins interacting with this lncRNA inside cells. From the output of this screen, we demonstrate that in MM cells, CRNDE interacts with and stabilises the deacetylase protein SIRT1, previously identified as an important mediator of the Hedgehog (Hh) signalling pathway in MM. We further show that CRNDE exerts downstream effects on MM cell survival, tumorigenic potential, and stem-like properties via an effect on the SIRT1/Hh signalling axis. Our findings add to the molecular understanding of the pro-tumorigenic activity of CRNDE in MM and could have wider implications in other malignant diseases.

Chronic inflammation that accompanies aging impairs the function of hematopoietic stem cells (HSCs) and promotes expansion of TET2 -mutated cells, leading to clonal hematopoiesis of indeterminate potential (CHIP). The molecular mechanisms underlying these processes are still unclear. We recently showed that age and stresses like irradiation reduce the heterochromatin mark H3K9me3, leading to the epigenetic derepression of transposable elements such as LINE-1/L1, and L1-induced HSC functional changes through DNA damage and transcriptomic alterations. However, the impact of TEs on the clonal expansion of Tet2-/- HSCs upon chronic inflammation is unknown. Here we show that in wild type (WT) HSC, chronic inflammation induced by repeated low doses of lipopolysaccharide (LPS) triggers H3K9me3 loss at L1. We further show that L1s are involved in LPS-induced DNA damage and reduction of HSC clonogenicity. In contrast, Tet2-/- HSCs resist LPS-induced L1 epigenetic derepression and associated DNA damage. As a result, WT HSCs show decreased competitiveness against Tet2-/- HSCs upon chronic inflammation. These findings identify epigenetic control of L1s as a key mediator of inflammation-induced HSC dysfunction and clonal expansion of Tet2 -mutant cells. ### Competing Interest Statement The authors have declared no competing interest. * HSC : hematopoietic stem cell CHIP : clonal hematopoiesis of indeterminate potential MDS : myelodysplastic syndromes AML : acute myeloid leukemia CMML : chronic myelomonocytic leukemia TE : transposable element LTR : long terminal repeat ERV : endogenous retrovirus LINE/SINE : long/short interspersed elements La Ligue Contre le Cancer, https://ror.org/00rkrv905, LNCC, Equipe labellisée EL2020 Fondation ARC pour la Recherche sur le Cancer, https://ror.org/0489qz649, ARCPGA2023110007352_7985, ARCPJA2022070005354 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-15-CE14-0003, ANR-23-CE14-0017, ANRJCJC20-CE14-0018-01 Fondation de France, https://ror.org/02zkxjz73, 00135490/ PR-151961 European Hematology Association

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist , in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti–nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

The developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map demonstrating that lymphoid specification follows independent direct or stepwise hierarchic routes converging toward the emergence of newly characterized CD117(lo) multi-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127(-) (NK/ILC/T) or CD127(+) (B) lymphoid pathways. While the differentiation of CD127(-) early lymphoid progenitors is mainly driven by Flt3 signaling, emergence of their CD127(+) counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their upstream precursors, including hematopoietic stem cells. Further, transcriptional mapping of differentiation trajectories reveals that whereas myeloid granulomonocytic lineages follow continuous differentiation pathways, lymphoid trajectories are intrinsically discontinuous and characterized by sequential waves of cell proliferation allowing pre-commitment amplification of lymphoid progenitor pools. Besides identifying new lymphoid specification pathways and regulatory checkpoints, our results demonstrate that NK/ILC/T and B lineages are under fundamentally distinct modes of regulation.

Recent data have shown that liver fibrosis can regress even at later stages of cirrhosis and shifting the immune response from pro-inflammatory towards a resolutive profile is considered as a promising option. The immune regulatory networks that govern the shift of the inflammatory phenotype and thus potential reversal of liver fibrosis are lesser known. Here we show that in precision-cut human liver slices obtained from patients with end-stage fibrosis and in mouse models, inhibiting Mucosal-Associated Invariant T (MAIT) cells using pharmacological or antibody-driven approaches, limits fibrosis progression and even regresses fibrosis, following chronic toxic- or non-alcoholic steatohepatitis (NASH)-induced liver injury. Mechanistic studies, combining RNA sequencing, in vivo functional studies (performed in male mice) and co-culture experiments indicate that disruption of the MAIT cell-monocyte/macrophage interaction results in resolution of fibrosis both by increasing the frequency of restorative Ly6Clo at the expenses of pro-fibrogenic Ly6Chi monocyte-derived macrophages and promoting an autophagic phenotype in both subsets. Thus, our data show that MAIT cell activation and the consequential phenotype shift of liver macrophages are important pathogenic features of liver fibrosis and could be targeted by anti-fibrogenic therapy.

PDF file - 143K, Table S1: Clinical and biological characteristics of 53 patients with MDS/sAML. Table S2: Primers and experimental conditions for FAS and BCL2L1 quantification by RT-qPCR. Table S3: Biotinylated oligonucleotide sequences for in vitro p65 pull-down and primers for DNA methylation assay. Table S4: Primers for chromatin immunoprecipitation at FAS, B2M, RAG1 and PAX6 promoters.

Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and post-natal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in eryth-ropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoi-esis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.

Chimeric antigen receptor T cells (CAR-T) have provided promising results in multiple myeloma (MM). However, many patients still relapse, pointing toward the need of improving this therapy. Here, we analyzed peripheral blood T cells from MM patients at different stages of the disease and investigated their phenotype and capacity to generate functional CAR-T directed against CS1 or B Cell Maturation antigen. We found a decrease in naive T cells and elevated frequencies of exhaustion markers in T cells from treated MM patients. Interestingly, individuals treated with daratumumab display elevated ratios of central memory T cells. CAR-T derived from patients at relapse show reduced in vitro expansion and cytotoxic capacities in response to MM cells compared to those produced at diagnosis. Of note, CAR-T from daratumumab treated patients display intermediate defects. Reduced anti-myeloma activity of CAR T cells from treated patients was also observed in a mouse model. Our findings suggest that T cell defects in MM patients, specifically during relapse, have a major impact on their capacity to generate efficient therapeutic CAR-T. Selecting naive or central memory T cell subsets to generate therapeutic T cells could improve the CAR-T therapy for MM.

The developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map that extends the current view of lymphoid development. Our results demonstrate that lymphoid specification follows independent direct or stepwise differentiation pathways converging toward the emergence of CD117lo multi-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127- (T/NK/ILC) or CD127+ (B) lymphoid pathways. While the emergence of CD127- early lymphoid progenitors is driven by Flt3 signaling, differentiation of their CD127+ counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their precursors. Single-cell mapping of lymphoid differentiation trajectories reveals that a dissociation between proliferation and differentiation phases allows amplification of the precursor pools prior to the onset of antigen receptor rearrangement. Besides demonstrating that B and T/NK/ILC lineages are subjected to differential cell-autonomous versus Flt3-inducible regulation, our results go a long way to reconciling human and mouse models of lymphoid architecture.

Multiple myeloma (MM) is a currently incurable malignancy of antibody-secreting plasma cells in the bone marrow. Taken together with its premalignant precursor stage disease (monoclonal gammopathy of undetermined significance), MM accounts for ~20% of all hematological malignancies, making it one of the most common blood cancers. One of the prominent features of MM is a high level of genetic and biological diversity, which underlies the significant heterogeneity in the rates of disease progression observed between patients. For more than a decade, short- and more recently long noncoding RNAs (ncRNAs) have been investigated in tumor plasma cells of MM patients to understand the contribution of these molecules to disease progression, treatment response, and prognosis. In this chapter, we describe the existing databases documenting the changes in ncRNA expression in MM, highlight important ncRNAs for whom the role in the disease has been characterized, and also describe the complex interactions between ncRNAs and the major signaling pathways involved in this disease. Finally, we discuss the use of ncRNAs as potential diagnostic and prognostic biomarkers for MM, as well as recent efforts to target ncRNAs that could eventually complement existing therapies to achieve durable relapse and eventual cure.

SUMMARYThe developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map that extends the current view of lymphoid development. Our results demonstrate that lymphoid specification follows independent direct or stepwise differentiation pathways converging toward the emergence of CD117lomulti-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127-(T/NK/ILC) or CD127+(B) lymphoid pathways. While the emergence of CD127-early lymphoid progenitors is driven by Flt3 signaling, differentiation of their CD127+counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their precursors. Single-cell mapping of lymphoid differentiation trajectories reveals that a dissociation between proliferation and differentiation phases allows amplification of the precursor pools prior to the onset of antigen receptor rearrangement. Besides demonstrating that B and T/NK/ILC lineages are subjected to differential cell-autonomousversusFlt3-inducible regulation, our results go a long way to reconciling human and mouse models of lymphoid architecture.

Over the past 15 years, long non-coding RNAs (lncRNA) have emerged as an important class of regulatory molecules. The currently accepted definition is that lncRNA refers to RNA molecules with little or no protein-coding potential, and which are greater than 200 nucleotides in length, a size cut-off chosen largely to distinguish them from the more-extensively characterised group of small non-coding regulatory RNAs, which includes micro (mi)RNAs, small inhibitory (si)RNAs and PIWI-interacting (pi)RNAs [1]. A recent compilation of annotations from diverse sources identified nearly 57,000 genes encoding lncRNAs in the human genome [2].

RNAs originating from transcription upstream and downstream of genes accumulate in the cytoplasm of a subset of senescent cells, suggesting an RNA alternative to cytoplasmic DNA in the triggering of senescence. Accumulation of senescent cells is an important contributor to chronic inflammation upon aging. The inflammatory phenotype of senescent cells was previously shown to be driven by cytoplasmic DNA. Here, we propose that cytoplasmic double-stranded RNA has a similar effect. We find that several cell types driven into senescence by different routes share an accumulation of long promoter RNAs and 3′ gene extensions rich in retrotransposon sequences. Accordingly, these cells display increased expression of genes involved in response to double stranded RNA of viral origin downstream of the interferon pathway. The RNA accumulation is associated with evidence of reduced RNA turnover, including in some cases, reduced expression of RNA exosome subunits. Reciprocally, depletion of RNA exosome subunit EXOSC3 accelerated expression of multiple senescence markers. A senescence-like RNA accumulation was also observed in cells exposed to oxidative stress, an important trigger of cellular senescence. Altogether, we propose that in a subset of senescent cells, repeat-containing transcripts stabilized by oxidative stress or reduced RNA exosome activity participate in driving and maintaining the permanent inflammatory state characterizing cellular senescence.

Haematopoietic stem cells (HSCs) occupy the apex of the haematopoietic system, and ensure the renewal of all mature blood cells throughout life. Studies in both mice and humans have demonstrated that the functional properties of HSCs change with age, altering their capacity to reconstitute the haematopoietic system, and diminishing their lymphoid differentiation potential, giving rise to a myeloid-biased haematopoiesis in aged individuals. A range of different events are now known to contribute to the decline of HSC function observed with age, including alterations within the HSC themselves, as well as changes occurring in the bone marrow microenvironment where HSC reside. Cell intrinsic changes include the accumulation of DNA damage and telomere erosion, as well as alterations in HSC metabolism which trigger stress-response mechanisms and cellular exhaustion. More recently, age-associated changes in the epigenetic state have also been implicated as an important driver of HSC functional decline. Within the bone marrow microenvironment, the onset of low-level chronic inflammation and remodelling of HSC niches which take place with age also influence HSC functional capacity and contribute to a myeloid-biased differentiation. HSC aging has important downstream consequences for the health of the elderly, contributing to the chronic inflammatory state and decline in adaptive immune function observed in aged individuals, and predisposing to the onset of myeloid malignancies. Understanding the mechanisms driving HSC aging has suggested numerous therapeutic strategies to rejuvenate HSC function, which have shown promising results in aging mice.

Multiple myeloma (MM) is a currently incurable malignancy of antibody-secreting plasma cells. Long non-coding RNAs (lncRNAs) have been recognised as an important class of regulatory molecules which are increasingly implicated in tumorigenesis. While recent studies have demonstrated changes in expression of lncRNAs in MM, the functional significance and molecular pathways downstream of these changes remain poorly characterised. In this study, we have performed CRISPR-mediated deletion of the locus encoding the lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE), a known oncogenic lncRNA that is overexpressed in plasma cells of MM patients and is a marker of poor prognosis. We found that CRISPR-mediated deletion of the CRNDE locus in MM cells decreases proliferation and adhesion properties, increases sensitivity to Dexamethasone and reduces tumour growth in an in vivo xenograft model. Transcriptomic profiling in CRNDE-deleted MM cells demonstrated that CRNDE activates expression of a number of genes previously implicated in the aetiology of MM, including IL6R. We further demonstrate that deletion of the CRNDE locus diminishes IL6 signalling and proliferative responses in MM cells. Altogether this study reveals the IL6 signalling pathway as a novel mechanism by which CRNDE impacts upon MM cell growth and disease progression.

Although a growing body of evidence indicates that phenotypic plasticity exhibited by glioblastoma cells plays a central role in tumor development and post-therapy recurrence, the master drivers of their aggressiveness remain elusive. Here we mapped the changes in active (H3K4me3) and repressive (H3K27me3) histone modifications accompanying the repression of glioblastoma stem-like cells tumorigenicity. Genes with changing histone marks delineated a network of transcription factors related to cancerous behavior, stem state, and neural development, highlighting a previously unsuspected association between repression of ARNT2 and loss of cell tumorigenicity. Immunohistochemistry confirmed ARNT2 expression in cell sub-populations within proliferative zones of patients' glioblastoma. Decreased ARNT2 expression was consistently observed in non-tumorigenic glioblastoma cells, compared to tumorigenic cells. Moreover, ARNT2 expression correlated with a tumorigenic molecular signature at both the tissue level within the tumor core and at the single cell level in the patients' tumors. We found that ARNT2 knockdown decreased the expression of SOX9, POU3F2 and OLIG2, transcription factors implicated in glioblastoma cell tumorigenicity, and repressed glioblastoma stem-like cell tumorigenic properties in vivo. Our results reveal ARNT2 as a pivotal component of the glioblastoma cell tumorigenic signature, located at a node of a transcription factor network controlling glioblastoma cell aggressiveness.

Multiple myeloma (MM) is a malignancy of antibody-secreting plasma cells which remains incurable, despite significant improvements in treatment and patient care. MM is characterized by a wide clinical and prognostic spectrum, even within groups bearing the same primary initiating cytogenetic event, for which the molecular mechanisms responsible remain poorly understood. Long non-coding RNAs (lncRNAs; broadly defined as non-coding RNAs of > 200 nt) have recently emerged as an important class of regulatory molecules, exercising diverse functions in normal cells and tissues, and are increasingly implicated in tumorigenesis and cancer progression. However at present the contribution of lncRNAs to the progression and clinical variability of MM is largely unknown.