Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche.

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.

Lack of VMP1 impairs hepatic lipoprotein secretion and promotes non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that regulates the formation of autophagosomes and lipid droplets. Recent evidence suggests that VMP1 plays a critical role in lipoprotein secretion in zebra fish and cultured cells. However, the pathophysiological roles and mechanisms by which VMP1 regulates lipoprotein secretion and lipid accumulation in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are unknown. METHODS: Liver-specific and hepatocyte-specific Vmp1 knockout mice as well as Vmp1 knock-in mice were generated by crossing Vmp1flox or Vmp1KI mice with albumin-Cre mice or by injecting AAV8-TBG-cre, respectively. Lipid and energy metabolism in these mice were characterized by metabolomic and transcriptome analyses. Mice with hepatic overexpression of VMP1 who were fed a NASH diet were also characterized. RESULTS: Hepatocyte-specific deletion of Vmp1 severely impaired VLDL secretion resulting in massive hepatic steatosis, hepatocyte death, inflammation and fibrosis, which are hallmarks of NASH. Mechanistically, loss of Vmp1 led to decreased hepatic levels of phosphatidylcholine and phosphatidylethanolamine as well as to changes in phospholipid composition. Deletion of Vmp1 in mouse liver also led to the accumulation of neutral lipids in the ER bilayer and impaired mitochondrial beta-oxidation. Overexpression of VMP1 ameliorated steatosis in diet-induced NASH by improving VLDL secretion. Importantly, we also showed that decreased liver VMP1 is associated with NAFLD/NASH in humans. CONCLUSIONS: Our results provide novel insights on the role of VMP1 in regulating hepatic phospholipid synthesis and lipoprotein secretion in the pathogenesis of NAFLD/NASH. LAY SUMMARY: Non-alcoholic fatty liver disease and its more severe form, non-alcoholic steatohepatitis, are associated with a build-up of fat in the liver (steatosis). However, the exact mechanisms that underly steatosis in patients are not completely understood. Herein, the authors identified that the lack of a protein called VMP1 impairs the secretion and metabolism of fats in the liver and could therefore contribute to the development and progression of non-alcoholic fatty liver disease.

Intestinal epithelial regeneration: active versus reserve stem cells and plasticity mechanisms.

The gastrointestinal system is arguably one of the most complicated developmental systems in a multicellular organism, as it carries out at least four major functions: digestion of food, absorption of nutrients, excretion of hormones, and defense against pathogens. Anatomically, the fetal gut has a tubular structure with an outer layer of smooth muscle derived from lateral splanchnic mesoderm and an inner lining of epithelium derived from the definitive endoderm. During morphogenesis of the gut tube, the definitive endoderm transforms into a primitive gut tube with a foregut, midgut, and hindgut. During the course of further development, the midgut gives rise to the small and proximal large intestine and the hindgut gives rise to the distal large intestine and rectum. The small intestine is subdivided into three parts: duodenum, jejunum, and ileum, whereas the large intestine is subdivided into the cecum, colon, and rectum.

Hematopoietic stem cells: self-renewal and expansion.

: Hematopoietic stem cells (HSCs) are a unique population of cells with the remarkable ability to replenish themselves through self-renewal and to give rise to differentiated cell lineages. Though having been discovered more than 50 years ago, and having been widely used in bone marrow transplantation to treat blood disorders including leukemia, expansion of HSCs remains an unmet task, thus affecting its more effective usage in clinical practice. PURPOSE OF REVIEW: The purpose of this review article is to summarize past efforts in ex-vivo HSC expansion and to compare recent advances in expanding murine and human HSCs by targeting the N-methyladenosine (mA) pathway. RECENT FINDINGS: Unlike past many efforts that mainly target single or limited pathways and often lead to lineage bias or expansion of progenitor cells or limited long-term HSCs (LT-HSCs), the blocking the degradation of mA pathway has an advantage of stabilizing hundreds of key factors required for maintaining HSCs, thus resulting in expansion of functional LT-HSCs. SUMMARY: The new approach of targeting the mA pathway has a promising application in clinical HSC-based transplantation.

Impaired TFEB-Mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-Induced Liver Injury and Steatosis in Mice.

BACKGROUND & AIMS: Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis. METHODS: We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfebflox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. RESULTS: Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues. CONCLUSIONS: We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.

Maternal imprinting at the H19-Igf2 locus maintains adult haematopoietic stem cell quiescence.

The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19-Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2-Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.

Cooperation between both Wnt/{beta}-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion.

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and ß-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of ß-catenin alone results in apoptosis of HSCs. However, together, the Wnt/ß-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, ß-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/ß-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity.

The regulatory niche of intestinal stem cells.

The niche constitutes a unique category of cells that support the microenvironment for the maintenance and self-renewal of stem cells. Intestinal stem cells reside at the base of the crypt, which contains adjacent epithelial cells, stromal cells and smooth muscle cells, and soluble and cell-associated growth and differentiation factors. We summarize here recent advances in our understanding of the crucial role of the niche in regulating stem cells. The stem cell niche maintains a balance among quiescence, proliferation and regeneration of intestinal stem cells after injury. Mesenchymal cells, Paneth cells, immune cells, endothelial cells and neural cells are important regulatory components that secrete niche ligands, growth factors and cytokines. Intestinal homeostasis is regulated by niche signalling pathways, specifically Wnt, bone morphogenetic protein, Notch and epidermal growth factor. These insights into the regulatory stem cell niche during homeostasis and post-injury regeneration offer the potential to accelerate development of therapies for intestine-related disorders.

PTEN-deficient intestinal stem cells initiate intestinal polyposis.

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN-deficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector beta-catenin. Akt phosphorylates beta-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of beta-catenin.

Leucine-rich repeat-containing G-protein-coupled Receptor 5 marks short-term hematopoietic stem and progenitor cells during mouse embryonic development.

Lgr5 is a marker for proliferating stem cells in adult intestine, stomach, and hair follicle. However, Lgr5 is not expressed in adult hematopoietic stem and progenitor cells (HSPCs). Whether Lgr5 is expressed in the embryonic and fetal HSPCs that undergo rapid proliferation is unknown. Here we report the detection of Lgr5 expression in HSPCs in the aorta-gonad-mesonephros (AGM) and fetal liver. We also found that a portion of Lgr5(+) cells expressed the Runx1 gene that is critical for the ontogeny of HSPCs. A small portion of Lgr5(+) cells also expressed HSPC surface markers c-Kit and CD34 in AGM or CD41 in fetal liver. Furthermore, the majority of Lgr5(+) cells expressed Ki67, indicating their proliferating state. Transplantation of fetal liver-derived Lgr5-GFP(+) cells (E12.5) demonstrated that Lgr5-GFP(+) cells were able to reconstitute myeloid and lymphoid lineages in adult recipients, but the engraftment was short-term (4-8 weeks) and 20-fold lower compared with the Lgr5-GFP(-) control. Our data show that Lgr5-expressing cells mark short-term hematopoietic stem and progenitor cells, consistent with the role of Lgr5 in supporting HSPCs rapid proliferation during embryonic and fetal development.

Detection of functional haematopoietic stem cell niche using real-time imaging.

Haematopoietic stem cell (HSC) niches, although proposed decades ago, have only recently been identified as separate osteoblastic and vascular microenvironments. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP(+)) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP(+) HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter-enhancer. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin(+) pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.

Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.

BACKGROUND & AIMS: Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS: We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS: CD44(+)CD24(lo)CD166(+) cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell-associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS: We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

FGF signaling facilitates postinjury recovery of mouse hematopoietic system.

Previous studies have shown that fibroblast growth factor (FGF) signaling promotes hematopoietic stem and progenitor cell (HSPC) expansion in vitro. However, it is unknown whether FGF promotes HSPC expansion in vivo. Here we examined FGF receptor 1 (FGFR1) expression and investigated its in vivo function in HSPCs. Conditional knockout (CKO) of Fgfr1 did not affect phenotypical number of HSPCs and homeostatic hematopoiesis, but led to a reduced engraftment only in the secondary transplantation. When treated with 5-fluorouracil (5FU), the Fgfr1 CKO mice showed defects in both proliferation and subsequent mobilization of HSPCs. We identified megakaryocytes (Mks) as a major resource for FGF production, and further discovered a novel mechanism by which Mks underwent FGF-FGFR signaling dependent expansion to accelerate rapid FGF production under stress. Within HSPCs, we observed an up-regulation of nuclear factor κB and CXCR4, a receptor for the chemoattractant SDF-1, in response to bone marrow damage only in control but not in Fgfr1 CKO model, accounting for the corresponding defects in proliferation and migration of HSPCs. This study provides the first in vivo evidence that FGF signaling facilitates postinjury recovery of the mouse hematopoietic system by promoting proliferation and facilitating mobilization of HSPCs.

Stress-protecting harbors for hematopoietic stem cells.

Hematopoietic stem cells (HSCs) rely on specialized microenvironments known as niches to maintain their self-renewal and multilineage potential to generate diverse types of blood cells continuously. Over the last two decades, substantial advancements have been made in unraveling the niche cell components and HSC localizations under homeostatic and stressed circumstances. Advances in imaging, combined with the discovery of phenotypic surface markers combinations and single cell sequencing, have greatly facilitated the systematic examination of HSC localizations. This review aims to present a summary of HSC localizations, highlighting potential distinctions between phenotypically and functionally defined HSCs, and explore the functionality of niches in ensuring the integrity and long-term maintenance of HSCs.

BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling.

In humans, mutations in BMPR1A, SMAD4 and PTEN are responsible for juvenile polyposis syndrome, juvenile intestinal polyposis and Cowden disease, respectively. The development of polyposis is a common feature of these diseases, suggesting that there is an association between BMP and PTEN pathways. The mechanistic link between BMP and PTEN pathways and the related etiology of juvenile polyposis is unresolved. Here we show that conditional inactivation of Bmpr1a in mice disturbs homeostasis of intestinal epithelial regeneration with an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling human juvenile polyposis syndrome. We show that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell self-renewal. Mechanistically, PTEN, through phosphatidylinosital-3 kinase-Akt, mediates the convergence of the BMP and Wnt pathways on control of beta-catenin. Thus, BMP signaling may control the duplication of intestinal stem cells, thereby preventing crypt fission and the subsequent increase in crypt number.

PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention.

Haematopoietic stem cells (HSCs) must achieve a balance between quiescence and activation that fulfils immediate demands for haematopoiesis without compromising long-term stem cell maintenance, yet little is known about the molecular events governing this balance. Phosphatase and tensin homologue (PTEN) functions as a negative regulator of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway, which has crucial roles in cell proliferation, survival, differentiation and migration. Here we show that inactivation of PTEN in bone marrow HSCs causes their short-term expansion, but long-term decline, primarily owing to an enhanced level of HSC activation. PTEN-deficient HSCs engraft normally in recipient mice, but have an impaired ability to sustain haematopoietic reconstitution, reflecting the dysregulation of their cell cycle and decreased retention in the bone marrow niche. Mice with PTEN-mutant bone marrow also have an increased representation of myeloid and T-lymphoid lineages and develop myeloproliferative disorder (MPD). Notably, the cell populations that expand in PTEN mutants match those that become dominant in the acute myeloid/lymphoid leukaemia that develops in the later stages of MPD. Thus, PTEN has essential roles in restricting the activation of HSCs, in lineage fate determination, and in the prevention of leukaemogenesis.

Phosphoinositide 3-kinase signaling mediates beta-catenin activation in intestinal epithelial stem and progenitor cells in colitis.

BACKGROUND & AIMS: Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation. METHODS: The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice named I-pik3r1KO. Acute inflammation was induced in mice and intestines were analyzed by biochemical and histologic methods. The effects of chemically blocking PI3K in colitic interleukin-10(-/-) mice were examined. Biopsy samples from patients were examined. RESULTS: Compared with wild-type, I-pik3r1KO mice had reduced T-cell-mediated Akt and beta-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K-Akt signaling increased nuclear total beta-catenin and P-beta-catenin(552) levels and reduced N-terminal beta-catenin phosphorylation, which is associated with degradation. PI3K inhibition in interleukin-10(-/-) mice impaired colitis-induced epithelial Akt and beta-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-beta-catenin(552) throughout expanded crypts and increased messenger RNA expression of beta-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples. CONCLUSIONS: PI3K-Akt signaling cooperates with Wnt to increase beta-catenin signaling during inflammation. PI3K-induced and Akt-mediated beta-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.

Atlas of the human intestine.

In this issue of JEM, Wang et al. (https://doi.org/10.1084/jem.20191130), using a single-cell RNA-seq approach, establish an atlas of human colon, rectum, and ileum epithelial cells. Their study reveals that different regions have specialized nutrient absorption preferences, microbe defenses, and endocrine function. They also identify new markers for a variety of cell types.

ß-Catenin and Associated Proteins Regulate Lineage Differentiation in Ground State Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. ß-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of ß-catenin in restraining germline and somatic lineage differentiation genes. We show that ß-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help ß-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of ß-catenin in preserving lineage differentiation integrity in ground state ESCs.

Overcoming Wnt-ß-catenin dependent anticancer therapy resistance in leukaemia stem cells.

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.