Bone Marrow (BM) Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated with Nilotinib

Abstract Introduction Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Download Full-text

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽

10.3324/haematol.2019.232793 ◽

2020 ◽

Vol 106 (1) ◽

pp. 111-122 ◽

Cited By ~ 4

Author(s):

Sandrine Jeanpierre ◽

Kawtar Arizkane ◽

Supat Thongjuea ◽

Elodie Grockowiak ◽

Kevin Geistlich ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Tyrosine Kinase ◽

Stromal Cells ◽

Kinase Inhibitor ◽

Bmp Signaling ◽

Myelogenous Leukemia ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Download Full-text

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia

Blood ◽

10.1182/blood.v86.1.60.bloodjournal86160 ◽

1995 ◽

Vol 86 (1) ◽

pp. 60-65 ◽

Cited By ~ 14

Author(s):

JT Holden ◽

RB Geller ◽

DC Farhi ◽

HK Holland ◽

LL Stempora ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Acute Leukemia ◽

Lymphoblastic Leukemia ◽

Blast Crisis ◽

Myelogenous Leukemia ◽

Acute Leukemias ◽

Hematopoietic Stem

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low- density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.

Download Full-text

Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽

10.1182/blood-2014-06-582924 ◽

2015 ◽

Vol 125 (17) ◽

pp. 2678-2688 ◽

Cited By ~ 64

Author(s):

Marisa Bowers ◽

Bin Zhang ◽

Yinwei Ho ◽

Puneet Agarwal ◽

Ching-Cheng Chen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Key Points ◽

Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Download Full-text

Mac-1 and F4/80 Surface Markers Are Present on Murine Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.1677.1677 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1677-1677

Author(s):

Toska J. Zomorodian ◽

Debbie Greer ◽

Kyle Wood ◽

Bethany Foster ◽

Delia Demers ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Muscle Fibers ◽

Stem Cell Marker ◽

Stem Cell Markers ◽

Surface Markers ◽

Hematopoietic Stem ◽

Cell Markers

Abstract Transplanted bone marrow donor cells with tissue specific phenotypes have been found in the brain, liver, heart, skin, lung, kidney, and gut of transplanted humans and mice. Such observations have led to the controversial hypothesis that hematopoietic stem cells (HSC) might be intrinsically plastic, and through transdifferentiation or fusion lead to the repair of damaged tissues throughout the body. Alternately, it is suggested that fusion of macrophages to the recipient cells may explain this phenomenon. We have shown recently that purified HSC are the cells responsible for GFP positive donor-derived muscle fibers in the recipient mice post bone marrow transplantation. However, further studies sorting for macrophage markers Mac-1 and F4/80 also resulted in donor-derived muscle fibers in the host. To address this discrepancy, we investigated subpopulations of Mac-1 and F4/80 positive cells, in the presence or absence of stem cell markers (Sca-1 and C-kit). We demonstrate that only the subpopulations of Mac-1 and F4/80 positive cells harboring stem cell markers, Sca-1 or c-kit, were capable of contributing to the regenerating muscle post transplantation. Furthermore, these same subpopulations demonstrated single cell High Proliferative Potential (HPP) (6–26%) in a 7 factor cytokine cocktail, compared to the Mac-1 or F4/80 cells with no stem cell markers (0%). Additionally, they demonstrated long-term engraftment in all three lineages at 1-year (average chimerism of 55% versus 0% in stem cell marker negative groups). These subpopulations were also evaluated for morphology using Hematoxylin/Eosin (H/E), Wright-Giemsa, and Nonspecific Esterase staining. In the Mac-1 and F4/80 positive groups, those negative for stem cell markers resembled differentiated cells of the myeloid origin (macrophages, granulocytes), while those with positive stem cell markers demonstrated stem cell characteristics. We did not observe any engraftability, donor-derived muscle fibers, or HPP potential for CD14 or cfms positive cells coexpressing stem cell markers, indicating that these markers are more appropriate for identifying macrophages. In conclusion, our studies demonstrate that both Mac-1 and F4/80 surface markers are present on HSC and therefore caution must be taken in the interpretation of data using these macrophage markers. It is reasonable to believe that the use of Mac-1 and/or F4/80 surface markers in a lineage depletion process may result in the loss of a subpopulation of stem cells, and other markers such as CD14 or c-fms may be more appropriate for eliminating differentiated macrophages.

Download Full-text

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.861.861 ◽

2006 ◽

Vol 108 (11) ◽

pp. 861-861 ◽

Cited By ~ 1

Author(s):

Viktor Janzen ◽

Heather E. Fleming ◽

Michael T. Waring ◽

Craig D. Milne ◽

David T. Scadden

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Caspase 3 ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

Download Full-text

Antibody-Based Depletion of Hematopoietic Stem Cells Empties Niches for Efficient Transplantation.

Blood ◽

10.1182/blood.v110.11.lb2.lb2 ◽

2007 ◽

Vol 110 (11) ◽

pp. LB2-LB2

Author(s):

Agnieszka Czechowicz ◽

Daniel L. Kraft ◽

Deepta Bhattacharya ◽

Irving L. Weissman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Conditioning Regimen ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Exact Function ◽

Potential Applications ◽

Conditioning Regimens

Abstract Hematopoietic stem cells (HSCs) are used therapeutically in bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) to correct hematolymphoid abnormalities. Upon intravenous transplantation, HSCs can home to specialized bone marrow niches, self-renew and differentiate and thus generate a new, complete hematolymphoid system. Unfortunately BMT has had limited applications, due to the risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy, that are deemed necessary for HSC engraftment. Elimination of these toxic conditioning regimens could expand the potential applications of BMT to include many non-malignant hematologic disorders, a wide variety of autoimmune disorders such as diabetes and multiple sclerosis, as well as in the facilitation of organ transplantation. The exact function of these traditional myeloablative conditioning regimens is not clear. To elucidate the barriers of HSC engraftment, we transplanted 50–1000 purified HSCs (Ckit+Lin−Sca1+CD34+CD150−) into immunodeficient, Rag2−/− or Rag2−/−gc−/− recipient mice and show that HSC engraftment levels rarely exceed 0.5% following transplantation without toxic conditioning, indicating that the immune system is not the only barrier to engraftment. Additionally, we did not observe a significant increase in HSC engraftment when HSC doses of >250 cells were transplanted. Even when up to 18000 HSC were transplanted, we did not see a linear increase in HSC engraftment, indicating that the increased doses of HSCs transplant inefficiently. We believe this is due to the naturally low frequency of available HSC niches, which we postulate may result from the physiologic migration of HSCs into circulation. Conversely, separation of the graft into small fractions and the subsequent time-delayed transplantation of these doses did result in increased engraftment due to the natural physiologic creation of new available HSC niches. When 1800 HSC were transplanted daily for seven days, the engraftment was 6.1-fold higher than transplantation of 12800 HSC in a single bolus. Here, we provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Through elimination of host HSCs we are able to increase available HSC niches for engraftment. We have developed a novel system where HSCs can be eliminated by targeting C-kit, a cell surface antigen that is highly expressed on the surface of HSCs. Cultivation of HSCs with ACK2, a depleting antibody specific for c-kit, prevented stem-cell factor (SCF) dependent HSC proliferation in vitro and resulted in cell death. Administration of ACK2 to mice led to the rapid and transient removal of >98% of endogenous HSCs in vivo thus resulting in equal numbers of available niches for engraftment. Following ACK2 clearance from serum, transplantation of these animals with donor HSCs led to chimerism levels of up to 90%, representing a 180-fold increase as compared to unconditioned animals. This non-myeloablative conditioning regimen had few side effects, other than temporary loss of coat color. The HSCs in even untransplanted animals rapidly recovered and animals remained healthy and fertile. This work redefines the way we approach BMT/HSCT, and places great emphasis on the necessity to create available HSC niches prior to transplantation. Extrapolation of these methods to humans may enable efficient yet mild conditioning regimens for transplantation, thus expanding the potential applications of BMT/HSCT.

Download Full-text

Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽

10.1182/blood.v120.21.sci-42.sci-42 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-42-SCI-42

Author(s):

Toshio Suda

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Metabolic Regulation ◽

Conflicts Of Interest ◽

Hypoxia Inducible Factor ◽

Ros Generation ◽

Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Zinc Finger Transcription Factor Growth Factor Independence 1b (Gfi1b) Regulates The Wnt/Beta-Catenin Signaling Pathway In Hematopoietic Stem Cells Through Interaction With Inhibitory Proteins

Blood ◽

10.1182/blood.v122.21.2417.2417 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2417-2417

Author(s):

Peiman Shooshtarizadeh ◽

Ryan Chen ◽

Tarik Moroy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Amino Acid ◽

Hematopoietic Stem Cells ◽

Signaling Pathway ◽

Beta Catenin ◽

Hematopoietic Stem ◽

Endosteal Niche ◽

Canonical Wnt

Abstract Hematopoietic stem cells (HSCs) reside in the bone marrow in specific niches at the border between bone cells and the bone marrow (endosteal niche) or around blood vessels (perivascular niche). In the endosteal niche, HSCs are maintained at low oxygen levels in a quiescent (dormant) state by adhesion to niche cells. We have previously shown that Gfi1b restricts the expansion and proliferation of HSCs as well as their mobilization or re-localization into peripheral blood. We have proposed that Gfi1b exerts this function by regulating the expression of surface molecules such as integrins on HSCs that are required to maintain them in their bone marrow niche at a quiescent state. The objective of this study was to gain more insight into the precise molecular mechanisms by which Gfi1b regulates HSCs dormancy and mobilization and to obtain insights that may be exploited in the future to improve stem cell therapies or the expansion of human hematopoietic stem cells for clinical use. Immune precipitation and mass spectrometry identified a series of Gfi1b interacting proteins, most notably a group of regulators of the canonical Wnt/beta-catenin pathway. Independent protein IP validation of these findings suggested that Gfi1b can interact with several inhibitors of the canonical Wnt/beta catenin pathway namely with APC (Adenomatous polyposis coli) a tumor suppressor protein and important factor in the beta-catenin destruction complex, with the DNA helicase and chromatin remodeling factor CHD8, which silences beta catenin mediated transcription, with CtBP which antagonizes beta-catenin activity and is part of the LSD1/CoRest histone demethylase complex and with the direct beta-catenin inhibitors TLE1 and TLE3 (also called Groucho). Of particular interest was that the interactions between the Groucho proteins and Gfi1b were dependent on a previously unidentified Groucho binding domain (GBD) in Gfi1b. This is a well-conserved six-amino acid stretch that is found in the middle part of the Gfi1b protein. In addition, the binding of CtBP was dependent on the presence of the 20 amino acid N-terminal SNAG domain in Gfi1b that also mediates LSD1 binding. Using luciferase reporter gene assays (TOP/FOP reporter assay), we found that Gfi1b was able to significantly up-regulates TCF/beta-catenin-dependent transcription upon activation by LiCl or Wnt3A in HEK293 cells. This activity of Gfi1b was dependent on both the presence of the SNAG domain and the newly identified Groucho binding domain. Also, Gfi1b was able to reverse partially the inhibitory effect of CtBP and TLE3 on beta-catenin activity in the TOP/FOP reporter assays. To obtain further evidence that Gfi1b is indeed implicated in regulating the Wnt/beta catenin signaling pathway in hematopoietic stem cells, we FACS sorted Lin-Kit1+Sca+ hematopoietic progenitors (LSK cells) from wt and Gfi1b deficient mice and tested them for expression of Wnt effector genes using a Wnt signaling specific PCR array. We observed that the majority of Wnt target genes were significantly down regulated in Gfi1 deficient LSKs compared to wt LSKs. Among the genes affected the most were typical Wnt targets such as Axin2, Frz7, Tcf4, Klf5, Vegfa and Ccnd1. To show that Gfi1b is able to regulate Wnt pathway effectors in vivo in HSCs, we crossed Gfi1b flox/flox, Mx-Cre mice with animals that carry a NLS-lacZ reporter gene under the control of the endogenous Axin2 promoter/enhancer region. Treatment with pIpC, which deletes Gfi1b correlated with a significant decrease of Axin2 expression in HSCs and MPP1, which are high Gfi1b expressing cells. The Axin2 reporter was not affected by Gfi1b deletion in MPP2 or GMPs, which express low levels or no Gfi1b. The canonical Wnt/b-catenin signaling pathway is recognized as one of the elements that are critically important in the regulation of HSC function. Here we have identified Gfi1b as a potential new player in the Wnt-beta catenin signaling pathway. Our data suggest that Gfi1b acts on at least two inhibitory complexes of this pathway, on the TLE family of Groucho proteins and the CtBP/LSD1 complex and regulates effectors of the Wnt/beta-catenin signaling cascade. We propose therefore that Gfi1b may titer the level of activation of the Wnt/beta-catenin signaling pathway in HSCs, which offers an explanation of the hematopoietic stem cell phenotype seen in mice lacking Gfi1b. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽

10.1182/blood.v124.21.350.350 ◽

2014 ◽

Vol 124 (21) ◽

pp. 350-350

Author(s):

Kyung-Hee Chang ◽

Amitava Sengupta ◽

Ramesh C Nayak ◽

Angeles Duran ◽

Sang Jun Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Negative Regulator ◽

Wild Type ◽

Hematopoietic Stem ◽

P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

Download Full-text

Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Blood ◽

10.1182/blood.v124.21.sci-20.sci-20 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-20-SCI-20

Author(s):

Margaret A. Goodell

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Stem Cell Differentiation ◽

Primary Myelofibrosis ◽

Differentiation Potential ◽

Hematopoietic Stem

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text