Regulatory Lineage Research Articles

PKC-Epsilon Overexpression Is Associated with Poor Outcomes in AML and Promotes Chemoresistance and Hematopoietic Stem Cell Quiescence

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies which are characterized by the accumulation of clonal, abnormally differentiated blasts in the bone marrow. For most patients, prognosis remains poor as a large proportion relapse following a period of complete remission. Protein kinase c epsilon (PKCε) is a pleiotropic regulator of cell proliferation, differentiation, apoptotic and survival signaling. Uniquely in the PKC family, PKCε also has demonstrated oncogenic properties and high expression of PKCε has been associated with poor patient outcomes, disease aggressiveness, and chemoresistance in solid cancers. A role for PKCε has also been implicated in AML; however, its clinical significance has not been elucidated. To address this, we first determined whether PKCε was aberrantly expressed in AML. Patient samples were collected from the MRC AML14 and AML15 clinical trials and quantitative estimation of PKCε at the protein and mRNA level was conducted. PKCε overexpression (>2SD of mean of controls) occurred in 32% (43/132) of patients based on mRNA expression and 37% (26/70) of samples based on protein expression, compared to normal human CD34+ cells. In addition, a positive correlation between PKCε mRNA and protein expression was established (R=0.64, p<0.01). We next evaluated the clinical impact of high PKCε expression in AML using the TCGA RNAseq dataset. High PKCε expression (>median), correlated with significantly worse overall survival (95% CI (34-54); p<0.05), with the median survival for being 11 months, compared with 24 months for patients with low PKCε expression. High PKCε expression did not correlate with age, molecular abnormality, gender, or WBC, and is therefore likely to be an independent prognostic factor. To study the mechanisms through which PKCε could contribute to poor outcomes in AML, the influence of PKCε overexpression on the chemosensitivity of AML cell lines was explored. In the AML cell lines investigated (U937, HEL, TF-1 and NOMO-1), PKCε overexpression was associated with significantly increased daunorubicin (DNR) resistance in U937 and HEL cells (2.6-fold, n=4; p<0.05 for both lines) without affecting their sensitivity to cytarabine, suggesting that PKCε can selectively promote resistance to DNR. The therapeutic mechanism of DNR is, in part, accounted for by the generation of reactive oxygen species (ROS) and we are currently investigating whether PKCε expression protects against ROS-generating agents as has previously been suggested. We next examined the effect of PKCε on normal human hematopoiesis. RNAseq data from both human and murine hematopoietic cells show that PKCε is endogenously expressed in normal hematopoietic cells, with the highest levels of expression occurring in the HSC compartment and monocytes. Overexpression of PKCε in human CD34+ progenitor cells promoted myeloid differentiation of unilineage monocyte and granulocyte progenitors which showed an increase in expression CD14 and CD15, with CD11b being significantly upregulated on monocytes and granulocytes by 1.4-fold±0.3 (n=4, p<0.05) and 1.6-fold±0.2 (n=3, p<0.05), respectively. Conversely, shRNA mediated PKCε knockdown in these cells reduced the expression of these myeloid markers, supporting a role for PKCε in developmental regulation of the myeloid lineage. We next examined the effect of PKCε overexpression on the self-renewal and hematopoietic activity of human HSC (CD34+CD38-). Transduced HSC were scored for colony formation, in addition to being seeded onto MS5 stroma and assessed for their capacity to generate cobblestone area forming cells (CAFC). PKCε had no significant impact on colony forming efficiency but ablated CAFC formation suggesting that PKCε selectively promotes quiescence of HSC (n=2). Since quiescence of leukemic stem cells (LSC) is widely thought to contribute to relapse in AML, this may represent a further mechanism through which PKCε expression confers a poor outcome in this disease and we are currently investigating this. In summary, we show that elevated expression of PKCε occurs frequently in AML and our data indicate it is an independent prognostic marker of poor outcome in this disease. Experimental models suggest that overexpression of PKCε may contribute to treatment resistance through promoting DNR resistance and potentially by promoting LSC quiescence. Disclosures Knapper: Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Honoraria; Tolero: Membership on an entity's Board of Directors or advisory committees.

Human Cortical Organoids Expose a Differential Function of GSK3 on Cortical Neurogenesis.

SummaryThe regulation of the proliferation and polarity of neural progenitors is crucial for the development of the brain cortex. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remains to be elucidated. We harnessed human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Chronic GSK3 inhibition increased the proliferation of neural progenitors and caused massive derangement of cortical tissue architecture. Single-cell transcriptome profiling revealed a direct impact on early neurogenesis and uncovered a selective role of GSK3 in the regulation of glutamatergic lineages and outer radial glia output. Our dissection of the GSK3-dependent transcriptional network in human corticogenesis underscores the robustness of the programs determining neuronal identity independent of tissue architecture.

Shape-dependent regulation of differentiation lineages of bone marrow-derived cells under cyclic stretch

Multipotent stem cells are considered as a key material in regenerative medicine, and the understanding of the heterogeneity in the differentiation potentials of bone marrow-derived cells is important in the successful regenerative tissue repair. Therefore, the present study has been performed to investigate how the differentiation of post-harvest, native bone marrow-derived cells is regulated by cyclic stretch in vitro. Bone marrow-derived cells were obtained from mouse femur of both hind limbs and categorized into the following five categories: amebocytes, round cells, spindle cells, stellate cells and others. The cells were seeded on a silicone-made stretch chamber, and subjected to cyclic stretch with an amplitude of 10% at a frequency of 1 Hz for 7 days for cell shape analysis and for 3 days for the analysis of the expression of marker proteins of osteogenic (osteocalcin), vascular smooth muscle (α-smooth muscle actin and smooth muscle myosin heavy chain) and neurogenic (neurofilament) differentiation. When disregarding the differences in the cell shapes, there was an overall trend that the application of 10% cyclic stretch inhibited osteogenic and neurogenic differentiation, but enhanced smooth muscle differentiation. Close examinations revealed that round cells were influenced the most by cyclic stretch (significant up- or down-regulation in all the four marker protein expressions) while amebocytes and spindle cells were only influenced by cyclic stretch for vascular smooth muscle and/or neurogenic differentiation. As far as the authors know, this is the first study reporting the shape-related differences in the fate decision criteria for mechanical strain in bone marrow-derived cells.

Comparative Evaluation of Hormones and Hormone-Like Molecule in Lineage Specification of Human Induced Pluripotent Stem Cells.

Background and ObjectivesProficient differentiation of human pluripotent stem cells (hPSCs) into specific lineages is required for applications in regenerative medicine. A growing amount of evidences had implicated hormones and hormone-like molecules as critical regulators of proliferation and lineage specification during in vivo development. Therefore, a deeper understanding of the hormones and hormone-like molecules involved in cell fate decisions is critical for efficient and controlled differentiation of hPSCs into specific lineages. Thus, we functionally and quantitatively compared the effects of diverse hormones (estradiol 17-β (E2), progesterone (P4), and dexamethasone (DM)) and a hormone-like molecule (retinoic acid (RA)) on the regulation of hematopoietic and neural lineage specification.Methods and ResultsWe used 10 nM E2, 3 μM P4, 10 nM DM, and 10 nM RA based on their functional in vivo developmental potential. The sex hormone E2 enhanced functional activity of hematopoietic progenitors compared to P4 and DM, whereas RA impaired hematopoietic differentiation. In addition, E2 increased CD34+CD45+ cells with progenitor functions, even in the CD43− population, a well-known hemogenic marker. RA exhibited lineage-biased potential, preferentially committing hPSCs toward the neural lineage while restricting the hematopoietic fate decision.ConclusionsOur findings reveal unique cell fate potentials of E2 and RA treatment and provide valuable differentiation information that is essential for hPSC applications.

Osteocyte Death and Bone Overgrowth in Mice Lacking Fibroblast Growth Factor Receptors 1 and 2 in Mature Osteoblasts and Osteocytes.

Fibroblast growth factor (FGF) signaling pathways have well-established roles in skeletal development, with essential functions in both chondrogenesis and osteogenesis. In mice, previous conditional knockout studies suggested distinct roles for FGF receptor 1 (FGFR1) signaling at different stages of osteogenesis and a role for FGFR2 in osteoblast maturation. However, the potential for redundancy among FGFRs and the mechanisms and consequences of stage-specific osteoblast lineage regulation were not addressed. Here, we conditionally inactivate Fgfr1 and Fgfr2 in mature osteoblasts with an Osteocalcin (OC)-Cre or Dentin matrix protein 1 (Dmp1)-CreER driver. We find that young mice lacking both receptors or only FGFR1 are phenotypically normal. However, between 6 and 12 weeks of age, OC-Cre Fgfr1/Fgfr2 double- and Fgfr1 single-conditional knockout mice develop a high bone mass phenotype with increased periosteal apposition, increased and disorganized endocortical bone with increased porosity, and biomechanical properties that reflect increased bone mass but impaired material properties. Histopathological and gene expression analyses show that this phenotype is preceded by a striking loss of osteocytes and accompanied by activation of the Wnt/β-catenin signaling pathway. These data identify a role for FGFR1 signaling in mature osteoblasts/osteocytes that is directly or indirectly required for osteocyte survival and regulation of bone mass during postnatal bone growth. © 2019 American Society for Bone and Mineral Research.

Regulation of Tfh cell development through STAT1 signaling

Abstract T follicular helper (Tfh) cells play a key role in providing help to B cells during germinal center (GC) reactions. The generation and function of Tfh cells is regulated by multiple checkpoints including their early priming stage in T zones and throughout the effector stage of differentiation in GCs. Signaling pathways activated downstream of cytokine and costimulatory receptors as well as consequent activation of subset-specific transcriptional factors are essential steps for Tfh cell generation. While to date early signaling events for fate committed differentiation of Tfh cells are sufficiently studied, mechanisms allowing Tfh cells to maintain their commitment/programming are still unclear. Here we report that signal transducer and activator of transcription 1 (STAT1) deficiency in CD4+ T cells leads to a diminished number of Tfh cells at day 7 and 14 days after antigen immunization, while at day 3–4, number of Tfh cells was comparable between STAT1-sufficient and STAT1 deficient mice, suggesting the role of STAT1 in Tfh lineage maintenance rather than in Tfh cell priming. In fact, in both mouse and human Tfh cells interleukin (IL)-21 mRNA and protein expression is dependent on the presence of STAT1. Functionally, STAT1 in cooperation with STAT3, Batf, and IRF4 triggers IL-21 production in Tfh cells by directly binding to and activation of the IL-21 gene locus. Moreover, utilizing gene knockout approach, we have determined that Batf is a crucial connector to form STAT1/STAT3/Batf/IRF4 active complex for triggering IL-21 transcription. Our results thus indicate functional contribution of STAT1 towards regulation of IL-21 expression and Tfh lineage maturation/maintenance.

The plant stomatal lineage at a glance.

Stomata are structures on the surfaces of most land plants that are required for gas exchange between plants and their environment. In Arabidopsis thaliana, stomata comprise two kidney bean-shaped epidermal guard cells that flank a central pore overlying a cavity in the mesophyll. These guard cells can adjust their shape to occlude or facilitate access to this pore, and in so doing regulate the release of water vapor and oxygen from the plant, in exchange for the intake of carbon dioxide from the atmosphere. Stomatal guard cells are the end product of a specialized lineage whose cell divisions and fate transitions ensure both the production and pattern of cells in aerial epidermal tissues. The stomatal lineage is dynamic and flexible, altering stomatal production in response to environmental change. As such, the stomatal lineage is an excellent system to study how flexible developmental transitions are regulated in plants. In this Cell Science at a Glance article and accompanying poster, we will summarize current knowledge of the divisions and fate decisions during stomatal development, discussing the role of transcriptional regulators, cell-cell signaling and polarity proteins. We will highlight recent work that links the core regulators to systemic or environmental information and provide an evolutionary perspective on stomata lineage regulators in plants.

Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation.

The differentiation of self-renewing progenitor cells requires not only the regulation of lineage- and developmental stage–specific genes but also the coordinated adaptation of housekeeping functions from a metabolically active, proliferative state toward quiescence. How metabolic and cell-cycle states are coordinated with the regulation of cell type–specific genes is an important question, because dissociation between differentiation, cell cycle, and metabolic states is a hallmark of cancer. Here, we use a model system to systematically identify key transcriptional regulators of Ikaros-dependent B cell–progenitor differentiation. We find that the coordinated regulation of housekeeping functions and tissue-specific gene expression requires a feedforward circuit whereby Ikaros down-regulates the expression of Myc. Our findings show how coordination between differentiation and housekeeping states can be achieved by interconnected regulators. Similar principles likely coordinate differentiation and housekeeping functions during progenitor cell differentiation in other cell lineages.

Abstract P4-05-01: Limb-Bud-and-Heart (LBH), a novel WNT effector in promoting basal-like breast cancer

Abstract BACKGROUND: Clinically aggressive basal-like breast cancers (BLBC) disproportionally contribute to cancer deaths, but lack effective treatment options due to absence of expression of key therapeutic targets (i.e. ER, PR, HER2). New evidence suggests BLBC may originate from luminal breast epithelial cells through luminal-to-basal cell lineage conversion and dedifferentiation. However, the factors that reprogram cell fate and differentiation states during BLBC development remain poorly understood. Our laboratory has identified a novel WNT/β-Catenin target transcriptional regulator, Limb-Bud-and-Heart (LBH), and shown LBH is majorly over expressed in aggressive BLBC harboring WNT hyperactivation. We previously showed in genetic mouse models in vivo that LBH is a key mammary stem cell and basal cell lineage regulator essential for normal mammary gland development. Using crosses between MMTV-Wnt1 transgenic mice and conditional LBH knockout mice, we tested if LBH is a critical effector of WNT-driven basaloid breast cancer, and whether its inhibition may prevent and/or attenuate BLBC formation and progression. RESULTS: In our in vivo mouse model, LBH inactivation in the basal mammary epithelium using a Keratin 14/K14-Cre deleter strain significantly attenuated MMTV-Wnt1-induced mammary gland hyperplasia and led to a delay in tumor onset. Surprisingly, tumor burden and tumor volumes were not changed, indicating that LBH is not required for WNT-driven tumorigenesis. However, LBH-deficient MMTV-Wnt1-transgenic tumors exhibited pronounced histopathological differences compared to LBH WT MMTV-Wnt1-transgenic tumors. Whereas MMTV-Wnt1+;K14CreLbh WT tumors were highly vascularized, disorganized, with mixed basal and luminal cell identity; tumors from MMTV-Wnt1+;K14CreLbh KO mice were more organized, differentiated, and predominantly luminal Keratin 8 positive. Since attenuation of mammary gland hyperplasia and tumor onset in this model suggests that LBH deficiency may negatively affect WNT-induced stem cell expansion, we next investigated the LBH effects on the tumor stem cell compartment. MMTV-Wnt1+;K14CreLbh KO mice were crossed with two different stem cell reporter lines: Lgr5-eGFP mice, which specifically mark WNT-responsive epithelial stem cells; and SHIP-GFP reporter mice, which mark activated, proliferating mammary stem cells but not quiescent or slow-cycling stem cells. These studies are ongoing and the latest findings will be discussed. CONCLUSION: Our data indicate that LBH is an essential cell fate regulator downstream of oncogenic WNT signaling that maintains basal lineage identity in baseloid breast cancers. LBH inhibition may be a potential novel strategy to treat basal subtype triple negative breast cancer patients through differentiation therapy. Citation Format: Briegel K, Ashad-Bishop K. Limb-Bud-and-Heart (LBH), a novel WNT effector in promoting basal-like breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-05-01.

Asymmetric Expression of LincGET Biases Cell Fate in Two-Cell Mouse Embryos

In early mammalian embryos, it remains unclear how the first cell fate bias is initially triggered and amplified toward cell fate segregation. Here, we report that a long noncoding RNA, LincGET, is transiently and asymmetrically expressed in the nucleus of two- to four-cell mouse embryos. Overexpression of LincGET in one of the two-cell blastomeres biases its progeny predominantly toward the inner cell mass (ICM) fate. Mechanistically, LincGET physically binds to CARM1 and promotes the nuclear localization of CARM1, which can further increase the level of H3 methylation at Arginine 26 (H3R26me), activate ICM-specific gene expression, upregulate transposons, and increase global chromatin accessibility. Simultaneous overexpression of LincGET and depletion of Carm1 no longer biased embryonic fate, indicating that the effect of LincGET in directing ICM lineage depends on CARM1. Thus, our data identify LincGET as one of the earliest known lineage regulators to bias cell fate in mammalian 2-cell embryos.

Integrated Analysis of Global DNA Methylation and Transcription Reveals a Leukemia Subtype with Extreme Hypermethylation Associated with Silencing of Regulators of Differentiation

Acute myeloid leukemia (AML) represents a highly heterogeneous myeloid stem cell disorder classified based on various genetic defects. Besides genetic alterations, epigenetic changes are recognized as an additional mechanism contributing to leukemogenesis, but insight into the latter process remains minor. Using a combination of Methyl-CpG-Immunoprecipitation (MCIp-chip) and MALDI-TOF analysis of bisulfite-treated DNA in a cohort of 196 AML patients we previously demonstrated that (cyto)genetically defined AML subtypes, including CBFB-MYH11, AML-ETO, NPM1-mut, CEBPA-mut or IDH1/2-mut subtypes, express specific DNA-methylation profiles (Gebhard et al, Leukemia, 2018). A fraction of AML patients (5/196) displayed a unique abnormal hypermethylation profile that was completely distinct from any other AML subtype. These patients present immature leukemia (FAB M0, M1) with various chromosomal aberrations but very few mutations (e.g. no IDH1/2, KRAS, DNMT3A) that might explain the CpG island methylator phenotype (CIMP) phenotype. The CIMP patients showed high resemblance with a recently reported CEBPA methylated subgroup (Wouters et al, 2007 and Figueroa et al, 2009), which we confirmed by MCIp-chip and MALDI-TOF analysis.To explore the whole range of epigenetic alterations in the CIMP-AML patients we performed in-depth global DNA methylation and gene expression analyses (MCIp-seq and RNA-seq) in 45 AML and 12 CIMP patients from both studies. Principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) revealed that CIMP patients express a unique DNA-methylation and gene-expression signature that separated them from all other AMLs. We could discriminate promoter methylation from non-promoter methylation by selecting MCIp-seq peaks within 3kb around TSS. Promoter hypermethylation was highly associated with repression of genes (PCC = -0.053, p-value = 0.00075). Hypermethylation of non-promoter regions was more strongly associated with upregulation of genes (PCC = 0.046, p-value = 4.613e-06). Interestingly, differentially methylated regions also showed a positive association with myeloid lineage CTCF binding sites (27% vs 18% expected, p-value < 2.2e-16 in a chi-square test of independence). Methylation of CTCF sites causes loss of CTCF binding, which has been reported to disrupt boundaries between so-called topologically associated domains (TADs), allowing enhancers located in a particular TAD to become accessible to genes in adjacent TADs and affect their transcription. Whether this is the case is under investigation. In this study we particularly focused on the role of hypermethylation of promoters in CIMP-AMLs.Promoters of many transcriptional regulators that are involved in the differentiation of myeloid lineages of which several are frequently mutated in AML were hypermethylated and repressed, including CEBPA, CEBPD, IRF8, GATA2, KLF4, MITF or MAFB. Notably, HMGA2, a critical regulator of myeloid progenitor expansion, exhibited the largest degree of CIMP promoter hypermethylation compared to the other AMLs, accompanied by a reduction in gene expression. Moreover, multiple members of the HOXB family and KLF1 (erythroid differentiation) were methylated and repressed as well.In addition, these patients frequently showed hypermethylation of many chromatin factors (e.g. LMNA, CHD7 or TET2). Hypermethylation of the TET2 promoter could result in a loss of maintenance DNA demethylation and therefore successive hypermethylation at CpG islands. We carried out regulome-capture-bisulfite sequencing on CIMP-AMLs compared to other AML samples and normal blood cell controls and confirmed methylation of the same transcription and chromatin factor promoters.We conclude that these leukemias represent very primitive HSCPs which are blocked in differentiation into multiple hematopoietic lineages, due to the absence of regulators of these lineages. Although the underlying cause for the extreme hypermethylation signature is still subject to ongoing studies, the consequence of promoter hypermethylation is silencing of key lineage regulators causing the differentiation arrest in these cells. We argue that these patients may particularly benefit from therapies that revert DNA methylation. DisclosuresEhninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN and TP53-Mutated Prostate Cancer.

Purpose: Deletions or mutations in PTEN and TP53 tumor suppressor genes have been linked to lineage plasticity in therapy-resistant prostate cancer. Fusion-driven overexpression of the oncogenic transcription factor ERG is observed in approximately 50% of all prostate cancers, many of which also harbor PTEN and TP53 alterations. However, the role of ERG in lineage plasticity of PTEN/TP53-altered tumors is unclear. Understanding the collective effect of multiple mutations within one tumor is essential to combat plasticity-driven therapy resistance.Experimental Design: We generated a Pten-negative/Trp53-mutated/ERG-overexpressing mouse model of prostate cancer and integrated RNA-sequencing with ERG chromatin immunoprecipitation-sequencing (ChIP-seq) to identify pathways regulated by ERG in the context of Pten/Trp53 alteration. We investigated ERG-dependent sensitivity to the antiandrogen enzalutamide and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib in human prostate cancer cell lines, xenografts, and allografted mouse tumors. Trends were evaluated in TCGA, SU2C, and Beltran 2016 published patient cohorts and a human tissue microarray.Results: Transgenic ERG expression in mice blocked Pten/Trp53 alteration-induced decrease of AR expression and downstream luminal epithelial genes. ERG directly suppressed expression of cell cycle-related genes, which induced RB hypophosphorylation and repressed E2F1-mediated expression of mesenchymal lineage regulators, thereby restricting adenocarcinoma plasticity and maintaining antiandrogen sensitivity. In ERG-negative tumors, CDK4/6 inhibition delayed tumor growth.Conclusions: Our studies identify a previously undefined function of ERG to restrict lineage plasticity and maintain antiandrogen sensitivity in PTEN/TP53-altered prostate cancer. Our findings suggest ERG fusion as a biomarker to guide treatment of PTEN/TP53-altered, RB1-intact prostate cancer. Clin Cancer Res; 24(18); 4551-65. ©2018 AACR.

Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells.

T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations1-3. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing4,5, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.

Abstract 141: HEY1-mediated inhibition of glioma stem cell proliferation is associated with restoration of glioma stem cell division asymmetry and transcriptional repression of PDGFRA

Abstract Emerging evidence suggests that deregulation of cell-fate regulatory mechanisms in subpopulations of stem-like tumor cells can contribute to the initiation, progression, and recurrence of glioma. In this study, we report that Hey1 expression suppresses the expansion of oligodendroglial progenitor (OPC)-like glioma stem cells, which are a putative cell of origin for high-grade gliomas. We found that Hey1 expression reduced the proliferation of primary neural progenitor cells isolated from mice without the tumor suppressor Tp53, and OPC-like glioma stem cell cultures isolated from a mouse model of glioma driven by an EGFR gain-of-function mutation and Tp53 loss (v-ErbB/p53-/-). Further analysis of these cultures revealed that Hey1-mediated reductions in proliferation were associated with suppression of self-renewal and a reduced percentage of symmetric cell divisions. Mechanistically, we found that HEY1 directly bound to novel promoter elements and repressed the mRNA expression of a key marker and mitogenic driver of OPCs, the PDGF Receptor A (Pdgfra). Furthermore, Hey1-mediated of Pdgfra expression was associated with inactivation of the canonical PDGF/PDGFRA downstream target, extracellular signal-regulated kinases (ERK), in v-ErbB/p53-/- glioma stem-like cells. Key findings made in the murine system were validated in patient-derived glioma stem-like cells. Moreover, analyzing publicly available databases, we found that the subset of glioma patients with high levels of HEY1 expression had improved survival compared to patients with tumors expressing low levels of HEY1. Further supporting a role for HEY1 in the regulation of the oligodendroglial lineage, ontologic analysis revealed that the subset of patients with high levels of HEY1 expression were also characterized by the expression of gene sets associated with oligodendroglial differentiation. These cellular, molecular and genetic findings provide support for a model in which HEY1 functions to suppress glioma progression by inhibiting the expansion of glioma stem cells. Citation Format: Damian A. Almiron Bonnin, Jaclyn Sullivan, Mark A. Israel, Matthew Havrda C. Havrda. HEY1-mediated inhibition of glioma stem cell proliferation is associated with restoration of glioma stem cell division asymmetry and transcriptional repression of PDGFRA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 141.

Abstract 534: Oncogenes alter mammary epithelial cell lineage identity prior to tumor formation

Abstract Background: Breast cancer is a heterogeneous disease encompassing multiple subtypes that differ in their molecular profiles, clinical course of progression, and response to therapeutics. Interestingly, breast cancers fall broadly into luminal and basal subtypes, which resemble the normal luminal and basal epithelial cells of the breast. This correlation suggests that the molecular mechanisms controlling epithelial cell lineage identity during normal mammary development may also influence tumor subtype during cancer formation. Since many breast cancers are driven by oncogenes that are important for normal mammary development, we hypothesize that dysregulated oncogene signaling may alter mammary epithelial cell lineage identity at an early step of cancer initiation, priming the breast tumor subtype that will be formed upon full tumorigenesis. Methods: Human breast cell lines, MCF10a and MCF10f, and primary human breast tissues were infected with virus expressing single breast cancer oncogenes. Flow cytometry and quantitative RT-PCR were performed on breast cell lines to determine changes in the balance of luminal to basal cells and expression of luminal or basal gene sets. Primary tissues were grown in hydrogels and stained for luminal and basal markers to determine the effect of oncogene expression on the growth of mammary ductal structures. Mouse tumor models, MMTV-Myc and MMTV-Wnt1, were investigated for changes in their mammary epithelial architecture by histology and flow cytometry at ages prior to tumor formation. Results: Oncogene overexpression in human breast cell lines causes shifts in basal and luminal cell balance that correlate with changes seen in mammary ductal growth in hydrogel cultures of primary human breast epithelial cells. In mouse models, individual oncogenes alter mammary epithelial architecture and cell balance at early stages prior to tumor formation. In both models, individual oncogenes are able to regulate mammary epithelial cell lineage identity in the absence of other tumor-initiating signals, indicating that oncogenes may influence breast cancer subtype through mammary epithelial cell lineage regulation at early stages of cancer progression. Citation Format: Jerrica Breindel, Felix Nampanya, Aditya Datye, Daniel H. Miller, Piyush Gupta, Charlotte Kuperwasser. Oncogenes alter mammary epithelial cell lineage identity prior to tumor formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 534.

Abstract 1138: LBH, a novel WNT effector in promoting basal-like breast cancer

Abstract BACKGROUND: Clinically aggressive basal-like breast cancers (BLBC) disproportionally contribute to cancer deaths, but lack effective treatment options due to absence of expression of key therapeutic targets (ER, PR, HER2). New evidence suggests BLBC may originate from luminal breast epithelial cells through luminal-to-basal cell lineage conversion and dedifferentiation. However, the factors that reprogram cell fate and differentiation states during BLBC development remain poorly understood. Our laboratory has identified a novel WNT/β-Catenin target transcriptional regulator, Limb-Bud-and-Heart (LBH), which is majorly overexpressed in aggressive BLBC harboring WNT hyperactivation. We previously showed in a conditional LBH knockout mouse model in vivo that LBH is a key stem cell and basal lineage regulator in normal mammary gland development. However, it's role in breast cancer remains elusive. Here, we crossed conditional LBH knockout (KO) mice with a mouse model for WNT-driven breast cancer (MMTV-Wnt1) to test if LBH is a critical effector of WNT-driven basaloid breast cancer, and whether its inhibition may affect BLBC tumorigenesis. RESULTS: In our in vivo mouse model, epithelial-specific LBH inactivation using a Keratin 14/K14-Cre significantly attenuated Wnt1-induced mammary gland hyperplasia and delayed tumor onset. Yet, tumor volumes and burden were not changed, indicating that LBH is not essential for WNT-induced tumorigenesis. However, LBH-deficient MMTV-Wnt1-transgenic tumors exhibited pronounced histopathological differences compared to LBH WT MMTV-Wnt1-transgenic tumors. Whereas Wnt1+;K14CreLbh WT tumors were highly vascularized, disorganized, with mixed basal and luminal cell identity; tumors from Wnt1+;K14CreLbh KO mice were more organized, differentiated, and predominantly luminal Keratin 8 positive. We are currently investigating if the attenuated hyperplasia and tumor onset caused by LBH loss may be due to a deficiency in cancer stem cells in this breast cancer model. CONCLUSION: Our data indicate that LBH is an essential cell fate regulator downstream of WNT signaling that maintains basal lineage identity and the undifferentiated nature of BLBC. Thus, LBH inhibition may be a novel strategy to reduce the high mortality of BLBC patients through differentiation therapy. Citation Format: Kilan C. Ashad-Bishop, Karoline J. Briegel. LBH, a novel WNT effector in promoting basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1138.

Transcriptome Profiling Analysis Reveals the Potential Mechanisms of Three Bioactive Ingredients of Fufang E'jiao Jiang During Chemotherapy-Induced Myelosuppression in Mice.

Although multiple bioactive components have been identified in Fufang E’jiao Jiang (FEJ), their hematopoietic effects and molecular mode of action in vivo are still not fully understood. In the current study, we analyzed the effects of martynoside, R-notoginsenoside R2 (R2), and 20S-ginsenoside Rg2 (Rg2) in a 5-fluorouracil-induced myelosuppression mouse model. Bone marrow nucleated cells (BMNCs) counts, hematopoietic progenitor cell colony-forming unit (CFU) assay, as well as flow cytometry analysis of Lin-/c-kit+/Sca-1+ hematopoietic stem cell (HSC) population were conducted, and bone marrow cells were subjected to RNA sequencing. The transcriptome data were processed based on the differentially expressed genes. The results of the analysis show that each of the three compounds stimulates BMNCs and HSC growth, as well as burst-forming unit-erythroid and colony-forming unit granulocyte-monocyte colony expansion. The most relevant transcriptional changes appeared to be involved in regulation of hematopoietic cell lineage, NF-κB and TNF-α signaling, inhibition of inflammation, and acceleration of hematopoietic cell recovery. Notably, the individual compounds shared similar but specified transcriptome profiles. Taken together, the hematopoietic effects for the three tested compounds of FEJ are confirmed in this myelosuppression mouse model. The transcriptome maps of these effects provide valuable information concerning their underlying mechanisms and provide a framework for the continued study of the complex mode of action of FEJ.

Perspective: Biophysical regulation of cancerous and normal blood cell lineages in hematopoietic malignancies.

It is increasingly appreciated that physical forces play important roles in cancer biology, in terms of progression, invasiveness, and drug resistance. Clinical progress in treating hematological malignancy and in developing cancer immunotherapy highlights the role of the hematopoietic system as a key model in devising new therapeutic strategies against cancer. Understanding mechanobiology of the hematopoietic system in the context of cancer will thus yield valuable fundamental insights that can information about novel cancer therapeutics. In this perspective, biophysical insights related to blood cancer are defined and detailed. The interactions with immune cells relevant to immunotherapy against cancer are considered and expounded, followed by speculation of potential regulatory roles of mesenchymal stromal cells (MSCs) in this complex network. Finally, a perspective is presented as to how insights from these complex interactions between matrices, blood cancer cells, immune cells, and MSCs can be leveraged to influence and engineer the treatment of blood cancers in the clinic.

Differential surface phenotype and context-dependent reactivity of functionally diverse NKT cells

Abstract Natural Killer T (NKT) cells are a functionally diverse population that recognize lipid-based antigens in association with the antigen-presenting molecule CD1d. Here we define a technique to separate the functionally distinct NKT1, NKT2 and NKT17 cell subsets by their surface expression of CD279 (ICOS) and the activation-associated glycoform of CD43, enabling the investigation of subset-specific effector-functions. We report that all three subsets express the transcription factor GATA-3 and the potential to produce IL-4 and IL-10 following activation. This questions the notion that NKT2 cells are the predominant source of IL-4 within the NKT cell pool, and suggests that IL-10-production may be more indicative of NKT cell plasticity than the existence of a distinct regulatory lineage or subset. We also show that many NKT17 cells are CD4+ and are biased towards Vβ8.3 TCR gene usage. Lastly, we demonstrate that the toll-like receptor (TLR) ligand lipopolysaccharide (LPS) can induce a NKT17 cell-biased response, even in the absence of exogenous antigen, and that combining LPS with α-GalCer resulted in enhanced IL-17A-production, and reduced levels of the immunosuppressive cytokine IL-10. This study provides a novel means to examine the context-dependent reactivity of the functionally heterogeneous NKT cell population and provides important new insight into the functional biology of these subsets.

Differential surface phenotype and context-dependent reactivity of functionally diverse NKT cells.

Natural Killer T (NKT) cells are a functionally diverse population that recognizes lipid-based antigens in association with the antigen-presenting molecule CD1d. Here, we define a technique to separate the functionally distinct thymic NKT1, NKT2 and NKT17 cell subsets by their surface expression of CD278 (ICOS) and the activation-associated glycoform of CD43, enabling the investigation of subset-specific effector-functions. We report that all three subsets express the transcription factor GATA-3 and the potential to produce IL-4 and IL-10 following activation. This questions the notion that NKT2 cells are the predominant source of IL-4 within the NKT cell pool, and suggests that IL-10-production may be more indicative of NKT cell plasticity than the existence of a distinct regulatory lineage or subset. We also show that many NKT17 cells are CD4+ and are biased toward Vβ8.3 TCR gene usage. Lastly, we demonstrate that the toll-like receptor (TLR) ligand lipopolysaccharide (LPS) can induce a NKT17 cell-biased response, even in the absence of exogenous antigen, and that combining LPS with α-GalCer resulted in enhanced IL-17A-production, and reduced levels of the immunosuppressive cytokine IL-10. This study provides a novel means to examine the context-dependent reactivity of the functionally heterogeneous NKT cell population and provides important new insight into the functional biology of these subsets.