Tumor-initiating stem cell shapes its microenvironment into an immunosuppressive barrier and pro-tumorigenic niche.

Tumor-initiating stem cells (TSCs) are critical for drug resistance and immune escape. However, the mutual regulations between TSC and tumor microenvironment (TME) remain unclear. Using DNA-label retaining, single-cell RNA sequencing (scRNA-seq), and other approaches, we investigated intestinal adenoma in response to chemoradiotherapy (CRT), thus identifying therapy-resistant TSCs (TrTSCs). We find bidirectional crosstalk between TSCs and TME using CellPhoneDB analysis. An intriguing finding is that TSCs shape TME into a landscape that favors TSCs for immunosuppression and propagation. Using adenoma-organoid co-cultures, niche-cell depletion, and lineaging tracing, we characterize a functional role of cyclooxygenase-2 (Cox-2)-dependent signaling, predominantly occurring between tumor-associated monocytes and macrophages (TAMMs) and TrTSCs. We show that TAMMs promote TrTSC proliferation through prostaglandin E2 (PGE2)-PTGER4(EP4) signaling, which enhances ß-catenin activity via AKT phosphorylation. Thus, our study shows that the bidirectional crosstalk between TrTSC and TME results in a pro-tumorigenic and immunosuppressive contexture.

Identification of stem cells from large cell populations with topological scoring.

Machine learning and topological analysis methods are becoming increasingly used on various large-scale omics datasets. Modern high dimensional flow cytometry data sets share many features with other omics datasets like genomics and proteomics. For example, genomics or proteomics datasets can be sparse and have high dimensionality, and flow cytometry datasets can also share these features. This makes flow cytometry data potentially a suitable candidate for employing machine learning and topological scoring strategies, for example, to gain novel insights into patterns within the data. We have previously developed a Topological Score (TopS) and implemented it for the analysis of quantitative protein interaction network datasets. Here we show that TopS approach for large scale data analysis is applicable to the analysis of a previously described flow cytometry sorted human hematopoietic stem cell dataset. We demonstrate that TopS is capable of effectively sorting this dataset into cell populations and identify rare cell populations. We demonstrate the utility of TopS when coupled with multiple approaches including topological data analysis, X-shift clustering, and t-Distributed Stochastic Neighbor Embedding (t-SNE). Our results suggest that TopS could be effectively used to analyze large scale flow cytometry datasets to find rare cell populations.

Author Correction: Suppression of m6A reader Ythdf2 promotes hematopoietic stem cell expansion.

In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.

Suppression of m6A reader Ythdf2 promotes hematopoietic stem cell expansion.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Retinoid-Sensitive Epigenetic Regulation of the Hoxb Cluster Maintains Normal Hematopoiesis and Inhibits Leukemogenesis.

Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes.

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

The Dlk1-Gtl2 Locus Preserves LT-HSC Function by Inhibiting the PI3K-mTOR Pathway to Restrict Mitochondrial Metabolism.

The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation.

A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes.

Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML's activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1.

Functional genomic analysis of the periodic transcriptome in the developing Drosophila wing.

The eukaryotic cell cycle, driven by both transcriptional and posttranslational mechanisms, is the central molecular oscillator underlying tissue growth throughout animals. Although genome-wide studies have investigated cell-cycle-associated transcription in unicellular systems, global patterns of periodic transcription in multicellular tissues remain largely unexplored. Here we define the cell-cycle-associated transcriptome of the developing Drosophila wing epithelium and compare it with that of cultured Drosophila S2 cells, revealing a core set of periodic genes and a surprising degree of context specificity in periodic transcription. We further employ RNAi-mediated phenotypic profiling to define functional requirements for more than 300 periodic genes, with a focus on those required for cell proliferation in vivo. Finally, we investigate uncharacterized genes required for interkinetic nuclear migration. Combined, these findings provide a global perspective on cell-cycle control in vivo, and they highlight a critical need to understand the context-specific regulation of cell proliferation.

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.

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.

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.

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.

DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.

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.

Kinetics of in vivo elimination of suicide gene-expressing T cells affects engraftment, graft-versus-host disease, and graft-versus-leukemia after allogeneic bone marrow transplantation.

Suicide gene therapy is one approach being evaluated for the control of graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). We recently constructed a novel chimeric suicide gene in which the entire coding region of HSV thymidine kinase (HSV-tk) was fused in-frame to the extracellular and transmembrane domains of human CD34 (DeltaCD34-tk). DeltaCD34-tk is an attractive candidate as a suicide gene in man because of the ensured expression of HSV-tk in all selected cells and the ability to rapidly and efficiently purify gene-modified cells using clinically approved CD34 immunoselection techniques. In this study we assessed the efficacy of the DeltaCD34-tk suicide gene in the absence of extended ex vivo manipulation by generating transgenic animals that express DeltaCD34-tk in the peripheral and thymic T cell compartments using the CD2 locus control region. We found that DeltaCD34-tk-expressing T cells could be purified to near homogeneity by CD34 immunoselection and selectively eliminated ex vivo and in vivo when exposed to low concentrations of GCV. The optimal time to administer GCV after allogeneic BMT with DeltaCD34-tk-expressing transgenic T cells was dependent on the intensity of the conditioning regimen, the leukemic status of the recipient, and the dose and timing of T cell infusion. Importantly, we used a controlled graft-vs-host reaction to promote alloengraftment in sublethally irradiated mice and provide a graft-vs-leukemia effect in recipients administered a delayed infusion of DeltaCD34-tk-expressing T cells. This murine model demonstrates the potential usefulness of DeltaCD34-tk-expressing T cells to control GVHD, promote alloengraftment, and provide a graft-vs-leukemia effect in man.

Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease.

Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (DeltaCD34-tk). High-efficiency transfer of DeltaCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC(50) approximately 3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vbeta family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the DeltaCD34-tk suicide gene. T cells modified with the DeltaCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control.