Dissecting RNA biology in hematopoietic stem cells: The long non-coding RNA Meg3 is dispensable for hematopoiesis and alternative polyadenylation orchestrates hematopoietic stem cell function

Hematopoietic stem cells (HSCs) reside at the top of a tightly regulated and hierarchically organized differentiation cascade. They are multipotent and able to self-renew, thereby ensuring life-long replenishment of mature blood cells. In-depth Omics analyses within the hematopoietic hierarchy, including analysis of HSCs and multipotent progenitor (MPP) cells, revealed not only differential expression of protein-coding genes, but also HSC-specific splicing variants and expression of long non-coding RNAs (lncRNAs) upon HSC commitment (Cabezas-Wallscheid et al., 2014; Klimmeck et al., 2014; Luo et al., 2015). Functional analyses revealed that non-coding RNAs and regulation of RNA biogenesis are crucial for HSC function and potency. In this thesis, the role of the lncRNA maternally expressed gene 3 (Meg3) in hematopoiesis is studied. In addition, I introduce the RNA regulatory mechanism alternative polyadenylation (APA) as a mechanism controlling HSC/MPP biology in homeostasis and during replicative stress response by extensive in silico, in vitro and in vivo analyses. Furthermore, I generated an inducible knockout mouse model, targeting the prominent APA regulator poly(A) binding protein 1 (Pabpn1). Thereby, I aimed to dissect the general role of lncRNAs and RNA regulatory mechanisms in hematopoiesis. Project 1: The role of the lncRNA Meg3 in HSCs The tumor suppressor lncRNA Meg3 is encoded in the imprinted Dlk1-Meg3 locus and expressed from the maternally inherited allele (Zhou et al., 2012). We and others found Meg3 to be highly and specifically expressed in the HSC compartment compared to MPP cells. In this thesis, I crossed an inducible Meg3 flox mouse model (Klibanski et al., unpublished) to MxCre mice, generating MxCre Meg3 mat flox/pat wt mice. Cre-induction deleted the maternal allele in the hematopoietic compartment and thereby completely abrogated the expression of Meg3 and its associated miRNA cluster in HSCs. Extensive in vivo and in vitro analyses of adult mice harboring a Meg3-deficient blood system surprisingly did not reveal any impairment of hematopoiesis or stem cell function. In addition, I performed serial transplantation assays to investigate the functional capacity of Meg3-deficient HSCs. Again, knockout cells did not exhibit altered blood contribution, even upon tertiary transplantation. Imprinting of the Dlk1- Meg3 locus has recently been reported to regulate fetal liver HSC function (Qian et al., 2016). To analyze effects of the hematopoiesis-specific Meg3 knockout in the developing embryo, we generated VavCre Meg3 mat flox/pat wt mice. Cre+ offspring were born and developed normally. In- depth analysis of adult animals revealed loss of Meg3 expression in HSCs, but again no hematopoietic impairments were detected. Next, I performed interferon-mediated stimulation in MxCre Meg3 mat flox/pat wt mice. During both activation and recovery phase, Meg3-deficient adult HSCs responded highly similar compared to controls. Taken together, my work shows that the highly expressed, imprinted lncRNA Meg3 is dispensable for the function of HSCs during adulthood and embryonic development. In the adult system, loss of Meg3 does not impair the performance in serial reconstitution assays or response to stress mediators. (Sommerkamp et al., 2019) Project 2: HSC function, differentiation and activation are regulated by APA The majority of mammalian genes have multiple different polyadenylation sites. The RNA editing mechanism APA controls the selection of these sites, thereby altering 3’-UTR length and isoform expression. Thus, APA modulates RNA stability, localization, protein output and even protein localization (Di Giammartino et al., 2011; Tian and Manley, 2017). So far, the role of APA in the regulation of the adult HSC/MPP compartment has not been studied. In this thesis, I show that the APA regulator Pabpn1 is essential for HSCs, as knockdown (KD) of Pabpn1 led to decreased HSC function in vitro and in vivo. To analyze the prevalence of APA at the top of the hematopoietic hierarchy, I established an ultra-low input 3’-Seq approach and performed analysis of HSCs, MPP cells and HSCs activated by inflammation. Bioinformatic analysis revealed dynamic APA patterns in numerous genes between HSCs and MPPs as well as during inflammation-induced HSC stress response. We observed global 3’-UTR shortening both upon HSC differentiation towards MPPs and HSC activation. Further, 3’-Seq analysis of Pabpn1 KD cells revealed that PABPN1 regulates APA in the HSC/MPP compartment. We observed an APA-mediated glutaminase (Gls) isoform switch upon exit of HSCs from quiescence. Gls isoform switching led to enhanced relative expression of the highly active GLS GAC isoform and overall increased GLS protein levels. In line, small molecule-mediated inhibition of glutaminolysis in vitro enhanced HSC maintenance by limiting proliferation. I could show that Gls isoform switching leading to increased glutaminolysis is mediated by the APA regulator NUDT21 and in turn is required for proper HSC function. KD of Nudt21 led to inhibition of Gls isoform switching, impaired HSC function and a partial block in HSC differentiation. In summary, my results install differential employment of APA and associated glutamine metabolism adaptations as novel layers in the regulation of the HSC-controlled hematopoietic hierarchy. (Sommerkamp et al., under revision) Project 3: Generation of Pabpn1flox mice To enable in-depth in vivo analysis of the role of APA in different tissues and disease settings in the future, we generated an inducible Pabpn1flox mouse model. Here, we used the Easi- CRISPR approach (Miura et al., 2018; Quadros et al., 2017) to generate transgenic animals. I performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency in vivo. Two different crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, were injected into one of the pronuclei of zygotes together with a long single-stranded DNA (ssDNA) template. By extensive genotyping, I identified 3 of 17 offspring animals to be correctly targeted. Homozygous offspring mice were successfully bred and can be used in the future to determine functionality of the Pabpn1flox mouse model and to functionally asses the role of APA in different biological settings

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1

Funder: Studienstiftung des Deutschen Volkes (German National Academic Foundation); doi: https://doi.org/10.13039/501100004350Funder: Heinrich F.C. Behr StiftungFunder: Dietmar Hopp Stiftung; doi: https://doi.org/10.13039/501100005941Abstract: Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1

Funder: Studienstiftung des Deutschen Volkes (German National Academic Foundation); doi: https://doi.org/10.13039/501100004350Funder: Heinrich F.C. Behr StiftungFunder: Dietmar Hopp Stiftung; doi: https://doi.org/10.13039/501100005941Abstract: Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal

Engineering human hematopoietic environments through ossicle and bioreactor technologies exploitation

The bone marrow microenvironment contains cellular niches that maintain the pool of hematopoietic stem and progenitor cells and support hematopoietic maturation. Malignant hematopoietic cells also co-opt normal cellular interactions to promote their own growth and evade therapy. In vivo systems used to study human hematopoiesis have been developed through transplantation into immunodeficient mouse models. However, incomplete cross-compatibility between the murine stroma and transplanted human hematopoietic cells limits the rate of engraftment and the study of relevant interactions. To supplement in vivo xenotransplantation models, complementary strategies have recently been developed, including the use of three-dimensional human bone marrow organoids in vivo, generated from bone marrow stromal cells seeded onto osteo-inductive scaffolds, as well as the use of ex vivo bioreactor models. These topics were the focus of the Spring 2020 International Society for Experimental Hematology New Investigator webinar. We review here the latest advances in generating humanized hematopoietic organoids and how they allow for the study of novel microenvironmental interactions

Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells

International audienceAbstract Hematopoietic stem cells (HSCs) and distinct multipotent progenitor (MPP) populations (MPP1-4) contained within the Lin−Sca-1+c-Kit+ (LSK) compartment have previously been identified using diverse surface-marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+CD135−CD48−CD150−). Upon transplantation, MPP5 supports initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. MPP5, capable of generating MPP1-4 but not HSCs, represents a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells, we performed RNA-sequencing (RNA-seq) analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with functional reconstitution activity, MPP5 is located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset

Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo.

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo

Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity

Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data , revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes

Differential phagocytosis rate of mouse M1 and M2 macrophages toward various human glioma cells upon CD47-SIRPα disruption.

<p>(A) Representative flow cytometric phagocytosis assay of mouse M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80^high live singlets was measured and compared between untreated and anti-CD47 antibody-treated co-cultures. (B) Representative flow cytometric phagocytosis assay of mouse M2 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD206^high live singlets was measured and compared between untreated (left column) and anti-CD47 antibody-treated (right column) co-cultures. (C) Bar graph demonstrating the change in phagocytosis rates by mouse M1 macrophages towards individual co-incubated cell lines (GBM1, GBM4 and PGBM1) -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests). (D) Bar graph demonstrating the change in phagocytosis rates by mouse M2 macrophages towards individual co-incubated cell lines (GBM1, GBM4 and PGBM1) -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests).</p

<i>In vivo</i> analysis of tumor-associated macrophage polarization upon anti-CD47 treatment; bioluminescence and survival analysis of treated mice.

<p>(A) Ratio of CD80 and CD206 positive cell count per total macrophage count in untreated and anti-CD47-treated mice (p = 0.0054 for CD80 and 0.164 for CD260, paired t-test). (B) Median fluorescent intensity (MFI) measurement of CD80-AF647 and CD206-PE (p = 0.0002 for CD80 and 0.423 for CD206, paired t-test). (C) Bioluminescence in vivo imaging data (left panel), photon flux values at days 21 and 50 (middle panel) and Kaplan-Meier analysis of mice grafted with GBM5 and treated with Hu5F9-G4 (right panel, 250 μg/dose, every other day, starting at week 3; n = 5 per group, p = 0.0018, log-rank analysis). Legend: blue shapes: mice engrafted with GBM4, black shapes: mice engrafted with GBM5.</p