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Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis (CH)-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative neoplasms (MPNs). While cooperating mutations can cause transformation, it remains unclear whether distinct bone marrow (BM) HSC niches can influence the growth and therapy response of HSCs carrying the same pathogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK-STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera (PV), and endosteal niche expansion in essential thrombocythemia (ET). MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same CH oncogenic driver.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. scRNA-seq and bulk RNA-seq data were deposited in ArrayExpress with accession number E-MTAB-10853 and ERP006862, respectively. Code used for single-cell RNA-seq data analysis was deposited in Zenodo and can be accessed as G. Shikha and P. Cristina (2022). scRNA-seq analysis of RUNX1-RUNX1T1(9a) preleukemia, Zenodo; https://doi.org/10.5281/zenodo.6584118. Supplementary Materials: This PDF file includes: Figs. S1 to S7 avilable at: https://www.science.org/doi/suppl/10.1126/sciadv.abn4886/suppl_file/sciadv.abn4886_sm.pdf (223.69 MB). Other Supplementary Material for this manuscript includes the following: Supplementary Files S1 to S9 available at: https://www.science.org/doi/suppl/10.1126/sciadv.abn4886/suppl_file/sciadv.abn4886_supplementary_files_s1_to_s9.zip (2.34 MB). View/request a protocol for this paper from Bio-protocol available at: https://en.bio-protocol.org/cjrap.aspx?eid=10.1126/sciadv.abn4886 Copyright © 2022 The Authors. Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of premalignancy to cancer. To investigate this, we initiated preleukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA sequencing of preleukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of preleukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. That is, transcriptional variability confers forward momentum to cell fate systems, with differential multistage impact throughout cancer evolution. Wellcome: WT098051; Leuka: John Goldman Fellowship; Lady Tata Memorial Trust: International Scholarship 2017-2021; Cancer Research UK: C22324/A23015; Kay Kendall Leukaemia Fund: KKL888; Wellcome Trust / University of Cambridge ISSF: Bridging Funding 2019.

Summary Hematopoietic stem cells (HSCs) are typically characterized by transplantation into irradiated hosts in a highly perturbed microenvironment. Here, we show that selective and temporally controlled depletion of resident HSCs through genetic deletion of Gata2 constitutes efficient recipient conditioning for transplantation without irradiation. Strikingly, we achieved robust engraftment of donor HSCs even when delaying Gata2 deletion until 4 weeks after transplantation, allowing homing and early localization to occur in a completely non-perturbed environment. When HSCs from the congenic strains Ly5.1 and Ly5.2 were competitively transplanted, we found that the more proliferative state of Ly5.2 HSCs was associated with superior long-term engraftment when using conditioning by standard irradiation, while higher CXCR4 expression and a better homing ability of Ly5.1 HSCs strongly favored the outcome in our inducible HSC depletion model. Thus, the mode and timing of recipient conditioning challenges distinct functional features of transplanted HSCs., Graphical abstract, Highlights • Inducible gene deletion of Gata2 rapidly and selectively depletes the HSC pool • Gata2 deletion constitutes efficient recipient conditioning for HSC transplantation • The model enables detection of HSC engraftment in a non-perturbed microenvironment • Transplantation without irradiation uniquely challenges homing properties of HSCs, In this study Larsson and colleagues show that selective and temporally controlled depletion of resident hematopoietic stem cells (HSCs) through genetic deletion of Gata2 constitutes efficient recipient conditioning for transplantation without irradiation. They demonstrate that engraftment in a non-perturbed microenvironment uniquely challenges properties affecting homing and early HSC localization, that otherwise are masked in irradiated recipients.

Funder: European Research Council, Clonal expansions driven by somatic mutations become pervasive across human tissues with age, including in the haematopoietic system, where the phenomenon is termed clonal haematopoiesis1-4. The understanding of how and when clonal haematopoiesis develops, the factors that govern its behaviour, how it interacts with ageing and how these variables relate to malignant progression remains limited5,6. Here we track 697 clonal haematopoiesis clones from 385 individuals 55 years of age or older over a median of 13 years. We find that 92.4% of clones expanded at a stable exponential rate over the study period, with different mutations driving substantially different growth rates, ranging from 5% (DNMT3A and TP53) to more than 50% per year (SRSF2P95H). Growth rates of clones with the same mutation differed by approximately ±5% per year, proportionately affecting slow drivers more substantially. By combining our time-series data with phylogenetic analysis of 1,731 whole-genome sequences of haematopoietic colonies from 7 individuals from an older age group, we reveal distinct patterns of lifelong clonal behaviour. DNMT3A-mutant clones preferentially expanded early in life and displayed slower growth in old age, in the context of an increasingly competitive oligoclonal landscape. By contrast, splicing gene mutations drove expansion only later in life, whereas TET2-mutant clones emerged across all ages. Finally, we show that mutations driving faster clonal growth carry a higher risk of malignant progression. Our findings characterize the lifelong natural history of clonal haematopoiesis and give fundamental insights into the interactions between somatic mutation, ageing and clonal selection.

Wyznacznikiem postaw człowieka, jego wyborów i sposobu oceniania zdarzeń jest system wyznawanych wartości. W dobie pluralizmu wartości współczesnego społeczeństwa szczególnego znaczenia nabierają wartości uniwersalne, które warunkują zachowanie ludzkiej egzystencji i trwanie świata. Do podstawowych wartości uniwersalnych należy prawo do życia oraz sprawiedliwość. Artykuł dotyczy kary śmierci, która wyklucza jednoczesne urzeczywistnianie tych wartości. Celem badań sondażowych, których wyniki przedstawiono w artykule, było poznanie opinii przyszłych pedagogów resocjalizacyjnych, studentów KUL i UMCS, na temat kary śmierci i sprawiedliwości jej orzekania.

Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of pre-malignancy to cancer. To investigate this, we initiated pre-leukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA-sequencing of pre-leukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of pre-leukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. In other words, transcriptional variability confers forward momentum to cell fate systems, with differential multi-stage impact throughout cancer evolution.One-sentence summaryLoss of Kat2a enhances transcriptional variability of ribosome biosynthetic programs and transiently accelerates pre-leukemia

SummaryHuman cells acquire somatic mutations throughout life, some of which can drive clonal expansion. Such expansions are frequent in the haematopoietic system of healthy individuals and have been termed clonal haematopoiesis (CH). While CH predisposes to myeloid neoplasia and other diseases, we have limited understanding of how and when CH develops, what factors govern its behaviour, how it interacts with ageing and how these variables relate to malignant progression. Here, we track 697 CH clones from 385 individuals aged 55 or older over a median of 13 years. We find that 92.4% of clones expanded at a stable exponential rate over the study period, with different mutations driving substantially different growth rates, ranging from 5% (DNMT3A,TP53) to over 50%/yr (SRSF2-P95H). Growth rates of clones with the same mutation differed by approximately +/−5%/yr, proportionately impacting “slow” drivers more substantially. By combining our time-series data with phylogenetic analysis of 1,731 whole genome-sequenced haematopoietic colonies from 7 older individuals, we reveal distinct patterns of lifelong clonal behaviour.DNMT3A-mutant clones preferentially expanded early in life and displayed slower growth in old age, in the context of an increasingly competitive oligoclonal landscape. By contrast, splicing gene mutations only drove expansion later in life, while growth ofTET2-mutant clones showed minimal age-dependency. Finally, we show that mutations driving faster clonal growth carry a higher risk of malignant progression. Our findings characterise the lifelong natural history of CH and give fundamental insights into the interactions between somatic mutation, ageing and clonal selection.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3–METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5–7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3–METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy. Treatment with a specific inhibitor of the N6-methyladenosine methyltransferase METTL3 leads to reduced growth of cancer cells, indicating the potential of approaches targeting RNA-modifying enzymes for anticancer therapy.

Hematopoietic stem cells (HSCs) are defined as the cells that have the ability of both multi-potency and self-renewal. This property of HSCs has traditionally been characterized by transplantation assays where the donor HSCs are transferred into the animal where the hematopoietic system has been ablated. In this assay, long-term sustained and multi-lineage reconstitution are the criteria to define the functional HSCs. However, recent technical advances that allow the monitoring of HSC kinetics in steady-state animals, have led to the discovery that steady-state hematopoiesis differs qualitatively from that assessed in the transplantation assay. In the ablated (irradiated) recipients, HSCs are forced to proliferate in order to sustain the hematopoietic system, while steady-state hematopoiesis is rather maintained by proliferating progenitors. It still remains unclear to what degree post-transplantation hematopoiesis reflects normal HSC physiology. To explore the HSC dynamics under different transplantation conditioning regimens, we developed the Mx1Cre-Gata2 mouse model where endogenous HSCs can be selectively depleted following inducible removal of Gata2 as a crucial factor for HSCs. In this manner the rest of the hematopoietic system and the microenvironment remains intact, and transplanted HSCs can still engraft efficiently. We found that the kinetics of transplanted HSCs are quite different compared to standard conditioning by irradiation, and that different properties of the HSCs are challenged in the two models. In our selective HSC depletion model, properties influencing homing and lodgement in the bone marrow influence the engraftment capacity to a greater extent, while proliferative capacity of the HSCs is more stringently assessed in the irradiation model. Thus, distinct aspects of fitness and function are measured when transplanted HSCs are challenged in different environments.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment., Y.-H.O. received fellowships from Alborada Scholarship (University of Cambridge), Trinity-Henry Barlow Scholarship (University of Cambridge), and R.O.C. Government Scholarship to Study Abroad (GSSA). A.G.G. received fellowships from the Ramón Areces Foundation and the LaCaixa Foundation. C.K. was supported by Marie Curie Career Integration (H2020-MSCA-IF-2015-70841). S.M.-F. was supported by Red TerCel (ISCIII-Spanish Cell Therapy Network). V.A. is supported by grants from the Spanish Ministerio de Economía, Industria y Competitividad (MEIC) with cofunding from the Fondo Europeo de Desarrollo Regional (FEDER, “Una manera de hacer Europa”) (SAF2016-79490-R), the Instituto de Salud Carlos III (AC16/00091 and AC17/00067), the Fundació Marató TV3 (122/C/2015), and the Progeria Research Foundation (Established Investigator Award 2014–52). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCIU), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This work was supported by core support grants from the Wellcome Trust and the MRC to the Cambridge Stem Cell Institute, MEIC (SAF-2011-30308), Ramón y Cajal Program Grant (RYC-2009-04703), ConSEPOC-Comunidad de Madrid (S2010/BMD-2542), National Health Service Blood and Transplant (United Kingdom), European Union’s Horizon 2020 research (ERC-2014-CoG-64765 and Marie Curie Career Integration grant FP7-PEOPLE-2011-RG-294096), and a Programme Foundation Award from Cancer Research UK to S.M.-F., who was also supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute.

The molecular principles regulating hematopoietic stem cells (HSCs) remain incompletely defined. In this review, we will discuss how RNA interference (RNAi) screening has emerged as a new and powerful tool to molecularly dissect various functional aspects of both normal and malignant HSCs, and how this may ultimately enable the discovery of novel therapeutic targets for clinical applications.Advances in RNAi technology and the creation of short hairpin RNA retroviral and lentiviral vector-libraries have provided tools to perform broad forward genetic screens in primary mammalian hematopoietic cells. Recent studies have identified novel fate determinants in murine HSCs as well as potential targets for ex-vivo expansion of human HSCs. RNAi screens have further unraveled tumor suppressor genes associated with hematopoietic neoplasms as well as candidate therapeutic targets in leukemic cells.RNAi screening is a feasible tool to discover novel molecules that regulate both normal and malignant HSCs, thus increasing our general understanding of the physiology and pathophysiology of hematopoiesis. Moreover, RNAi technology holds great promise for the discovery of specific targets for therapeutic interventions.

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. New evidence has revealed an important role for RNA modifications in cancer development and maintenance via the catalytic function of RNA-modifying enzymes. We and others have recently shown that METTL3, the RNA methyltransferase responsible for the deposition of N-6-methyl groups on adenosine (m6A) in mRNA, is a promising therapeutic target for AML1,2. Here we present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3 as an effective therapeutic strategy in AML. Recently, we generated a comprehensive catalogue of RNA-modifying enzymes that are essential for AML cells using CRISPR-Cas9 recessive screens and characterised METTL3 as a novel therapeutic candidate through its effects on mRNA translational efficiency of key leukemia oncogenes1. Using a structure-guided medicinal chemistry platform we developed and optimised small molecule inhibitors of METTL3 from 2 distinct chemical series. Here we demonstrate that compounds 1 and 2 show biochemical inhibition of METTL3 enzyme with single digit nanomolar potency, while direct binding to METTL3 was confirmed by Surface Plasmon Resonance (SPR) analysis with comparable potency between compounds. Additionally, we developed compound 3 as an inactive analog which was confirmed inactive in enzyme assays (>50 µM IC50). Importantly, we verified that compounds 1 and 2 are selective for METTL3 and do not inhibit a panel of other RNA, DNA or protein methyltransferases tested (>10 µM IC50). Cellular target engagement was confirmed by demonstrating that compounds 1 and 2 reduced m6A levels and inhibited the protein expression of METTL3-dependent m6A substrates in mouse and human AML models, including SP1, with nanomolar potency. Furthermore, treatment of MOLM13 cells with compounds 1 and 2 inhibited their proliferation with comparable potency to SP1 inhibition. The same anti-proliferative effect was observed using a large panel of human AML cell lines. In addition, polyribosome profiling in MOLM13 cells treated with compounds 1 and 2 revealed enhanced blocking of mRNA translation, mirroring the effects derived from the genetic inhibition of METTL3. Notably, all of the above effects were not observed when the inactive analog (compound 3) was used, further highlighting the specificity and sensitivity of our active candidates. We subsequently performed in vivo characterisation of compound 1. This compound exhibited excellent bioavailability after oral or intraperitoneal administration with good dose-proportional exposure in mice and a half-life of 3.5 hours. It also appeared to be well-tolerated with no body weight loss or clinical signs of toxicity. We also evaluated its anti-tumor effects in patient derived xenotransplantation experiments (PDX) as well as transplantation experiments using an MLL-AF9 driven primary murine AML model. Daily dosing of 30 mg/kg significantly inhibited AML expansion and reduced spleen weight compared to vehicle control, indicating a pronounced anti-tumor effect in vivo. Target engagement was confirmed in bone marrow and spleen as measured by the reduction of METTL3-dependent m6A targets. Importantly, we went on to demonstrate that, while the pharmacological inhibition of METTL3 is required for AML cell survival, it was dispensable for normal hematopoiesis. Collectively, we describe the detailed characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrate their activity and utility using biochemical, cellular and in vivo systems. We show that inhibition of METTL3 by small molecules in vivo leads to strong anti-tumor effects in physiologically and clinically relevant models of AML. To our knowledge, this is the first study demonstrating in vivo activity of inhibitors of an RNA methyltransferase, hence providing proof of concept that RNA modifying enzymes represent a new target class for anti-cancer therapeutics. References Barbieri, I. et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature552, 126-131, doi:10.1038/nature24678 (2017). Vu, L. P. et al. The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med23, 1369-1376, doi:10.1038/nm.4416 (2017). Disclosures Yankova: STORM THERAPEUTICS: Employment. Fosbeary:STORM THERAPEUTICS: Employment. Hendrick:STORM THERAPEUTICS: Employment. Leggate:STORM THERAPEUTICS: Employment. Ofir-Rosenfeld:STORM THERAPEUTICS: Employment. Sapetschnig:STORM THERAPEUTICS: Employment. Albertella:STORM THERAPEUTICS: Employment. Blackaby:STORM THERAPEUTICS: Employment. Rausch:STORM THERAPEUTICS: Employment. Vassiliou:Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding. Kouzarides:STORM THERAPEUTICS: Equity Ownership.

Microenvironment-targeted therapies are emerging as possible complementary approaches to treat malignancies. However, progress is hindered by the complexity of the interactions between malignant cells and their microenvironment. In leukemia, this complexity is further emphasized by a highly dynamic behavior of leukemic cells recently reported in Nature. Hawkins et al.1 analyze leukemic cell localization relative to microenvironmental cells in T-cell acute lymphoblastic leukemia (T-ALL) through real-time intravital imaging. Contrasting previous studies, Lo Celso and colleagues report a highly motile behavior and lack of stable interactions with specific microenvironmental cell types during disease progression or after chemotherapy. This study challenges some current concepts on microenvironmental dependence of leukemia.

Different MPNs have distinct rates of malignant transformation (PMF>PV>ET). Although PV and ET can arise from hematopoietic stem/progenitor cells (HSPCs) with similarly activated JAK-STAT oncogenic pathway, the transformation rate into secondary myelofibrosis and leukemia is higher for PV than for ET. However, the underlying reasons are not fully clear. Whereas in some cases secondary mutations might cause transformation, it remains unclear whether distinct bone marrow (BM) microenvironments can influence the progression of MPNs or any preleukemic disorder. Previous studies have suggested that normal BM niches close to bone (endosteal) promote HSPC quiescence, whereas non-endosteal vessels permit transmigration of activated HSPCs. We hypothesized that PV and ET HSPCs might expand in different BM niches, which could thereby influence disease progression. To address this question, we performed combined two-photon and confocal real-time intravital microscopy in the skull BM of WT mice transplanted with HSPCs from WT mice or MPN mice carrying the same driver mutation (JAK2V617F) but showing phenotypically distinct diseases (ET or PV). ET HSPCs resembled WT HSPCs in their homing and engraftment near bone (22±15 and 26±13 μm, respectively) 3 days after i.v. injection into lethally-irradiated WT mice. In contrast, PV HSPCs located significantly further (31±21 μm) from bone, which was independently confirmed in a distinct PV model. The different homing of ET and PV HSPCs was similarly observed in non-irradiated WT recipients and was therefore independent of myeloablative conditioning. Following engraftment, ET and PV HSPCs preferentially expanded in endosteal and non-endosteal locations, respectively, over 7 months' follow up. Importantly, the asymmetric expansion of HSPCs in ET and PV was confirmed in human BM trephines. Human CD34+ HSPCs were significantly closer to bone in ET than in PV patients (86±2 vs. 109±6 μm). Together, these results suggest that mutated HSPCs preferentially occupy distinct BM niches in ET and PV. Murine ET and PV HSPCs also differed in their dynamic interactions with the microenvironment. ET HSPCs migrated significantly faster (1 μm/min) than PV HSPCs (0.8 μm/min) and covered longer tracks after 1 h (57±2 and 35±1 μm, respectively). Moreover, ET HSPCs (but not PV HSPCs) migrated faster when closer to bone, suggesting an exploratory strategy of ET HSPCs to find endosteal niches. Separation of endosteal and non-endosteal BM fractions revealed increased abundance of integrin β3+ HSPCs in the endosteal BM of ET mice carrying JAK2V617F or CALRdel52/del52 mutations, but not in different PV models (despite generally sharing oncogenic JAK-STAT activation). Furthermore, competitive gravity adhesion assay and interference reflection microscopy showed that ET HSPCs are more adhesive to ECM substrates of integrin β3, suggesting that integrin β3 might trigger endosteal lodging of ET HSPCs. Asymmetric HSPC expansion caused differential microenvironmental remodeling possibly explaining differences in the pathophysiology and secondary outcomes of ET and PV. Non-endosteal sinusoids were dilated in different PV (but not ET) models, whereas CD31hiSca1hi arterioles and aberrant bone-forming integrin β1+ blood vessels increased only in ET mice. Similarly, sinusoidal vessels showed increased coverage by the integrin β1 ligand laminin α4 only in ET mice. Consequently, increased bone (μCT), osteoblasts and osteoclasts were found in ET but not PV mice. Increased expression of vascular-derived bone-forming factors (such as Bmp1 and Dll4) downstream of endothelial laminin α4/integrin β1 signaling might trigger osteosclerosis in ET mice. Finally, we tested whether HSPC location might directly impact ET progression. In a separate study we found that β3-adrenergic receptor (AR) signaling regulates Cxcl12-dependent BM HSPC localization (ASH abstract ID 116015). Therefore, we transplanted WT or ET donor BM cells into WT and β3-AR KO mice. Measurement of peripheral blood counts over 28 weeks showed that the microenvironment lacking β3-AR significantly worsened thrombocytosis and leukocytosis in ET, which correlated with redistribution of HSPCs and their progeny away from bone towards central BM. Altogether, these results suggest that differential interactions with the microenvironment might impact disease progression in MPNs and possibly in other preleukemic disorders. Disclosures Mead: Cell Therapeutics: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Research Funding; Elstar: Research Funding; Evotek: Research Funding.

Background: BM HSC accumulate during aging but are functionally impaired; however, it remains debated whether this aging results from HSC-intrinsic and/or -extrinsic mechanisms. Myeloid malignancies are more frequent in the elderly, but whether the aged microenvironment predisposes to these malignancies is unclear. Myeloid-biased HSC and especially platelet-primed HSC expand in aged mice. However, both platelet-primed and unprimed old HSCs exhibit myeloid bias, possibly suggesting a microenvironmental participation. Moreover, some hallmarks of murine hematopoietic aging (like increased platelets) are present in premature aging (Hutchinson-Gilford progeria syndrome, HGPS) and are exacerbated in age-related myeloproliferative neoplasms (MPN), where we found that niche heterogeneity can influence disease progression (ASH abstract ID 113495). Given that aging increases myelopoiesis in physiology, HGPS and MPN (despite their heterogenous genetic background), we hypothesized that shared microenvironmental alterations influence HSC lineage bias during aging Results: HSCs, perisinusoidal megakaryocytes (Mk) and myelopoietic cytokines increase in the BM of HGPS mice, resembling normally aged mice. However, WT recipients of HGPS BM cells do not reproduce myeloid bias, indicating that premature aging affects hematopoiesis in HGPS in a non-HSC-autonomous manner. We have investigated niche contributions to lineage bias during physiological aging because HGPSmice do not tolerate myeloablation. Consistently with recent findings (Nat Med 2018;24:782-91) HSC niches decrease near bone (endosteal BM) and expand further from bone in aged mice, but we observed different vascular changes mainly consisting of halved endosteal transition zone vessels with associated Nes-GFP+ cells and 4-fold-increased non-endosteal capillaries with Nes-GFP+ cells. However, contrasting the afore mentioned study but in agreement with other reports, we found aging-related increase of BM noradrenergic innervation, which promotes megakaryopoiesis in WT mice, but not in mice lacking β2 and β3 adrenergic receptors (ARs). Yet, these receptors exhibit opposite effects on microenvironmental regulation of myelopoiesis. β2-AR-agonist promotes Mk differentiation from human CD34+ HSCs co-cultured with MS-5 cells or in WT (but not IL-6 KO) primary murine BM culture. Consequently, adult β2-AR KO mice exhibit decreased Mk-lineage cells, which correlated with reduced BM IL-6. In contrast, adult β3-AR KO mice exhibit increased mature Mk near sinusoids, which correlated with tripled Cxcl12 mRNA expression in non-endosteal BM. Mechanistically β3-AR regulates Cxcl12-dependent HSPC and megakaryocyte localization, nitric oxide (NO) production and expression of myelopoietic cytokines. In vitro, β3-AR-agonist-treated stromal cells decrease human and murine HSPC differentiation into Mk. This effect requires Nos1-dependent NO production, since Nos1-/- mice have high circulating platelets and β3-AR agonist specifically increases lymphoid-biased HSCs in primary BM cultures in a Nos1-dependent manner. Finally, Mk increased in β3-AR KO mice transplanted with MPN BM cells, whereas Mk decreased in MPN mice treated with β3-AR agonist. To test the clinical relevance of our findings, we measured plasma nitrates and myelopoietic cytokines in MPN patients (median age=62) chronically treated only with β3-AR agonist, who show improved myelofibrosis correlated by rescued nestin+ niches (Blood 2016;128:3108), which is consistent with previous results in mice (Nature 2014;512:78-81). We found inverse correlations between the concentration of nitrates and myelopoietic cytokines Conclusions: microenvironmental aging promotes myeloid bias through similar cytokines (e.g. IL1β, IL6) during premature or physiological aging. In the latter, HSC supporting niches decrease near bone but expand further from bone. Increased BM sympathetic noradrenergic innervation promotes β2-AR-IL-6-dependent myeloid bias. Reduction of endosteal niches decreases β3-AR-NO-dependent inhibition of myelopoietic cytokines. However, chronic treatment with β3-AR-agonist does not rejuvenate overall hematopoiesis but decreases exacerbated megakaryopoiesis in mice and humans with MPNs. Therefore, niche aging promotes myeloid bias and might represent a therapeutic target in age-related myeloproliferative disorders Disclosures No relevant conflicts of interest to declare.

We report the case of a 40-year old man diagnosed with a scleroderma-like disease. Clinical similarities with graft versus host disease prompted initial testing for chimerism employing fluorescence in situ hybridization (FISH). Female cells were observed within peripheral blood mononuclear cells from the patient. Because maternal cells have been detected in healthy immunologically competent adults and patients with autoimmune conditions, we hypothesized that these cells were of maternal origin. Contrary to our expectations, HLA-specific quantitative PCR (QPCR) ruled out maternal microchimerism. However, HLA-specific QPCR testing was positive for the paternal HLA haplotype that the patient did not inherit. We reasoned that the most likely origin of chimerism with non-inherited paternal HLA alleles was from an unrecognized "vanished" twin. The patient had never received a blood transfusion. This report suggests that cells from a vanished twin are a possible source of chimerism. The frequency of chimerism fr...

Objectives. Male microchimerism (Mc) persisting from pregnancy has been found at greater frequencies and/or higher quantities in women with scleroderma (SSc) compared with controls, suggesting a possible role in disease development. Moreover, women with an HLA-compatible child have a higher risk to develop SSc. We tested the hypothesis, on our French SSc cohort, that women with lcSSc and dcSSc, two distinct clinical subsets, have a different profile in terms of Mc and HLA compatibility in families. Methods. We studied 98 women (52 lcSSc and 46 dcSSc) for male Mc, by real-time PCR, in their whole blood and/or peripheral blood mononuclear cells (PBMC). Similarly, 91 matched healthy women were analysed. Complete HLA-DRB1 typing was obtained for 58 SSc and 68 control families (proband/children). Results. Women with lcSSc (N ¼ 50) had male Mc more often in their whole blood than women with dcSSc (N ¼ 40, 20 vs 5%, P ¼ 0.038), but not significantly more than controls. By contrast, women with dcSSc (N ¼ 36) hold Mc more often in PBMC (25 vs 9%), but not significantly and have greater quantities than controls (N ¼ 82, P ¼ 0.048). This contrast is also visible in feto-maternal HLA-DRB1 compatibility, which was increased only among women with lcSSc (N ¼ 33) compared with controls (N ¼ 68, P ¼ 0.003). Conclusion. For the first time, we showed that women with lcSSc and dcSSc hold male Mc in different blood compartments. Furthermore, a distinct pattern between the two SSc subtypes is observed for feto-maternal HLA-DRB1 compatibility. These results suggest a different mechanism behind each type of disease.

The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors that serve to restrict HSC differentiation. In the present work, we identify ETS (E-twenty-six)-related gene (ERG) as a critical factor protecting HSCs from differentiation. Specifically, loss of Erg accelerates HSC differentiation by >20-fold, thus leading to rapid depletion of immunophenotypic and functional HSCs. Molecularly, we could demonstrate that ERG, in addition to promoting the expression of HSC self-renewal genes, also represses a group of MYC targets, thereby explaining why Erg loss closely mimics Myc overexpression. Consistently, the BET domain inhibitor CPI-203, known to repress Myc expression, confers a partial phenotypic rescue. In summary, ERG plays a critical role in coordinating the balance between self-renewal and differentiation of HSCs.

Rheumatoid arthritis, a chronic inflammatory joint disease, is strongly associated with HLA-DRB1*01 and *04 alleles that have in common similar 5-amino acid motifs in the third hypervariable region of DRB1 (QKRAA, QRRAA, RRRAA), the so called shared epitope (SE). Most patients with RA carry 1 or 2 doses of the SE, with particular genetic combinations at higher risk. In recent work we provided evidence that patients who lack HLA-DRB1*01 and/or *04 alleles can acquire RA susceptibility through fetal, maternal or iatrogenic microchimerism. We also discuss how Mc carrying HLA-DRB1*04 alleles is more likely to be present in the peripheral blood of RA patients compared to Mc carrying HLA-DRB1*01 alleles. We further analyze our results in light of the hierarchy for RA risk with different combinations of the SE. How Mc could contribute to RA susceptibility and whether it also contributes to the hierarchy of risk observed with particular combinations of SE-containing alleles is certainly the beginning of an intrig...

Many sources of foreign or semi foreign cells, known as microchimerism (Mc), can be found in healthy individuals. We have recently shown in women with end stage renal disease (ESRD) that Mc frequencies and levels are exacerbated prior to kidney transplantation. Is Mc arising from pregnancy a protective factor for renal diseases explaining lower incidence in women? Is Mc helpful in slowing down disease progression? However, natural Mc is not the only actor as post blood transfusion Mc is also found at high levels in women with ESRD. The difficulty is therefore to distinguish the different types of Mc and this is made even more complicated when the recipient receives a potentially chimeric organ. What part does each source of chimerism play in disease and transplant fate, and can one decipher each role knowing that one chimerism may hide another?

Objective Rheumatoid arthritis (RA) is an autoimmune disease that affects mostly women and is associated with HLA–DRB1 genes having in common a shared epitope sequence. In parallel, cells and/or DNA originating from pregnancy (microchimerism) persist for decades and could contribute to autoimmunity. The aim of this study was to examine whether microchimerism may be a source of the shared epitope among women with RA. Methods Women with RA and healthy women who lacked RA-associated genes such as HLA–DRB1*01 (n = 33 and n = 46, respectively) and/or HLA–DRB1*04 (n = 48 and n = 64, respectively), were tested for DRB1*01 or DRB1*04 microchimerism by HLA-specific quantitative polymerase chain reaction assays. As controls, alleles not associated with RA (DQB1*02 and DRB1*15/16) were also analyzed. Results Compared with healthy women, women (42% with RA had a higher frequency and higher levels of DRB1*04 microchimerism versus 8%; P = 0.00002) as well as DRB1*01 microchimerism (30% versus 4%; P = 0.0015). Moreover, no difference in microchimerism was observed for alleles not associated with RA. Conclusion Women with RA had microchimerism with RA-associated HLA alleles, but not with non–RA-associated HLA alleles, more often and at higher levels compared with healthy women. These observations are the first to indicate that microchimerism can contribute to the risk of an autoimmune disease by providing HLA susceptibility alleles.

Although many studies have analyzed HLA allele frequencies in several ethnic groups in patients with scleroderma (SSc), none has been done in French Caucasian patients and none has evaluated which one of the common amino acid sequences, (67)FLEDR(71), shared by HLA-DRB susceptibility alleles, or (71)TRAELDT(77), shared by HLA-DQB1 susceptibility alleles in SSc, was the most important to develop the disease. HLA-DRB and DQB typing was performed for a total of 468 healthy controls and 282 patients with SSc allowing FLEDR and TRAELDT analyses. Results were stratified according to patient's clinical subtypes and autoantibody status. Moreover, standardized HLA-DRß1 and DRß5 reverse transcriptase Taqman PCR assays were developed to quantify ß1 and ß5 mRNA in 20 subjects with HLA-DRB1*15 and/or DRB1*11 haplotypes. FLEDR motif is highly associated with diffuse SSc (χ(2) = 28.4, p

Patients with end stage renal diseases (ESRD) are generally tested for donor chimerism after kidney transplantation for tolerance mechanism purposes. But, to our knowledge, no data are available on natural and/or iatrogenic microchimerism (Mc), deriving from pregnancy and/or blood transfusion, acquired prior to transplantation. In this context, we tested the prevalence of male Mc using a real time PCR assay for DYS14, a Y-chromosome specific sequence, in peripheral blood mononuclear cells (PBMC) from 55 women with ESRD, prior to their first kidney transplantation, and compared them with results from 82 healthy women. Male Mc was also quantified in 5 native kidney biopsies obtained two to four years prior to blood testing and in PBMC from 8 women collected after female kidney transplantation, several years after the initial blood testing. Women with ESRD showed statistically higher frequencies (62%) and quantities (98 genome equivalent cells per million of host cells, gEq/M) of male Mc in their PBMC than healthy women (16% and 0.3 gEq/M, p

Adhesion is a key component of hematopoietic stem cell regulation mediating homing and retention to the niche in the bone marrow. Here, using an RNA interference screen, we identify cytohesin 1 (CYTH1) as a critical mediator of adhesive properties in primary human cord blood–derived hematopoietic stem and progenitor cells (HSPCs). Knockdown of CYTH1 disrupted adhesion of HSPCs to primary human mesenchymal stroma cells. Attachment to fibronectin and ICAM1, 2 integrin ligands, was severely impaired, and CYTH1-deficient cells showed a reduced integrin β1 activation response, suggesting that CYTH1 mediates integrin-dependent functions. Transplantation of CYTH1-knockdown cells to immunodeficient mice resulted in significantly lower long-term engraftment levels, associated with a reduced capacity of the transplanted cells to home to the bone marrow. Intravital microscopy showed that CYTH1 deficiency profoundly affects HSPC mobility and localization within the marrow space and thereby impairs proper lodgment into the niche. Thus, CYTH1 is a novel major regulator of adhesion and engraftment in human HSPCs through mechanisms that, at least in part, involve the activation of integrins.