The role of proto-oncogene GLI1 in pituitary adenoma formation and cell survival regulation

The Hedgehog (Hh) pathway is an important regulator of early tissue patterning and stem cell propagation. It was found to be aberrantly activated in numerous types of human cancer and might be relevant in cancer stem cells. The identification of adult stem cells in the pituitary raised the question if tumor-initiating cells and Hh signaling are involved in pituitary adenoma formation. The present study aimed at the evaluation of Hh signaling in relation to stem cell and cell cycle markers in 30 human pituitary adenomas and in cultured murine adenoma cells. Therefore, expression levels of components of the Hh pathway, stem cell marker SOX2, cell cycle regulator tumor-protein 53 (TP53), proliferation marker Ki67 (MKI67) and superoxide dismutase 1 (SOD1) were evaluated in 30 human pituitary adenomas in comparison to control tissue. Modulation of cell function and target gene expression by the inhibition and activation of the Hh pathway were studied in murine adenoma cells. We show that transcription factor glioma-associated oncogene 1 (GLI1) is overexpressed in 87% of all pituitary adenomas. The expression of GLI1 significantly correlated with that of SOX2, TP53, MKI67 and SOD1. Inhibition of GLI1 resulted in the downregulation of the above genes and severe cell death in mouse adenoma cells. On the other hand, activation of the Hh pathway increased cell viability and target gene expression. In conclusion, our findings point toward an alternative, ligand-independent Hh pathway activation with GLI1 playing a major role in the cell survival of pituitary adenoma cells.

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miR-26a Plays an Important Role in Cell Cycle Regulation in ACTH-Secreting Pituitary Adenomas by Modulating Protein Kinase Cδ

Endocrinology ◽

10.1210/en.2012-2070 ◽

2013 ◽

Vol 154 (5) ◽

pp. 1690-1700 ◽

Cited By ~ 44

Author(s):

Erica Gentilin ◽

Federico Tagliati ◽

Carlo Filieri ◽

Daniela Molè ◽

Mariella Minoia ◽

...

Keyword(s):

Cell Cycle ◽

Pituitary Adenoma ◽

Protein Kinase ◽

Pituitary Adenomas ◽

Viable Cell Number ◽

Pituitary Cells ◽

Human Pituitary ◽

Human Pituitary Adenoma ◽

Acth Secreting ◽

Protein Kinase Cδ

Abstract The functional aftermath of microRNA (miRNA) dysregulation in ACTH-secreting pituitary adenomas has not been demonstrated. miRNAs represent diagnostic and prognostic biomarkers as well as putative therapeutic targets; their investigation may shed light on the mechanisms that underpin pituitary adenoma development and progression. Drugs interacting with such pathways may help in achieving disease control also in the settings of ACTH-secreting pituitary adenomas. We investigated the expression of 10 miRNAs among those that were found as most dysregulated in human pituitary adenoma tissues in the settings of a murine ACTH-secreting pituitary adenoma cell line, AtT20/D16v-F2. The selected miRNAs to be submitted to further investigation in AtT20/D16v-F2 cells represent an expression panel including 5 up-regulated and 5 down-regulated miRNAs. Among these, we selected the most dysregulated mouse miRNA and searched for miRNA targets and their biological function. We found that AtT20/D16v-F2 cells have a specific miRNA expression profile and that miR-26a is the most dysregulated miRNA. The latter is overexpressed in human pituitary adenomas and can control viable cell number in the in vitro model without involving caspase 3/7-mediated apoptosis. We demonstrated that protein kinase Cδ (PRKCD) is a direct target of miR-26a and that miR26a inhibition delays the cell cycle in G1 phase. This effect involves down-regulation of cyclin E and cyclin A expression via PRKCD modulation. miR-26a and related pathways, such as PRKCD, play an important role in cell cycle control of ACTH pituitary cells, opening new therapeutic possibilities for the treatment of persistent/recurrent Cushing's disease.

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Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Blood ◽

10.1182/blood.v122.21.4199.4199 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4199-4199 ◽

Cited By ~ 1

Author(s):

Carolina L. Bigarella ◽

Pauline Rimmele ◽

Rebeca Dieguez-Gonzalez ◽

Raymond Liang ◽

Brigitte Izac ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Dna Repair ◽

Stem Cell ◽

Gene Expression Profile ◽

Double Mutant ◽

Self Renewal ◽

Hematopoietic Stem ◽

Anti Oxidant

Abstract Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures: No relevant conflicts of interest to declare.

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Gene Expression Profiling in Quiescent Stem Cells from Normal and Chronic Myeloid Leukaemia Patients.

Blood ◽

10.1182/blood.v104.11.2962.2962 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2962-2962

Author(s):

Susan M. Graham ◽

Gerry J. Graham ◽

Tessa L. Holyoake

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Chronic Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Stem Cell Marker ◽

Quiescent Cells ◽

Dividing Cells ◽

Quiescent Stem Cells

Abstract Earlier studies have shown that Ph+ quiescent cells exist in chronic myeloid leukaemia (CML) (Blood (1999)94:2056) and we have previously shown that these cells are primitive in that they express the stem cell marker CD34. We have also shown that quiescent CML stem cells are insensitive to the effects of imatinib (IM Novartis Pharma) (Blood (2002) 99:319) and may present a possible source for relapse. This quiescent population therefore represents a potentially significant clinical problem and thus studies aimed at developing methods for eradicating this population are timely. In an effort to identify molecular markers of this population that may allow it to be specifically targeted during therapy, we have set out to investigate the transcriptional differences between quiescent and cycling stem cells. To this end, we have used specific stem cell enrichment and sorting protocols. Leukapheresis products from CML patients (N=5) in chronic phase at diagnosis and mobilised peripheral blood from allogeneic donors (N=3), were selected for CD34+ cells. Hoechst 33342 and Pyronin Y were used to discriminate the quiescent (G0) cells identified as Hoechstlo/Pyroninlo from the cycling cells. In combination with propidium iodide for dead cell exclusion we were able to sort 4–9x105 viable, quiescent stem cells and 4–11x106 cycling cells, which were processed for microarrays. Affymetrix gene chips (U133A) were used for the analysis and the data obtained was analysed using GeneSpring. Number of Genes Changed in Each Comparison 3 Fold 4 Fold 5 Fold CML G0 V CML Div 37 21 10 Norm G0 V Norm Div 188 92 47 CML G0 V Norm G0 168 85 49 CML Div V Norm Div 49 27 8 Initial analysis indicates that the greatest differences in gene expression are between the normal quiescent cells (G0) and normal dividing cells (Div) and between the normal quiescent cells and CML quiescent cells. A large percentage of the genes differentially expressed between the quiescent and cycling normal cells encode regulators of the cell cycle confirming the success of the sorting strategy for quiescent and cycling cells A selection of Genes Up-Regulated in Normal Cycling Cells Compared to G0 Gene Fold Up-regulation PCNA 3 CDC2 8 CCNB2 5 CCN1 3.5 CDC20 6 CDC25A 3.5 MCM5 3 In addition, many of the genes identified in our analysis are consistent with other published expression profiles for haemopoietic cells. Curiously, we have identified unanticipated changes in expression of cell cycle genes in the CML quiescent cells, which merit further investigation. We have also identified a number of unexpected genes as being more than 5 fold changed in the quiescent cells compared to dividing cells for both normal and CML samples. Specifically, there is a large cohort of genes preferentially expressed in quiescent normal or CML cells, which encode members of the chemokine family of proteins. Work is ongoing to establish the relevance, if any, of these genes to stem cell quiescence.

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Marrow Stem Cells Shift Gene Expression and Engraftment Phenotype with Cell Cycle Transit

Journal of Experimental Medicine ◽

10.1084/jem.20030031 ◽

2003 ◽

Vol 197 (11) ◽

pp. 1563-1572 ◽

Cited By ~ 57

Author(s):

Jean-François Lambert ◽

Meng Liu ◽

Gerald A. Colvin ◽

Mark Dooner ◽

Christina I. McAuliffe ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Cell Phenotype ◽

Phenotypic Change ◽

Hematopoietic Stem ◽

Differential Display Analysis ◽

Cell Gene Expression ◽

Major Shift

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3′ end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from “stem cell genes” being highly expressed at 0 h and turned off at 48 h, while “cell division” genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

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Circadian Rhythms of Whole Bone Marrow and Lineage Negative Stem Cells of the Mouse.

Blood ◽

10.1182/blood.v108.11.4188.4188 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4188-4188

Author(s):

Galatia Politopoulou ◽

Mark S. Dooner ◽

Gerald A. Colvin ◽

Deborah Greer ◽

Delia Demers ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Circadian Rhythm ◽

Circadian Rhythms ◽

Stem Cell Factor ◽

Clock Genes ◽

Circadian Variation

Abstract Circadian rhythms underlie most biological processes. In mammals circadian control of physiology and behavior is mediated via a central master oscillator, in the supra-schismatic nuclei of the hypothalamus. At the cellular level this oscillator is composed of an auto-regulatory transcription-translation loop of clock genes. The Period2 (Per2) gene is one of the clock genes which plays a key role in controlling the circadian rhythm in mammals. Mice with mutations in Per2 become arrhythmic. Expression of clock genes is also present in many peripheral tissues, including the bone marrow. Stem cell engraftment has been shown to vary with cell cycle transit (Habibian et al, 1998). A diurnal circadian variation in the ability of bone marrow to engraft sub-lethally irradiated mice has been previously shown by our laboratory. An increase in numbers of progenitors in S-phase underlined the engraftment nadirs. The host’s ability to accept incoming cells did not show circadian variation. To further study the interplay of circadian rhythm with cell cycle in bone marrow and populations of engraftable stem cells, we utilized a transgenic mouse model for the Per2 gene. The mouse Period2 (mPer2) real time gene expression reporter of circadian dynamics, constructed by Takahashi et al., was employed for these studies. In this reporter a Luciferin (Luc) gene was fused in frame to the 3′ of the promoter of the endogenous mPer2 gene. This system allows for detection of Per2 gene expression in the presence of luciferase, by recording light given off, during the luciferase catalyzed conversion of Luciferin to Oxyluciferin. We have detected circadian rhythm in whole bone marrow and Lineage negative cells i.e. whole bone marrow mononuclear cells depleted of B220, Ter119, GR1, CD4, CD8 and CD11b, with one peak every 24 hours for up to 14 days, from as few as 500,000 cells. Dissociated lung cells also show a circadian rhythm as do Lineage Negative Sca+ marrow stem cells. The later show an intermittent rhythm, for up to 10 days. The best rhythms were obtained from cells grown on a 12mm dish bathed in 4 mls of media or a 1 ml drop of media, the later covered with mineral oil. The media was DMEM with L Glutamine, low glucose no phenol red, 1% Penicillin, 10,000 U/ml/streptomycin, 4.18mM NaHCO3, 10mM Hepes, 0.019mM D-glucose pH 7.2 supplemented with 1×B27, 5–15% HIFCS and either stem cell factor alone (50ng/ml) or IL3, IL6, IL11 and stem cell factor (steel) (50 ng or units/ml). Feeding of the cells after 7 days increased the amplitude of the rhythm. Absence of cytokines dampened the rhythm, especially for Lineage Negative Sca+ cells. Steel in the presence of HIFCS induces some rhythm, but is not as effective as a cocktail of IL3, IL6, IL11 and steel, together with HIFCS. Given work from our laboratory on synchronized progenitor cells entering and progressing through cell cycle in cytokine cocktails, including IL3, IL6, IL11 and steel, and the correlation of engraftment potential with cell cycle phase and adhesion molecule phenotype, the appearance of the best circadian rhythm in proliferating cytokine cocktails, in this system, is intriguing. The cell cycle kinetics of the marrow cells exhibiting circadian rhythmicity, are being explored further, in this culture system. The links between expression of cell cycle control molecules and adhesion molecules in Lineage Negative Sca+ cells and circadian rhythms for engraftment are also under investigation.

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Selective Persistence of Distinct Stem/B Leukaemic Stem Cells In Childhood Acute Lymphoblastic Leukaemia In Clinical Remission.

Blood ◽

10.1182/blood.v116.21.1585.1585 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1585-1585

Author(s):

Christoph Lutz ◽

Petter Woll ◽

Anders Castor ◽

Helen Ferry ◽

Christina Jensen ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

B Cells ◽

B Cell ◽

Quiescent State ◽

Childhood All ◽

Response To Chemotherapy ◽

Leukaemic Cells

Abstract Abstract 1585 Recent studies utilising surrogate leukaemic stem cell (LSC) assays have suggested that LSCs in acute lymphoblastic leukaemias (ALLs) might be neither rare, nor phenotypically or functionally distinct. However, studies of candidate LSCs in surrogate assays might not recapitulate the full leukaemic potential of candidate LSCs in patients, and in particular their responsiveness and resistance to therapeutic targeting. Therefore, we have investigated the identity, molecular and functional properties, and persistence of different subsets of candidate LSCs in childhood ALL, at diagnosis and during the course of clinical and molecular remissions in response to chemotherapy, and their relationship to subsequent relapses. First, we investigated 6 patients diagnosed with “good prognosis” TEL-AML1+ ALL, and at diagnosis we found TEL-AML1+ leukaemic cells within the immature B cell progenitor compartment (proB: 34+38+19+), mature B-cells (34-19+), as well as in a population expressing an aberrant combination of stem cell (34+38-/lo) and B-cell (19+) cell surface markers. These stem/B (34+38-/lo19+) cells were all TEL-AML1+ and not present in age-matched normal bone marrow controls. In contrast, haematopoietic stem cells (HSC: 34+38-19-) were not part of the TEL-AML1+ leukaemic clone in any of the patients. 15 days into chemotherapy, all TEL-AML1+ mature B-cells were eliminated in all patients, and this was followed by a clearance of leukaemic proB cells by day 28 of treatment. In striking contrast, leukaemic stem/B cells were still detectable at day 28, but in all TEL-AML1 patients, at later stages all leukaemic cells including the stem/B cells were undetectable, and at the same time these patients went into complete remission with less than 1 leukaemic cell in 10e4 cells detectable. A similar pattern was observed in a case of “high risk” BCR-ABL+ ALL: BCR-ABL+ proB and B-cells were efficiently eliminated by day 90 of the course of chemotherapy, and up to 180 days into the treatment only 34+38-/lo19+ stem/B cells remained part of the BCR-ABL+ clone. In agreement with the persistence of BCR-ABL+ 34+38-/lo19+ stem/B cells, this patient relapsed 17 months after the initiation of chemotherapy. In order to understand the underlying mechanisms of the observed functional and therapeutic heterogeneity seen in leukaemic subpopulations, we performed comparative gene-expression analysis of diagnostic leukaemic stem/B and proB cells of TEL-AML1+ patients. This analysis revealed a differential gene expression pattern between leukaemic stem/B and proB cells, with positive regulators of cell cycle being the most distinctly up regulated genes in leukaemic proB cells. In agreement with this, cell cycle analysis of 3 diagnostic TEL-AML1+ cases also showed proB cells to be more actively cycling compared to the more quiescent state of the leukaemic stem/B compartment (proB: G0 42%; G1 40%; S,G2,M 18% vs. stem/B: G0 81%; G1 18%; S,G2,M 1%), providing a potential mechanistic basis for the relative therapy resistance of ALL stem/B cells. Taken together the present studies suggest that quiescent 34+38-/lo19+ stem/B cells are selectively resistant to chemotherapy, and most likely the origin of relapses when these occur in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

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Single-Cell Gene Expression Analysis Reveals Gene Regulatory Networks Driving Proliferation in Pituitary Stem and Endocrine Cells

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1042 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A509-A510

Author(s):

Leonard Cheung ◽

Alexandre Daly ◽

Michelle Brinkmeier ◽

Sally Ann Camper

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Single Cell ◽

Regulatory Networks ◽

Endocrine Cells ◽

Postnatal Period ◽

Pituitary Cells ◽

Cell Gene

Abstract A fundamental question for pituitary development and disease is to understand the mechanisms that regulate proliferation, quiescence, and differentiation of stem cells and endocrine lineage-committed precursor cells. Pituitary stem cells, marked by expression of SOX2, are highly proliferative during development and the early postnatal period. This pool becomes quiescent over time, but stem cells retain the ability to re-enter the cell cycle and differentiate into nascent endocrine cells of all lineages in response to physiological demands. The rodent pituitary gland undergoes substantial growth in the postnatal period, and much of this increase in size is due to proliferation of committed progenitors, such as Pou1f1-expressing cells. We performed single-cell RNA transcriptomics analyses of over 8,000 male and female 4-day-old mouse pituitary cells in order to assess stem cell heterogeneity and to identify novel pituitary stem cell biomarkers. We identified a number of factors enriched in pituitary stem cells relative to differentiating cells, including the transcription factors TGIF1 and NR4A3 as well as several members of the nuclear factor I family (NFIA, NFIB, NFIX). We also detected stem cell-specific expression of the cortisol synthesizing enzyme HSD11B1 and folate receptor FOLR1, suggesting novel roles for these pathways in pituitary stem cells. There were few transcriptomic differences between proliferating and non-proliferating stem cells. However, proliferating stem cells and proliferating committed progenitors shared expression of cell-cycle associated genes and novel transcription factors such as BRCA1 and E2F1. Furthermore, single-cell gene network inference and clustering (SCENIC) analyses demonstrated activation of common gene regulatory networks in both proliferating stem and proliferating endocrine populations, including both the E2f1 and Brca1 regulons. RNA velocity, trajectory, and phylogenetic analyses and find that proliferating stem and endocrine cells are likely independent cellular states. Instead, they support the idea that proliferating stem cells become quiescent and transition to committed progenitors that re-enter the cell cycle and then subsequently activate hormone gene expression. In conclusion, our single-cell gene expression analyses of early postnatal pituitary cells have shed light on the developmental trajectory from proliferating stem cell to quiescent differentiated hormone-producing cells.

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Characterisation of the Stem and Progenitor Cell Hierarchy in Patients with CMML

Blood ◽

10.1182/blood.v124.21.1896.1896 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1896-1896

Author(s):

Onima Chowdhury ◽

Petter S Woll ◽

Una Kjällquist ◽

Helen Doolittle ◽

Rikard Erlandsson ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Dna Sequencing ◽

Progenitor Cell ◽

Conflicts Of Interest ◽

Mutation Screening ◽

Recurrent Mutations

Abstract Background Identification and characterisation of tumour-propagating cells in distinct haematological malignancies may prove decisive in the development of effective therapies. However, conventional human stem cell assays might fail to uncover the true tumour-propagating potential of different clonal cell populations. To circumvent this, we recently integrated bone marrow cellular hierarchy analysis with in vivogenetic fate mapping, to provide evidence that low and intermediate risk myelodysplastic syndromes (MDS) are only propagated by Lin-CD34+CD38-CD90+ stem cells (Woll et al Cancer Cell 2014). We have now performed similar studies of patients with chronic myelomonocytic leukaemia (CMML). Methods Flow cytometry was used to identify and prospectively purify phenotypic stem cells (SCs; CD34+CD38-CD90+CD45RA-) and progenitor cells (GMPs; CD34+CD38+CD123+CD45RA+; MEPs CD34+CD38+CD123-CD45RA-; ProB cells CD34+CD19+). In vitrostem and progenitor assays, gene expression profiling and targeted DNA sequencing of the coding region of 88 genes recurrently mutated in myeloid malignancies were then performed to characterise the stem and progenitor cell hierarchy in 10 patients with CMML. Results CMML patients retained phenotypically distinct stem and progenitor compartments at frequencies not significantly different to that observed in normal age-matched controls. Stem cells, GMPs and MEPs were all highly clonally involved, as evidenced by high variant allele frequency for all detected mutations. Myeloid and erythroid gene expression signatures and functional capacity were restricted to the GMP and MEP compartments, respectively. A functional and transcriptional stem cell signature was restricted to the stem cell compartment of CMML patients. Candidate CMML stem cells also retained a high degree of cell cycle quiescence, whereas CMML progenitors resided predominantly in G1 and S/G2/M phases of the cell cycle. Taken together, these findings establish CMML SCs, GMPs and MEPs as highly clonally involved but phenotypically, molecularly and functionally distinct cell compartments. Targeted DNA sequencing of bone marrow mononuclear cells revealed recurrent mutations in splice factors (SRSF2 and ZRSR2), epigenetic regulators (TET2, ASXL1 and EZH2), transcription factors (RUNX1, GATA2) and cell signaling molecules (CBL and NRAS). The SRSF2 P95 mutation was identified in 7/10 patients. Mutations in TET2 (6/10) and ASXL1 (3/10) were the second and third most recurrently mutated genes. Notably two patients with the SRSF2 P95 mutation also had a recurrent mutation in EZH2, which some studies have reported as being mutually exclusive. Targeted mutation screening of purified stem cells and single colonies from long-term cultures established that all the mutations identified in the bulk bone marrow could be traced back to the phenotypically and functionally defined CMML stem cells in 9/10 patients. Conclusion Similar to our previous studies in low and intermediate-1 risk MDS cases, our investigation of CMML patients provides compelling evidence that CD34+CD38-CD90+ stem cells are the disease-propagating cells in most CMML patients. Disclosures No relevant conflicts of interest to declare.

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Cell Cycle Related Stem Cell Gene Expression.

Blood ◽

10.1182/blood.v104.11.4155.4155 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4155-4155

Author(s):

Gerri J. Dooner ◽

Gerald A. Colvin ◽

Mark S. Dooner ◽

Peter J. Quesenberry

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Rank Order ◽

Continuum Theory ◽

Surface Expression ◽

Stem Cell Regulation ◽

Cell Gene Expression ◽

Cell Gene

Abstract We have previously reported that marrow stem cells show changes in engraftment (Habibian, et al J Ex. Hem, 188:393–398, 1998), homing (Cerny et al., J Hematother Stem Cell Res11:913–922, 2002) and differentiation (Colvin et al., J Cell Phys199:20–31, 2004) phenotype as they transit a cytokine-driven cell cycle. mRNA and surface expression of adhesion proteins also change (Becker et al., Exp Hematol27:533–541, 1999). We have evaluated gene expression by Real-time PCR of murine lineage negative, Sca+ (Lin-Sca+) stem cells stimulated by Il-3, Il-6, Il-11 and Steel factor (at 0, 24 and 48h) and lineage negative Rhodamine low, Hoescht low (LRH) stem cells stimulated by TPO, Flt-3 and Steel at various points in cell cycle transit (0,32,40,48h). In Lin-Sca+ cells (4experiments, time 0) expression of the following genes in descending order was as follows: IKAROS, L-selectin, Pu-1, Gata-2, Pecam, Cd84, Rock-1, c-fms, FOG, Cxcr4, c-kit, Cd4. The following were either not expressed or expressed at very low levels: Il-11, Ccr4, Sdf-1, Gata-1, P-selectin and Vecam. A pattern of depressed gene expression in S-phase (24h) with subsequent recovery (48h) was seen with c-fms and c-kit. With LRH cells (2 experiments, time 0) approximate descending rank order of gene expression was Cd45r, Cd34, G-CSFR, Mac-1, GM-CSFR and Flt-3. Il7r was not detected. With cycle progression Cd34 and Sca-1 were markedly elevated while Mac-1 and c-mpl were decreased. The expression of GM-CSFR, G-CSFR, Cd45r and Cd4 showed variable fluctuation. Il-7r was negative throughout. These data show that primitive marrow stem cells express a wide variety of “hematopoietic genes”, that expression modulates with cell cycle transit and perhaps most importantly that observed changes in gene expression are reversible. This is consistent with the continuum theory of stem cell regulation.

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Immune-Mediated Reprogramming of Intestinal Stem Cells Drives STAT1-Dependent Myc Expression and Epithelial Regeneration in GI-Gvhd

Blood ◽

10.1182/blood-2021-152647 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 86-86

Author(s):

Shuichiro Takashima ◽

Roshan Sharma ◽

Anastasiya Egorova ◽

Jason Kuttiyara ◽

Takahiro Ito ◽

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Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

T Cell ◽

Target Gene ◽

Gene Set Enrichment Analysis ◽

Intestinal Stem Cells ◽

Immunofluorescent Staining ◽

Dependent Manner ◽

Stat1 Signaling

Abstract Crypt apoptosis and regeneration are characteristic findings in GI-GVHD. Intestinal stem cells (ISCs) are critical for maintaining the intestinal epithelium, but their frequencies are reduced in experimental GVHD, and the mechanisms driving crypt regeneration in this context are poorly understood. To better understand the impact of GVHD on individual epithelial components, we performed single cell RNA sequencing (scRNAseq) of purified small intestine crypts from B6 mice during homeostasis and five days after B6 into B6 syngeneic (syn) or B10.Br into B6 allogeneic (allo) BMT. Sequenced cells were first partitioned into distinct clusters using PhenoGraph, and the cluster identities were annotated based on their gene expression profiles. Unsupervised clustering indicated multiple subpopulations within the various recognized crypt components (Fig 1A). While secretory lineages clustered similarly across experimental conditions, there was highly distinct clustering among ISC populations and striking dissimilarity between allo and syn ISCs. Quantification by computing the subpopulation's phenotypic distance, a composite score integrating both the number and amplitude of differentially expressed genes, indicated that ISCs demonstrated the greatest transcriptional change in response to GVHD among all crypt lineages (Fig 1B). Gene Set Enrichment Analysis (GSEA) highlighted activation of the interferon-γ (IFNγ) pathway in allo ISCs, and differential gene expression indicated that STAT1 was their most highly upregulated transcription factor. We then performed MHC-mismatched allo-BMT into STAT1-floxed x villin-Cre recipients. Consistent with a role in IFNγ-related toxicity, STAT1-deficient recipients initially demonstrated reduced GVHD pathology, as well as a reduction in proliferating Ki67 + cells (Fig 2A). However, the pathology reduction in STAT1-deficient recipients was transient, while reduction in crypt proliferation persisted. STAT1-deficient recipients ultimately demonstrated increased mortality after allo-BMT, indicating a complex response to epithelial STAT1 signaling in GVHD. While IFNγ can induce epithelial apoptosis and kill organoid cultures, organoid exposure to IFNγ augmented size even at IFNγ concentrations that did not impair viability in a STAT1-dependent manner, and co-culture with allo T cells augmented organoid size (Fig 2B, C). Moreover, cell cycle analysis showed augmentation of cell cycle in ISCs after IFNγ treatment in association with upregulation of cyclin D1 (Fig 2D), and human organoids also showed increased size in response to IFNγ treatment, further suggesting that this growth promotion was not simply a secondary response to tissue injury. In addition to the IFNγ signaling, GSEA of allo ISCs indicated activation of the Myc pathway. scRNAseq data showed specific upregulation of Myc in allo ISCs (Fig 3A). Myc expression within individual ISCs indicated that greater STAT1 expression and IFNγ signaling directly correlated with Myc expression in the same ISCs, providing a potential direct link between T-cell-derived IFNγ and ISC-dependent regeneration (Fig 3B). Additionally, intestinal organoid qPCR showed that Myc expression was upregulated after IFNγ treatment, and scRNAseq of IFNγ-treated organoids indicated this Myc upregulation was restricted to the ISC/progenitor compartment. Although Myc is downstream of Wnt signaling, which is critical for ISC maintenance, expression of the representative Wnt target gene Axin2 was downregulated after IFNγ treatment, and Irf1, a representative IFNγ/STAT1 target gene, was upregulated, suggesting that IFNγ/STAT1 signaling could replace Wnt/β-catenin as a driver of ISC Myc expression. We next examined Myc function and found that treatment with the Myc inhibitor 10058-F4 suppressed IFNγ-dependent organoid growth (Fig 3C). Finally, immunofluorescent staining showed Myc protein expression in intestinal crypts after allo-BMT in a STAT1-dependent manner (Fig 3D), and Myc inhibitor treatment in vivo suppressed crypt epithelial proliferation in mice with GVHD. In summary, we found that epithelial STAT1 contributes to crypt regeneration in GVHD by transmitting T-cell-derived JAK/STAT cytokine signaling to activate Myc expression in ISCs. Clinical use of JAK inhibitors in GVHD may inhibit this regenerative response, necessitating concurrent interventions aimed at restoring it. Figure 1 Figure 1. Disclosures Hanash: Evive Biotech: Ended employment in the past 24 months.

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