Higher-Level Pathway Objectives of Epigenetic Therapy: A Solution to the p53 Problem in Cancer

Searches for effective yet nontoxic oncotherapies are searches for exploitable differences between cancer and normal cells. In its core of cell division, cancer resembles normal life, coordinated by the master transcription factor MYC. Outside of this core, apoptosis and differentiation programs, which dominantly antagonize MYC to terminate cell division, necessarily differ between cancer and normal cells, as apoptosis is suppressed by biallelic inactivation of the master regulator of apoptosis, p53, or its cofactor p16/CDKN2A in approximately 80% of cancers. These genetic alterations impact therapy: conventional oncotherapy applies stress upstream of p53 to upregulate it and causes apoptosis (cytotoxicity)—a toxic, futile intent when it is absent or nonfunctional. Differentiation, on the other hand, cannot be completely suppressed because it is a continuum along which all cells exist. Neoplastic evolution stalls advances along this continuum at its most proliferative points—in lineage-committed progenitors that have division times measured in hours compared with weeks for tissue stem cells. This differentiation arrest is by mutations/deletions in differentiation-driving transcription factors or their coactivators that shift balances of gene-regulating protein complexes toward corepressors that repress instead of activate hundreds of terminal differentiation genes. That is, malignant proliferation without differentiation, also referred to as cancer “stem” cell self-renewal, hinges on druggable corepressors. Inhibiting these corepressors (e.g., DNMT1) releases p53-independent terminal differentiation in cancer stem cells but preserves self-renewal of normal stem cells that express stem cell transcription factors. Thus, epigenetic-differentiation therapies exploit a fundamental distinction between cancer and normal stem cell self-renewal and have a pathway of action downstream of genetic defects in cancer, affording favorable therapeutic indices needed for clinical progress.

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Essential Role of Jun Family Transcription Factors in PU.1-Induced Leukemic Stem Cell Transformation.

Blood ◽

10.1182/blood.v106.11.463.463 ◽

2005 ◽

Vol 106 (11) ◽

pp. 463-463 ◽

Cited By ~ 4

Author(s):

Ulrich Steidl ◽

Frank Rosenbauer ◽

Roel G.W Verhaak ◽

Xuesong Gu ◽

Hasan H. Otu ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Transcription Factors ◽

Malignant Transformation ◽

Molecular Mechanisms ◽

Target Cells ◽

Leukemic Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Transcriptional Pattern

Abstract Knockdown of the expression of the myeloid master regulator PU.1 leads to the development of an immature acute myeloid leukemia (AML) in mice. Recent reports suggest that functional inactivation of PU.1 might also play a role in human AML. However, the molecular mechanisms underlying PU.1-mediated malignant transformation are unknown. We examined leukemic PU.1 knockdown mice and found a 3-fold expansion of lin-, c-kit+, Sca1+ (KLS) hematopoietic stem cells (HSC) as compared to wildtype controls, which was not observed during the preleukemic phase. When we transplanted double-sorted leukemic KLS-HSC into NOD-SCID mice the recipients developed AML after 9–12 weeks indicating that the leukemic stem cells derive from the HSC compartment. This finding prompted us to examine the transcriptome of PU.1 knockdown preleukemic HSC to identify early transcriptional changes underlying their malignant transformation. After lineage-depletion and FACS sorting of preleukemic KLS-HSC we performed linear amplification of RNA by 2 cycles of RT-IVT and hybridized the cRNA with Affymetrix Mouse Genome 430 2.0 arrays. Principal component analysis as well as hierarchical cluster analysis clearly distinguished PU.1 knockdown and wildtype HSC. Several in-vitro targets of PU.1 such as c-Fes, BTK, TFEC, CSF2R, and Ebi3 were downregulated demonstrating that those are also affected in HSC in vivo. Differential expression of 16 genes was corroborated by qRT-PCR. Strikingly, several Jun family transcription factors including c-Jun and JunB were downregulated. Retroviral restoration of c-Jun expression in bone marrow cells of preleukemic mice rescued the PU.1-initiated myelomonocytic differentiation block in this early phase. To target cells in the leukemic stage we applied lentiviral vectors expressing c-Jun or JunB. While c-Jun did not affect leukemic proliferation, lentiviral restoration of JunB led to an 80% reduction of clonogenic growth and a loss of leukemic self-renewal capacity in serial replating assays. Expression analysis of 285 patients with AML confirmed the correlation between PU.1 and JunB downregulation and suggests its relevance in human disease. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1 knockdown HSC and demonstrate that downregulation of c-Jun and JunB contribute to the development of PU.1-induced AML by blocking differentiation (c-Jun) and increasing self-renewal (JunB). Therefore, examination of disturbed gene expression in preleukemic HSC can identify genes whose dysregulation is essential for leukemic stem cell function and are potential targets for therapeutic interventions.

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Mathematical modeling of plant cell fate transitions controlled by hormonal signals

10.1101/830190 ◽

2019 ◽

Author(s):

Filip Z. Klawe ◽

Thomas Stiehl ◽

Peter Bastian ◽

Christophe Gaillochet ◽

Jan U. Lohmann ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

Transcription Factors ◽

Cell Division ◽

Cell Fate ◽

Plant Architecture ◽

Two Dimensional ◽

Cell Dynamics ◽

Non Linear

AbstractCoordination of fate transition and cell division is crucial to maintain the plant architecture and to achieve efficient production of plant organs. In this paper, we analysed the stem cell dynamics at the shoot apical meristem (SAM) that is one of the plant stem cells locations. We designed a mathematical model to elucidate the impact of hormonal signaling on the fate transition rates between different zones corresponding to slowly dividing stem cells and fast dividing transit amplifying cells. The model is based on a simplified two-dimensional disc geometry of the SAM and accounts for a continuous displacement towards the periphery of cells produced in the central zone. Coupling growth and hormonal signaling results in a non-linear system of reaction-diffusion equations on a growing domain with the growth velocity depending on the model components. The model is tested by simulating perturbations in the level of key transcription factors that maintain SAM homeostasis. The model provides new insights on how the transcription factor HECATE is integrated in the regulatory network that governs stem cell differentiation.SummaryPlants continuously generate new organs such as leaves, roots and flowers. This process is driven by stem cells which are located in specialized regions, so-called meristems. Dividing stem cells give rise to offspring that, during a process referred to as cell fate transition, become more specialized and give rise to organs. Plant architecture and crop yield crucially depend on the regulation of meristem dynamics. To better understand this regulation, we develop a computational model of the shoot meristem. The model describes the meristem as a two-dimensional disk that can grow and shrink over time, depending on the concentrations of the signalling factors in its interior. This allows studying how the non-linear interaction of multiple transcription factors is linked to cell division and fate-transition. We test the model by simulating perturbations of meristem signals and comparing them to experimental data. The model allows simulating different hypotheses about signal effects. Based on the model we study the specific role of the transcription factor HECATE and provide new insights in its action on cell dynamics and in its interrelation with other known transcription factors in the meristem.

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STEM-14. THE WRAD COMPLEX REPRESENTS A THERAPEUTIC TARGET FOR CANCER STEM CELLS IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.088 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi23-vi24

Author(s):

Kelly Mitchell ◽

Joseph Alvarado ◽

Christopher Goins ◽

Steven Martinez ◽

Jonathan Macdonald ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Transcription Factors ◽

Cancer Stem Cells ◽

Molecular Mechanisms ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Self Renewal ◽

Stem Cell Maintenance ◽

Cell Maintenance

Abstract Glioblastoma (GBM) progression and resistance to conventional therapies is driven in part by cells within the tumor with stem cell properties including quiescence, self-renewal and drug efflux potential. It is thought that eliminating these cancer stem cells (CSCs) is a key component to successful clinical management of GBM. However, currently, few known molecular mechanisms driving CSCs can be exploited for therapeutic development. Core transcription factors such as SOX2, OLIG2, OCT4 and NANOG maintain the CSC state in GBM. Our laboratory recently uncovered a self-renewal signaling axis involving RBBP5 that is necessary and sufficient for CSC maintenance through driving expression of these core stem cell maintenance transcription factors. RBBP5 is a component of the WRAD complex, which promotes Lys4 methylation of histone H3 to positively regulate transcription. We hypothesized that targeting RBBP5 could be a means to disrupt epigenetic programs that maintain CSCs in stemness transcriptional states. We found that genetic and pharmacologic inhibition of the WRAD complex reduced CSC growth, self-renewal and tumor initiation potential. WRAD inhibitors partially dissembled the WRAD complex and reduced H3K4 trimethylation both globally and at the promoters of key stem cell maintenance transcription factors. Using a CSC reporter system, we demonstrated that WRAD complex inhibition decreased growth of SOX2/OCT4 expressing CSCs in a concentration-dependent manner as quantified by live imaging. Overall, our studies assess the function of the WRAD complex and the effect of WRAD complex inhibitors in preclinical models and specifically on the stem cell state for the first time in GBM. Studying the functions of the WRAD complex in CSCs may improve understanding of GBM pathogenesis and elucidate how CSCs survive despite aggressive chemotherapy and radiation. Our ongoing studies aim to develop brain penetrant inhibitors targeting the WRAD complex as an anti-CSC strategy that could potentially synergize with standard of care treatments.

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Purine Metabolism and Hematopoietic Stem and Progenitor Stem Cells Under Stress

Blood ◽

10.1182/blood-2018-99-109515 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-19-SCI-19

Author(s):

Toshio Suda

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Cell Division ◽

Cellular Metabolism ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Mitochondria Pathway ◽

Quiescent Hscs

Abstract Hematopoietic stem cells (HSCs) play a key role in the lifelong maintenance of hematopoiesis through self-renewal and multi-lineage differentiation. Adult HSCs reside within a specialized microenvironment of the bone marrow (BM), called "niche", in which they are maintained in a quiescent state in cell cycle. Most of HSCs within BM show quiescence under the hypoxic niche. Since the loss of HSC quiescence leads to the exhaustion or aging of stem cells through excess cell division, the regulation of quiescence in HSCs is essential for hematopoietic homeostasis. On the other hand, cellular metabolism has been suggested to play a critical role in many biological processes including the regulation of stem cell properties and functions. However, the metabolic condition and adaptation of stem cells remain largely unaddressed. First, we have analyzed HSC metabolism using metabolomics approaches. With step-wise differentiation of stem cells, the cell metabolism associated with each differentiation stage may be different. A feature of quiescent HSCs is their low baseline energy production; quiescent HSCs rely on glycolysis and exhibit low mitochondrial membrane potential (ΔΨm). Likewise, HSCs with a low ΔΨm show higher reconstitution activity in BM hematopoiesis, compared to cells with high ΔΨm. By contrast, upon stress hematopoiesis, HSCs actively divide and proliferate. However, the underlying mechanism for the initiation of HSC division still remains unclear. In order to elucidate the mechanism underlying the transition of cell cycle state in HSCs, we analyzed the change of mitochondria activity in HSCs after BM suppression induced by 5-fluoruracil (5-FU). Upon 5-FU treatment, cycling progenitors are depleted and then quiescent HSCs start to divide. We found that HSCs initiate cell division after exhibiting enhanced ΔΨm, as a result of increased intracellular Ca2+ level. We hypothesize that extracellular adenosine, derived from hematopoietic progenitors, inhibits the calcium influx and mitochondrial metabolism. While further activation of Ca2+-mitochondria pathway led to loss of the stem cell function after cell division, the appropriate suppression of intracellular Ca2+ level by nifedipine, a blocker of L-type voltage-gated Ca2+ channels, prolonged cell division interval in HSCs, and simultaneously achieved both cell division and HSC maintenance (self-renewal division). Thus, our results indicate that the adenosine-Ca2+-mitochondria pathway induces HSC division critically to determine HSC cell fate. Next, to examine the mitochondria oxidative metabolism and purinergic pathways, we introduced the study on a tumor suppressor, Folliculin (FLCN). Conditional deletion of FLCN in HSC compartment using the Mx1-Cre or Vav-iCre system disrupted HSC quiescence and BM homeostasis dependently on the lysosomal stress response induced by TFE3. Together all, we propose that the change in cellular metabolism involving mitochondria is crucial for HSC homeostasis in the stress settings. Disclosures No relevant conflicts of interest to declare.

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The abrogation of condensin function provides independent evidence for defining the self-renewing population of pluripotent stem cells

10.1101/143339 ◽

2017 ◽

Cited By ~ 1

Author(s):

Alvina G. Lai ◽

Nobuyoshi Kosaka ◽

Prasad Abnave ◽

Sounak Sahu ◽

A. Aziz Aboobaker

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Division ◽

Cell Function ◽

Giant Cells ◽

The Self ◽

Stem Cell Population ◽

Self Renewal ◽

Independent Evidence

AbstractHeterogeneity of planarian neoblast stem cells has been categorised on the basis of single cell expression analyses and subsequent experiments to demonstrate lineage relationships. Some data suggest that despite gene expression heterogeneity amongst cells in the cell cycle, in fact only one sub-population, known as sigma neoblasts, can self-renew. Without the tools to perform live in vivo lineage analysis, we instead took an alternative approach to provide independent evidence for defining the self-renewing stem cell population. We exploited the role of highly conserved condensin proteins to functionally assay neoblast self-renewal properties. Condensins are involved in forming properly condensed chromosomes to allow cell division to proceed during mitosis, and their abrogation inhibits mitosis and can lead to repeated endoreplication of the genome in cells that make repeated attempts to divide. We find that planarians possess only the condensin I complex, and that this is required for normal stem cell function. Abrogation of condensin function led to rapid stem cell depletion accompanied by the appearance of giant cells with increased DNA content. Using previously discovered markers of heterogeneity we show that enlarged cells are always from the sigma-class of the neoblast population and we never observe evidence for endoreplication for the other neoblast subclasses. Overall, our data establish that condensins are essential for stem cell maintenance and provide independent evidence that only sigma-neoblasts are capable of multiple rounds of cell division and hence self-renewal.

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Acquisition of CD34 Correlates with Increased Hematopoietic and Self Renewal Activity of CD34−CD133+ Cord Blood Cells.

Blood ◽

10.1182/blood.v104.11.4143.4143 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4143-4143

Author(s):

Katharina S. Götze ◽

Matthias Schiemann ◽

Stefanie Marz ◽

Christian Peschel ◽

Robert A.J. Oostendorp

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Division ◽

Cord Blood ◽

Blood Cells ◽

Self Renewal ◽

Cd34 Cells ◽

Cord Blood Cells ◽

Cd34 Expression ◽

Hematopoietic Activity

Abstract CD34 is a sialomucin expressed on hematopoietic cells, endothelial cells and muscle satellite cells. Within the hematopoietic system, CD34 expression has been associated with very immature progenitor cells as well as hematopoietic stem cells (HSC), and it is widely used to assess stem cell activity in clinical protocols. In the past, HSC activity was thought to be retained exclusively in the subset of cells expressing CD34. This view has been challenged by recent observations in mice in which HSC activity was also found in the CD34-negative fraction. These findings have since been reproduced using human marrow and cord blood cells. However, the exact relationship between CD34+ and CD34− stem cells remains unclear. We investigated the regulation of CD34 expression as dependent on cell division history. To follow cell division, human cord blood cells were labeled with the fluorescent dye CFSE. Lin-CD34−CD133+CFSE+ (CD34−) and CD34+ populations were almost indistinguishable in their ability to produce CAFCweek6 content. After three days of serum-free culture with stem cell factor, Flt3 ligand and thrombopoietin, almost all initially CD34− cells had acquired expression of CD34, including all undivided cells. We found that, in cultures initiated from CD34− cells, virtually all CAFCweek6 were produced from the divided, now CD34+ cells, indicating these cells had self-renewed. In contrast, similar cultures from initially CD34+ cells demonstrated that hematopoietic activity associated with the undivided cell fraction. We did not find any hematopoietic activity in the cell fraction that remained CD34− or the fraction that lost CD34 after division. Analysis of mRNA expression showed that CD34− and CD34+ cells expressed almost equal levels of CD34, AC133, Flt1, Flk1 and Flt4, while CD34− cells expressed significantly lower levels of Tie1 and Tie2 than CD34+ cells. The expression of CD34 message in CD34− cells was explained by our observation that these cells contained intracellular CD34, indicating that they are “primed” to express the antigen on their cell surface. In conclusion, Lin−CD34−CD133+ cells acquire expression of CD34, even in the absence of cell divisions. These CD34− cells self-renew more rapidly in vitro than cells initially expressing CD34, and self-renewal is preceded by acquisition of CD34 antigen.

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Asymmetric cell division of mammary stem cells

Cell Division ◽

10.1186/s13008-021-00073-w ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Shaan N. Chhabra ◽

Brian W. Booth

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Stem Cell ◽

Cell Division ◽

Asymmetric Cell Division ◽

Mammary Stem Cells ◽

Self Renewal ◽

Symmetric Division ◽

Daughter Cells ◽

Mammary Stem

AbstractSomatic stem cells are distinguished by their capacity to regenerate themselves and also to produce daughter cells that will differentiate. Self-renewal is achieved through the process of asymmetric cell division which helps to sustain tissue morphogenesis as well as maintain homeostasis. Asymmetric cell division results in the development of two daughter cells with different fates after a single mitosis. Only one daughter cell maintains “stemness” while the other differentiates and achieves a non-stem cell fate. Stem cells also have the capacity to undergo symmetric division of cells that results in the development of two daughter cells which are identical. Symmetric division results in the expansion of the stem cell population. Imbalances and deregulations in these processes can result in diseases such as cancer. Adult mammary stem cells (MaSCs) are a group of cells that play a critical role in the expansion of the mammary gland during puberty and any subsequent pregnancies. Furthermore, given the relatively long lifespans and their capability to undergo self-renewal, adult stem cells have been suggested as ideal candidates for transformation events that lead to the development of cancer. With the possibility that MaSCs can act as the source cells for distinct breast cancer types; understanding their regulation is an important field of research. In this review, we discuss asymmetric cell division in breast/mammary stem cells and implications on further research. We focus on the background history of asymmetric cell division, asymmetric cell division monitoring techniques, identified molecular mechanisms of asymmetric stem cell division, and the role asymmetric cell division may play in breast cancer.

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