Loss Of Self-renewal Capacity Research Articles

MUC1-C integrates aerobic glycolysis with suppression of oxidative phosphorylation in triple-negative breast cancer stem cells

Activation of the MUC1-C protein promotes lineage plasticity, epigenetic reprogramming, and the cancer stem cell (CSC) state. The present studies performed on enriched populations of triple-negative breast cancer (TNBC) CSCs demonstrate that MUC1-C is essential for integrating activation of glycolytic pathway genes with self-renewal and tumorigenicity. MUC1-C further integrates the glycolytic pathway with suppression of mitochondrial DNA (mtDNA) genes encoding components of mitochondrial Complexes I-V. The repression of mtDNA genes is explained by MUC1-C-mediated (i)downregulation of the mitochondrial transcription factor A (TFAM) required for mtDNA transcription and (ii) induction of the mitochondrial transcription termination factor 3 (mTERF3). In support of pathogenesis that suppresses mitochondrial ROS production, targeting MUC1-C increases (i)mtDNA gene transcription, (ii) superoxide levels, and (iii) loss of self-renewal capacity. These findings and scRNA-seq analysis of CSC subpopulations indicate that MUC1-C regulates self-renewal and redox balance by integrating activation of glycolysis with suppression of oxidative phosphorylation.

ETV6 Represses TNF during Stress Hematopoiesis and Regulates HSC Self Renewal

INTRO: ETS Variant Transcription Factor 6 (ETV6), formerly known as Translocation ETS Leukemia (TEL), functions primarily as a transcriptional repressor and is highly expressed in mouse and human HSPCs where it is essential for the development and maintenance of adult hematopoiesis. Nevertheless, it has remained poorly understood how ETV6 regulates the HSC compartment. In 2015, our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies (most often B-acute lymphoblastic leukemia), a condition now known as thrombocytopenia 5 (T5). METHODS: To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis and promote leukemogenesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human T5-associated variant (R359X). We used complementary in vitro and in vivo assays to comprehensively characterize the numbers and functions of Etv6R355X/+ and Etv6+/+ hematopoietic progenitors. RESULTS:Etv6R355X/+ mice display several of the cardinal features of T5, including abnormally rounded platelets with increased area compared to platelets from wild-type Etv6+/+ littermates. Further, Etv6R355X/+ platelets exhibit significantly delayed and incomplete clot formation as measured through clot retraction assays. Together, these findings indicate that this novel model will serve as a viable means to study the human disease. We observe that 3-month-old Etv6R355X/+mice maintain normal frequencies and numbers of lineage-sca1+cKit+ (LSK) cells in the bone marrow (BM); however, at 12 months of age, they exhibit a significant decrease in frequency in this compartment. At each of these timepoints, there are variable shifts in the proportions and/or numbers of certain HSC subpopulations. To examine whether the Etv6R355X/+mutation impacts HSC function, we carried out in vitrosEtv6R355X/+BM cells generate more colonies than wild-type cells in the first two rounds of plating, but significantly fewer or no colonies by the fourth plating. Similarly, Etv6R355X/+LSK cells exhibit significantly impaired engraftment when challenged via competitive transplantation, with near complete failure to reconstitute irradiated recipients by the tertiary transplant. Etv6R355X/+LSKs home to the bone marrow comparably to Etv6+/+cells, indicating that homing does not account for the defect in repopulation capacity. To investigate how ETV6 functions in HSCs, we performed RNA-seq of Etv6R355X/+LSKs and flow cytometry-purified HSPC subpopulations from 12-month-old mice, which revealed upregulation of genes associated with an active stem cell phenotype, downregulation of genes supporting stem cell quiescence, and upregulated expression of Tnf with enrichment for the Hallmark TNFa Signaling via NFKB geneset. Subsequent experiments using CUT&RUN of Etv6+/+LSK cells revealed several known and novel ETV6 target genes, including Tnf, a cytokine implicated in regulating stress hematopoiesis. The sites bound by ETV6 at the Tnf locus aligned with ATAC-seq peaks generated from HPC5 cells, a mouse bone marrow progenitor cell line, suggesting that this is a region of open chromatin and active transcriptional regulation. Corroborating these findings, examination of Etv6R355X/+HSPCs at 6 weeks post-transplant revealed increased cell cycling and intracellular TNF production. CONCLUSION: From these studies, we conclude that ETV6 regulates HSC maintenance. Further, it binds to a regulatory sequence within the Tnf locus, whereby it represses TNF expression. The loss or reduction of this repression in Etv6R355X/+HSCs is associated with increased cell cycling under conditions of hematopoietic stress and a loss of self-renewal capacity. Studies are ongoing to determine how these changes promote leukemogenesis and contribute to the other features of T5.

Comprehensive Evaluation of Soil Substrate Improvement Based on the Minimum Data Set Method

Long-term transitional grazing on the northern slopes of the Tianshan Mountains in Xinjiang has led to severe vegetation degradation, loss of self-renewal capacity and regional ecological degradation in the region. This study was conducted to improve the soil quality and vegetation restoration efficiency in the foreland zone of the northern slope of the Tianshan Mountains (Xiangyataizi slope) using xanthic acid, bentonite, a green plant growth regulator (GGR) and high amounts of mulch as improvement materials, and eight sets of experiments were conducted. Fifteen physical and chemical indicators were selected as the total data set (TDS), and the minimum data set (MDS) was constructed using principal component analysis (PCA) combined with norm values to evaluate the soils in the study area by nonlinear (NL) and linear (L) evaluation methods. The results showed that the soil quality evaluation indexes of the MDS included effective phosphorus, organic matter, percentage of powder, total potassium and total salt for the Xiangyataizi slope of the Tianshan Mountains. The SQI was (p < 0.05). The Ⅵ treatment significantly improved soil quality; that is, plastic mulch applied to soil with 250 g of fulvic acid, 1000 g of bentonite and 15 g of GGR (mixed with 100 kg of water) was the best treatment. Additionally, since the nonlinear soil quality evaluation method (SQI-NL) had a smaller variation interval and coefficient of variation of the soil quality index compared with linear soil quality evaluation method (SQI-L), the coefficient of determination between the MDS and TDS was 0.873 and 0.811 under the SQI-NL and SQI-L evaluation methods, respectively. The nonlinear soil quality evaluation method had better applicability in this region, and the minimum data set was more accurate for soil quality evaluation.

1021 – SWI/SNF CHROMATIN REMODELING COMPLEXES GOVERN LYMPHOID LINEAGE COMMITMENT

Lymphocyte development from hemopoietic stem cells (HSCs) is accompanied by a loss of self-renewal capacity and a progressive restriction of developmental potential. Although key transcriptional pathways controlling lymphoid development have been identified, detailed molecular mechanisms explaining how multipotent HSCs initially commit to the lymphoid lineage are still lacking. Work from our laboratory indicates that combinatorial assembly of SWI/SNF chromatin remodeling complexes is a key epigenetic mechanism controlling cell fate decisions in the hemopoietic tissue. Single-cell RNA-seq analyses in bone marrow cells revealed that expression of the Smarcd1 subunit is enriched in lymphoid-primed multipotent progenitors. Using a conditional knock-out mouse model, we showed that Smarcd1 is specifically required for the development of the lymphoid lineage. Acute deletion of Smarcd1 in adult hematopoiesis causes lymphopenia with near complete absence of lymphoid-primed multipotent progenitors, indicating a role in lymphoid lineage commitment. Genome-wide studies revealed a functional collaboration between Smarcd1 and the bHLH transcription factor E2A in activating the lymphoid program of gene expression. Mechanistically, we show that Smarcd1 binds to and regulates chromatin accessibility of E2A at H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hemopoietic stem cells. Altogether, these studies identify Smarcd1 as a chromatin remodeler that collaborates with E2A to determine the lymphoid cell fate during hemopoiesis. Lymphocyte development from hemopoietic stem cells (HSCs) is accompanied by a loss of self-renewal capacity and a progressive restriction of developmental potential. Although key transcriptional pathways controlling lymphoid development have been identified, detailed molecular mechanisms explaining how multipotent HSCs initially commit to the lymphoid lineage are still lacking. Work from our laboratory indicates that combinatorial assembly of SWI/SNF chromatin remodeling complexes is a key epigenetic mechanism controlling cell fate decisions in the hemopoietic tissue. Single-cell RNA-seq analyses in bone marrow cells revealed that expression of the Smarcd1 subunit is enriched in lymphoid-primed multipotent progenitors. Using a conditional knock-out mouse model, we showed that Smarcd1 is specifically required for the development of the lymphoid lineage. Acute deletion of Smarcd1 in adult hematopoiesis causes lymphopenia with near complete absence of lymphoid-primed multipotent progenitors, indicating a role in lymphoid lineage commitment. Genome-wide studies revealed a functional collaboration between Smarcd1 and the bHLH transcription factor E2A in activating the lymphoid program of gene expression. Mechanistically, we show that Smarcd1 binds to and regulates chromatin accessibility of E2A at H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hemopoietic stem cells. Altogether, these studies identify Smarcd1 as a chromatin remodeler that collaborates with E2A to determine the lymphoid cell fate during hemopoiesis.

Integrated Single-Cell Bioinformatics Analysis Reveals Intrinsic and Extrinsic Biological Characteristics of Hematopoietic Stem Cell Aging.

Hematopoietic stem cell (HSC) aging, which is accompanied by loss of self-renewal capacity, myeloid-biased differentiation and increased risks of hematopoietic malignancies, is an important focus in stem cell research. However, the mechanisms underlying HSC aging have not been fully elucidated. In the present study, we integrated 3 independent single-cell transcriptome datasets of HSCs together and identified Stat3 and Ifngr1 as two markers of apoptosis-biased and inflammatory aged HSCs. Besides, common differentially expressed genes (DEGs) between young and aged HSCs were identified and further validated by quantitative RT-PCR. Functional enrichment analysis revealed that these DEGs were predominantly involved in the cell cycle and the tumor necrosis factor (TNF) signaling pathway. We further found that the Skp2-induced signaling pathway (Skp2→Cip1→CycA/CDK2→DP-1) contributed to a rapid transition through G1 phase in aged HSCs. In addition, analysis of the extrinsic alterations on HSC aging revealed the increased expression levels of inflammatory genes in bone marrow microenvironment. Colony formation unit assays showed that inflammatory cytokines promoted cellular senescence and that blockade of inflammatory pathway markedly rejuvenated aged HSC functions and increased B cell output. Collectively, our study elucidated the biological characteristics of HSC aging, and the genes and pathways we identified could be potential biomarkers and targets for the identification and rejuvenation of aged HSCs.

Inflammation, Aging and Hematopoiesis: A Complex Relationship.

All vertebrate blood cells descend from multipotent hematopoietic stem cells (HSCs), whose activity and differentiation depend on a complex and incompletely understood relationship with inflammatory signals. Although homeostatic levels of inflammatory signaling play an intricate role in HSC maintenance, activation, proliferation, and differentiation, acute or chronic exposure to inflammation can have deleterious effects on HSC function and self-renewal capacity, and bias their differentiation program. Increased levels of inflammatory signaling are observed during aging, affecting HSCs either directly or indirectly via the bone marrow niche and contributing to their loss of self-renewal capacity, diminished overall functionality, and myeloid differentiation skewing. These changes can have significant pathological consequences. Here, we provide an overview of the current literature on the complex interplay between HSCs and inflammatory signaling, and how this relationship contributes to age-related phenotypes. Understanding the mechanisms and outcomes of this interaction during different life stages will have significant implications in the modulation and restoration of the hematopoietic system in human disease, recovery from cancer and chemotherapeutic treatments, stem cell transplantation, and aging.

Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.

The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.

The Role of Lysophosphatidic Acid in Adult Stem Cells.

Stem cells are undifferentiated multipotent precursor cells that are capable both of perpetuating themselves as stem cells (self-renewal) and of undergoing differentiation into one or more specialized types of cells. And these stem cells have been reported to reside within distinct anatomic locations termed “niches”. The long-term goals of stem cell biology range from an understanding of cell-lineage determination and tissue organization to cellular therapeutics for degenerative diseases. Stem cells maintain tissue function throughout an organism’s lifespan by replacing differentiated cells. To perform this function, stem cells provide a unique combination of multilineage developmental potential and the capacity to undergo self-renewing divisions. The loss of self-renewal capacity in stem cells underlies certain degenerative diseases and the aging process. This self-renewal regulation must balance the regenerative needs of tissues that persist throughout life. Recent evidence suggests lysophosphatidic acid (LPA) signaling pathway plays an important role in the regulation of a variety of stem cells. In this review, we summarize the evidence linking between LPA and stem cell regulation. The LPA-induced signaling pathway regulates the proliferation and survival of stem cells and progenitors, and thus are likely to play a role in the maintenance of stem cell population in the body. This lipid mediator regulatory system can be a novel potential therapeutics for stem cell maintenance.

CD9 Marks Flt3+ Multipotent Hematopoietic Progenitors within Lsk Cells

Hematopoietic Stem Cells (HSCs) that give rise to all lineages of the blood are defined as a lineage-negative (Lin-), c-Kit+, Sca-1+(LSK) population in mouse. The expression of Flt3 on LSK cells is accompanied by loss of self-renewal capacity, and transition to lymphoid-primed multipotent progenitors (LMPPs). The magnitude of Flt3 surface abundance has been shown to be correlated with lineage potential, where megakaryocyte/erythroid (MegE) potential is incompatible with high Flt3 levels (Buza-Vidas et al. 2011). However, it is unclear whether MegE capacity is marked by Flt3 itself or restricted to a specific population within the Flt3 spectrum. Interestingly, it has been shown that commitment to MegE lineages is an early event in HSC differentiation and that MegE progenitors share several cell-surface markers with HSCs. One such marker is CD9, which is highly expressed on megakaryocyte progenitors (Nakorn, Miyamoto, and Weissman 2003)as well as on the most primitive stem cells in mouse (Karlsson et al. 2013). In fact, we have recently shown that CD9 captures all long-term reconstitution activity of the murine LSK population, even in cell fractions that lacks some of the most established HSC markers .To compare CD9- and Flt3 expression we performed FACS analysis of the LSK compartment and plotted CD9 high- and low cells on top of the CD34/Flt3 expression profile. Interestingly we found that even if CD9 expression were predominantly high in LT-(CD34-Flt3-) and ST-HSCs (CD34+Flt3-), CD9highcells reached all the way into the Flt3+population but were lost in LMPPs, as defined by the 25% highest Flt3 expressing cells. To addresswhether CD9highcells represent a distinct subpopulation within Flt3intcells, LSKCD34+Flt3int(Flt3int) and LSKCD34+FLT3intCD9high(CD9high) populations, were subjected to single-cell RNA-Sequencing analysis. Interestingly,our results demonstratethatCD9highcells are enriched in the most primitive cluster of the Flt3intpopulation. This was further validated in transplantation assays where only CD9highFlt3intcells reconstituted irradiated recipients. However, long-term reconstitution was limited to lymphoid lineages, while myeloid engraftment was only observed at early time points and self-renewal activity was absent. To address the clonal capacity of CD9highcells, we designed a protocol to assess the potential of single-cells to generate B-, Myeloid (My)- and Erythroid (Er) cells in vitro.In these conditions 68% of clones generated from CD9highcells were bipotent (55% My/B and 13% My/Er). In contrast, the majority of CD9lowcells were unipotent (32% My- and 52% B cells).Moreover,the colony forming capacity for the CD9highpopulation was significantly higher compared to CD9lowcells and when assayed for colony-forming unit-megakaryocyte (CFU-MK) capacity, Meg potential was exclusively observed in LT-HSCs, ST-HSCs as well as the CD9highFlt3intpopulation, while neither CD9lownor LMPPs generated any megakaryocyte colonies. In an attempt to improve the efficiency of MegE differentiation, single-cell clonogenic assays were performed in suspension cultures. Here,multipotency including MegE potential was gradually down regulated with increasing Flt3 cell surface expression, and decreasing CD9 expression. Together our results shows thatCD9 captures the most primitive cells within the LSKFlt3+population, including multipotent cells with MegE differentiation potential, thus resolving the heterogeneity of Flt3+hematopoietic progenitors and identifying a multipotent LSKCD9+Flt3+population distinct from LMPPs. Disclosures No relevant conflicts of interest to declare.

Abstract 4996: MLL3 haploinsufficiency preserves self-renewal capacity in HSCs with extensive cumulative division histories

Abstract KMT2C/MLL3 is one of the most commonly mutated tumor suppressor genes in human cancer. Haploinsufficiency at the KMT2C/MLL3 locus often occurs in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as a result of mono-allelic 7q deletions. Mll3 haploinsufficiency has been shown to accelerate AML in mice, but it is not clear how it regulates normal hematopoietic progenitors or why it is required to suppress leukemogenesis. We used a novel loss-of-function mouse allele to characterize hematopoiesis in Mll3-deficient fetal livers and adult bone marrow. Mll3 deletion caused a modest increase in fetal liver hematopoietic stem cells (HSCs) and a modest decrease in lineage restricted progenitors (HPCs). In contrast, Mll3 heterozygosity caused significant expansion of the HSC population in adult bone marrow without affecting more committed progenitor populations. Mll3+/- and Mll3-/- HSCs were able to repopulate lethally irradiated mice, and repopulating activity was enhanced in secondary and tertiary transplants relative to wild type HSCs. Thus, Mll3 haploinsufficiency enhances HSC self-renewal capacity. We next tested whether Mll3 regulates the balance between self-renewal and commitment in non-transplanted HSCs. We used H2B-GFP pulse-chase assays to assess the division histories of HSCs over time. After a 6-week chase, wild type HSCs retained high levels of H2B-GFP expression, consistent with prior studies showing that adult HSCs divide infrequently. In contrast, almost all Mll3+/- HSCs had low to moderate H2B-GFP expression levels, indicative of an extensive division history. This difference was not associated with an increase in the percentage of cycling (S/G2/M) HSCs, and it was not associated with a loss of self-renewal capacity that occurs with cumulative divisions in wild type HSCs. Thus, Mll3 haploinsufficiency allows cumulative HSC divisions to accrue without compromising self-renewal. Mll3 encodes a histone methyltransferase that binds active enhancers and promotes transcription. This raised the question of whether Mll3 deletion results in a loss of commitment-related gene expression, increased self-renewal gene expression or altered epigenetic landscapes. To our surprise, gene expression was indistinguishable between wild type and Mll3+/- HSCs. However, ATAC-seq analysis revealed patterns of nucleosome depletion in Mll3+/- HSCs that closely resembled wild type HPCs. No differences were observed in the ATAC-seq profiles of wild type and Mll3+/- HPCs. These observations suggest that as Mll3+/- HSCs self-renew, they remodel their epigenetic landscapes to resemble committed progenitors, yet they do not activate commitment related genes, and they do exit the stem cell pool. The data suggest that stress conditions that drive HSCs into cycle, such as the chemotherapy regimens that often precede 7q- MDS, may select for Mll3 haploinsufficient HSC clones. Citation Format: Theresa Owuor, Shaina Porter, Cluster Andrew, Riddhi Patel, Jeffrey Magee. MLL3 haploinsufficiency preserves self-renewal capacity in HSCs with extensive cumulative division histories [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 4996.

Roles of autophagy in controlling stem cell identity: a perspective of self-renewal and differentiation.

Autophagy is crucial for the removal of dysfunctional organelles and protein aggregates and for maintaining stem cell homeostasis, which includes self-renewal, cell differentiation and somatic reprogramming. Loss of self-renewal capacity and pluripotency is a major obstacle to stem cell-based therapies. It has been reported that autophagy regulates stem cells under biological stimuli, starvation, hypoxia, generation of reactive oxygen species (ROS) and cellular senescence. On the one hand, autophagy is shown to play roles in self-renewal by co-function with the ubiquitin-proteasome system (UPS) to promote pluripotency-associated proteins (NANOG, OCT4 and SOX2) in human embryonic stem cells (hESCs). On the other hand, autophagy activity acts as cell reprogramming processes that play an important role for clearance fate determination and upregulates neural and cardiac differentiation. Deregulation of autophagy triggers protein disorders such as neurodegenerative cardiac/muscle diseases and cancer. Therefore, understanding of the roles of the autophagy in stem cell renewal and differentiation may benefit therapeutic development for a range of human diseases.

Abstract PR14: HIF-2alpha regulates self-renewal of MYC dependent cancer stem cells

Abstract Numerous studies report that both hematopoietic and some solid tumors are organized in a hierarchical manner where tumor growth is driven by a small subset of cancer stem cells (CSCs). However, the distinct molecular identity of CSC versus non-CSC is not yet clearly known, which limit our understanding on how CSC self-renewal pathways operate to drive tumorigenicity. Here, we report that MYC and HIF-2alpha co-operate to maintain distinct identity and self-renewal capacity of CSCs. In a transgenic mouse model of conditional MYC driven thymic lymphoma, we identified a Sca-1+ cell population that showed tumor stemness (self-renewal and undifferentiation state) phenotype, including high expression of Nanog, Sox2 and HIF-2alpha. When injected to congenic mice, Sca-1+ cells but not Sca-1- cells exhibited hierarchical organization including self-renewal capacity. ChIP assay revealed direct MYC binding of HIF-2alpha promoter region in Sca-1+ but not in the Sca-1- cells. Inhibition of HIF-2alpha led to increase ROS generation, decrease of GSH synthesis, differentiation, and loss of self-renewal capacity of Sca-1+ cells. In contrast, inhibition of HIF-2alpha had no phenotypic effect on Sca-1- cells. Thus, we have identified a distinct CSC population in MYC dependent cancer cells, where MYC preferentially binds to HIF-2alpha to maintain self-renewal capacity. Citation Format: Bikul Das, Bikul Das, Hong Li, Hong Li, Rashmi Bhuyan, Dean W. Felsher. HIF-2alpha regulates self-renewal of MYC dependent cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR14.

Cardioprotection by Targeting the Pool of Resident and Extracardiac Progenitors.

The adult heart has the capacity to generate new myocytes that are markedly enhanced in acute and chronic heart failure of ischemic and non-ischemic origin. In addition, a pool of blood trafficking progenitor cells able to sense myocardial damage may home to the sites of injury participating to cardiac repair. This new view of myocardial biology leads to an expanding long-term research and therapeutic goals for cardioprotection. A fundamental concept to be analyzed is whether cardiac diseases are influenced by changes in the properties of tissue specific and circulating progenitors. Loss of self-renewal capacity, impaired growth or increased susceptibility to death may lead to a reduction of progenitors and leave myocardial damage unrepaired. Cardiac progenitors generate all myocardial cell lineages, thus impairment in their growth is expected to be critically involved in the structural and functional modifications of the heart. The fact that, in addition to well known effects of anthracyclines, also new drugs that target molecular pathways implicated in cell death and growth can be cardiotoxic further supports our hypothesis. Understanding the role of resident and extracardiac progenitors in the pathogenesis of cardiomyopathies of different etiology will provide not only a better comprehension of cardiac homeostasis but will also open new avenues for therapeutic interventions. The progress toward effective myocardial regeneration based on exploiting the self-renewal potential of the myocardium and the systemic pool of cardiogenic cells should advance the likelihood of efficient cardioprotection and restoration of cardiac function.

Abstract 993: The relationship between autophagy, senescence, and DNA repair

Abstract Radiotherapy is used along with other modalities such as surgery, chemotherapy, and immunotherapy to either shrink tumors before surgery or kill surviving tumor cells post surgery. Although radiation (and chemotherapy) induce different modes of cell death, recurrence of cancer may occur. It has been shown that DNA damage can induce cells to undergo both autophagy and senescence where both responses can function either as a pro-survival mechanism or in promoting loss of self-renewal capacity in the tumor cell. The relationship between autophagy, senescence, and the DNA repair system has not been well-characterized. The primary aims of this work were to understand whether autophagy and senescence might be permissive for DNA repair, thereby allowing the tumor cells to overcome the persistent DNA damage and recover using HCT116 WT and HCT116 Ligase IV (-/-) human colon carcinoma cells. Irradiation caused HCT116 Ligase IV (-/-) cells to lose their capacity to proliferate followed by delayed cell death, whereas HCT116 WT cells showed proliferative recovery 72 hours post-treatment, consistent with deficiencies in DNA repair in the Ligase IV (-/-) cells. Irradiation of both cell lines resulted in enlargement in cell size and accumulation of acidic vacuoles, indicative of the induction of autophagy. Inhibition of autophagy by Bafilomycin and Choroquine resulted in radio-sensitization in HCT116 WT cells but not in HCT116 Ligase IV (-/-) cells, indicating that radiation-induced autophagy in HCT116 WT cells is likely to be cytoprotective. Both repair competent HCT116 colon carcinoma cells and Ligase IV deficient cells demonstrated significant increases in senescent populations upon irradiation based on β-galactosidase staining and enlargement of treated cells. The PARP inhibitor, Olaparib, enhanced the efficacy of radiation through interference with DNA repair and the promotion of both autophagy and senescence but not apoptosis. Taken together, it appears that both autophagy and senescence may facilitate DNA repair during exposure to genotoxic stress, and that autophagy and senescence are closely linked responses in irradiated tumor cells. Citation Format: Moureq R. Alotaibi, Lawrence Povirk, Daivd Gewirtz. The relationship between autophagy, senescence, and DNA repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2015-993

Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures.

Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.

KLF4 Regulates Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Repressing Gbl Expression and Altering mTORC2 Activity

Abstract Tyrosine kinase inhibitors (TKIs) are the standard treatment for eradicating BCR-ABL-positive progenitor cells in chronic myeloid leukemia (CML); however, disease often relapses upon drug discontinuation because TKIs do not effectively eliminate leukemic stem cells (LSC). The development of novel strategies aimed at eradicating LSC without harming normal hematopoietic stem cells (HSC) is essential for the cure of CML patients. The generation of LSC-directed therapy relies on the identification of novel molecular pathways that selectively regulate LSC function independent of BCR-ABL. The Krüppel-like factor 4(KLF4) is a transcription factor that can either activate or repress gene transcription acting as an oncogene or a tumor suppressor depending on the cellular context. Analysis of a published dataset from chronic phase CML patients revealed elevated levels of KLF4 in LSC compared to progenitor cells indicating that KLF4 is likely implicated in LSC regulation. To study the role of KLF4 in LSC function, we used a CML mouse model combining somatic deletion of the Klf4 gene and retroviral transduction and transplantation of HSC. In contrast to mice receiving BCR-ABL-transduced Klf4fl/fl HSC that developed and succumbed to CML, mice transplanted with BCR-ABL-transduced Klf4Δ/Δ (Klf4fl/fl Vav-iCre+) HSC showed a progressive loss of leukemia despite an initial expansion of myeloid leukemic cells, which led to increased overall survival. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSC in bone marrow and the spleen. Furthermore, deletion of KLF4 impaired the ability of LSC to recapitulate leukemia in secondary recipients suggesting a loss of self-renewal capacity. In contrast to LSC, KLF4 deletion led to increased self-renewal of normal HSC assessed by serial competitive transplantation. To identify KLF4 target genes involved in LSC self-renewal, we performed a global gene expression analysis using Klf4Δ/Δ LSC purified by cell sorting from leukemic mice. Analysis of gene expression in Klf4Δ/Δ LSC revealed significant upregulation of GβL, a component of mTOR complexes. Finally, we identified that KLF4 binds to GβL promoter by Chip-Seq analysis and that silencing resulted in inhibition of mTORC2 but not mTORC1 activity in 32D-BCR-ABL-positive CML cells. Our findings suggest that KLF4 transcriptionally represses GβL expression in LSC and that mTORC2 inhibition has the potential to completely eradicate LSC and induce treatment-free remission. Disclosures No relevant conflicts of interest to declare.

The Linker Protein GAS7 Negatively Regulates Pre-B Cell Differentiation and Amplifies Proliferation and Survival Signals in Acute Lymphoblastic Leukemia

Background:Growth arrest-specific gene 7 (Gas7) functions as an adaptor for SH2- and SH3-containing proteins, in particular in cells that undergo growth arrest. Gas7 is abundantly expressed in the brain and is involved in neuronal differentiation. Interestingly, MLL-GAS7 fusion molecules resulting from the t(11;17)(q23;p13) chromosomal translocation have been reported in treatment-related acute myeloid leukemia (AML; Megonigal et al., 2000) and in a pediatric acute lymphoblastic leukemia (ALL). While the function of MLL has been extensively studied, the role of its fusion partner GAS7 in normal hematopoiesis and leukemia has not been elucidated.Results: Studying gene expression changes during normal B cell development, we identified Gas7 as the gene with the strongest relative increase at the pre-B cell receptor checkpoint. At the transition from IL7-dependent Fraction C’ to IL7-independent small resting pre-B cells (Fraction D), GAS7 mRNA levels were upregulated by >13-fold in both human and mouse B cell progenitors. Withdrawal of IL7 cytokine signaling and Cre-mediated conditional deletion of Stat5ab recapitulated the strong increase of GAS7 expression under cell culture conditions. These finding suggest that GAS7 is part of an adaptive response of differentiating pre-B cells to attenuation of cytokine/Stat5 signaling. Consistent with this scenario, we found that Gas7-/-pre-B cells undergo accelerated differentiation, including spontaneous Ig κ light chain gene recombination and loss of Stat5-signaling. Conversely, overexpression of GAS7, reduced responsiveness of pre-B cells to normal differentiation stimuli. These findings suggest that the linker molecule GAS7 is a negative regulator of pre-B cell differentiation.Likewise, we found that tyrosine kinase inhibitor treatment of human Ph+ ALL cells resulted in a strong increased of GAS7 expression, in parallel with loss of Stat5 function. To elucidate the function of Gas7 in B cell lineage leukemia, we transformed bone marrow pre-B cells from Gas7-/- mice with BCR-ABL1. Gas7 deficient Ph+ ALL cells showed decreased proliferation with reduced S phase and increased apoptosis. In agreement with effects of Stat5 on the sensitivity of Ph+ ALL cells against tyrosine kinase inhibitors (TKIs), Gas7 deficient Ph+ ALL cells showed massively increased susceptibility to Imatinib-induced apoptosis. In addition, absence of Gas7 caused loss of self-renewal capacity and failure to form colonies in methylcellulose assay. Co-immunoprecipitation experiments with flag tagged GAS7 in patient-derived Ph+ALL cells revealed that GAS7 physically interacts with STAT5 and retains STAT5-Y694 in an active conformation.Thereby, GAS7 can propagate even weak Stat5 activity and maintain residual cytokine or BCR-ABL1 oncogenic signaling in normal and malignant pre-B cells.Conclusions: Here show that GAS7 functions as an important positive regulator of Stat5 downstream of cytokine receptors in normal pre-B cells and downstream of BCR-ABL1 and other oncogenes in leukemia. Owing to the GAS7-dependent reinforcement of Stat5-dependent survival and proliferation signaling, normal and leukemic pre-B cells can survive periods of reduced cytokine/oncogene signaling. These findings suggest that the interaction interface between GAS7 and Stat5 represents a potential target for small molecule scaffolds and peptides. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2385: Involvement of autophagy and senescence in DNA repair in irradiated tumor cells

Abstract Radiotherapy is used along with other modalities such as surgery, chemotherapy, and immunotherapy to either shrink tumors before surgery or kill surviving tumor cells post surgery. Although radiation may induce different modes of cell death, recurrence of cancer may occur. It has been shown that DNA damage can induce cells to undergo both autophagy and senescence where both responses can function either as a pro-survival mechanism or in promoting loss of self-renewal capacity in the tumor cell. The relationship between autophagy, senescence, and the DNA repair system has not been well-characterized. The primary aims of this work were to understand whether autophagy and senescence might be permissive for DNA repair thereby allowing the tumor cells to overcome the persistent DNA damage and recover. Studies were performed using M059K (DNA-PK proficient) and M059J (DNA-PK deficient) human glioma cells to assess the kinetics of DNA repair after radiation in the absence and presence of autophagy inhibitors. M059J cells were observed to lose their capacity to proliferate as they undergo growth arrest followed by delayed cell death, whereas M059K cells showed proliferative recovery 72 hours post-treatment. Both irradiated cells showed an enlargement in cell size and accumulation of acidic vacuoles, indicating the induction of autophagy. Both cell lines also demonstrated reduced p62 level after radiation, indicative of autophagic flux. Inhibition of autophagy by 3-MA showed a radio-sensitization in M059K cells but not in M059J cells, indicating that radio-induced autophagy in M059K cells might be cytoprotective. Additional studies were performed in HCT116 cells that lack ligase IV to demonstrate a correspondence between the extent of drug or radiation induced senescence and the persistence of DNA damage foci. Ligase IV deficient cells were more sensitive to low radiation doses (2 Gy), whereas the ligase proficient cells showed recovery 96 hrs post-treatment. Both parental HCT116 colon carcinoma cells and Ligase IV deficient cells demonstrated significant increases in senescent populations at low doses of radiation based on β-galactosidase staining and enlargement and extensions of treated cells; senescence was more pronounced in the Ligase IV deficient cells. HCT116 cells were then treated with a dose of radiation (8 Gy) that induces ∼75% of senescence after 96 hrs and then reirradiated with half of that dose (4 Gy) to monitor the ability of senescent cells to repair the DNA damage. Although the majority of the cells were in a state of senescence, we found that these cells were able to repair the DNA damage induced by the second dose.Taken together, it seems that autophagy has an important function in the enhancement of DNA repair during exposure to genotoxic stress, but the role of autophagy may differ according to the status of DNA repair. Also, persistent DNA damage could be a determinant for senescence, and senescent cells might be able to repair these breaks. Citation Format: Moureq R. Alotaibi, Daivd Gewirtz, Lawrence Povirk. Involvement of autophagy and senescence in DNA repair in irradiated tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2014-2385

THOR METHYLATION PROVIDES INSIGHT INTO THE TELOMERE MAINTENANCE LANDSCAPE OF MALIGNANT GLIOMAS

BACKGROUND: Gliomas are a deadly group of childhood and adult cancers associated with high relapse rate following current therapies. Limitless self-renewal is a hallmark of cancer recurrence and is controlled by telomerase activation and telomere maintenance. We have recently uncovered THOR (TERT Hypermethylated Oncological Region) which is paradoxically hypermethylated in gliomas with telomerase activation. In order to further explore the biological impact of THOR hypermethylation on self renewal and telomere maintenance of gliomas we undertook a stepwise approach. METHODS: RESULTS: Mapping of the human TERT promoter reveals that THOR spans 432 BP and comprises 52 CG sites. In contrast, the area where mutations in TERT promoter were uncovered is permanently hypomethylated. Promoter driven expression was analysed through luciferase assays and unveiled a repressive effect of THOR on the TERT promoter. Moreover, TERT mutations promoted hyperactivation of the reporter gene providing explanations for the methylation pattern observed in malignant gliomas. THOR methylation increases in gliomas as they transform from low to high grade and from primary tumor to established cell lines (p < 0.001). Analysis of allelic Tert expression reveals that THOR is initally methylated in the mutant allele and throughout tumor progression, the other allele eventually becomes methylated. This correlates with higher TERT expression. In contrast, tumors utilizing alternative lengthening of telomeres (ALT) lack THOR methylation and TERT mutations. Glioma stem cells (n = 32) are addicted to telomerase and have higher THOR methylation than the bulk tumor. Strikingly, glioblastomas which activate ALT lack this phenotype in their stem cells compartment. Whole exome sequencing reveals multiple ALT related mutations (TP53 and ATRX) which are present in the mature tumor cells subpopulation and absent in the TERT expressing stem cell subpopulation. THOR demethylation with Decitabine results in loss of telomerase activation only in tumor cells and not in normal and embryonic stem cells which lack THOR methylation. Combining telomerase inhibition with decitabine result in permanent loss of self renewal capacity of patient derived cell lines in vitro and lack of tumor formation in vivo. CONCLUSIONS: We offer a model and a novel classification of gliomas based on THOR hypermethylation and alterations in the telomere maintenance pathway. We also suggest that intratumoral heterogeneity in telomere maintenance might be the result of secondary hits in the non-stem cell compartment Finally, we propose that THOR demethylation can be a safe and viable option for exhausting the self renewal capacity of gliomas. SECONDARY CATEGORY: Pediatrics.

Noncanonical NOTCH Signaling Limits Self-Renewal of Human Epithelial and Induced Pluripotent Stem Cells through ROCK Activation

NOTCH plays essential roles in cell fate specification during embryonic development and in adult tissue maintenance. In keratinocytes, it is a key inducer of differentiation. ROCK, an effector of the small GTPase Rho, is also implicated in keratinocyte differentiation, and its inhibition efficiently potentiates immortalization of human keratinocytes and greatly improves survival of dissociated human pluripotent stem cells. However, the molecular basis for ROCK activation is not fully established in these contexts. Here we provide evidence that intracellular forms of NOTCH1 trigger the immediate activation of ROCK1 independent of its transcriptional activity, promoting differentiation and resulting in decreased clonogenicity of normal human keratinocytes. Knockdown of NOTCH1 abrogated ROCK1 activation and conferred sustained clonogenicity upon differentiation stimuli. Treatment with a ROCK inhibitor, Y-27632, or ROCK1 silencing substantially rescued the growth defect induced by activated NOTCH1. Furthermore, we revealed that impaired self-renewal of human induced pluripotent stem cells upon dissociation is, at least in part, attributable to NOTCH-dependent ROCK activation. Thus, the present study unveils a novel NOTCH-ROCK pathway critical for cellular differentiation and loss of self-renewal capacity in a subset of immature cells.