A p53-Dependent, DNA Damage Response Mediates Ineffective Erythropoiesis Caused by Deregulated Cyclin E Activity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 74-74
Author(s):  
Yanfei Xu ◽  
Alexander C. Minella
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Dna Damage Response ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
Wild Type ◽  
P53 Expression ◽  
Damage Response

Abstract Abstract 74 Cyclin E is a critical regulator of cell cycle progression that is frequently found over-expressed in human cancers. We previously described a knock-in mouse model that permitted study of the physiologic consequences of cyclin E deregulation, via abrogation of the ubiquitin-proteasome pathway controlled by the Fbw7 tumor suppressor. We found that erythroid progenitor cells of cyclin ET74A T393A knock-in mice exhibit abnormally increased proliferation, increased apoptosis, impaired maturation, and dysplastic morphologies. To investigate further the molecular mechanisms by which deregulated cyclin E activity impairs normal erythropoiesis, we have now compared gene expression profiles of CD71high/Ter119high erythroid precursors from the cyclin E knock-in and wild-type, littermate control bone marrows. Most prominent among the gene expression abnormalities identified in the cyclin E knock-in erythroid cells were up-regulated expression of multiple target genes of the p53 tumor suppressor, indicative of p53 pathway activation. We confirmed these findings using both quantitative real-time RT-PCR and western blot, the latter of which identified increased phosphorylation on p53 at serine 15, which is phosphorylated by kinases activating the DNA damage response. These findings were previously identified in cultured primary fibroblasts expressing high cyclin E by ectopic expression, and our data now demonstrate that impaired cyclin E degradation activates p53 in a cell type-specific context in vivo. To test directly the importance of these gene expression abnormalities to the erythroid maturation defects in cyclin E knock-in cells, we utilized an in vitro system for erythroid differentiation of primary bone marrow progenitor cells. We first recapitulated the erythroid differentiation defect identified in vivo in cultured cyclin ET74A T393A knock-in bone marrow cells. Then, to determine the functional importance of p53 activation in cyclin E knock-in erythroid progenitors, we used small hairpin RNAs to knock-down p53 expression in hematopoietic progenitors. We found that reduced p53 expression rescued the erythroid differentiation defects in cyclin E knock-in progenitor cells, while not having a significant effect in wild-type cells. Together, our findings suggest that oncogenic activation of p53 in bone marrow progenitor cells can directly impair erythroid differentiation. Disclosures: No relevant conflicts of interest to declare.

Slug Plays an Essential Role in the Radioprotection of Hematopoietic Progenitors In Vivo by Antagonizing p53-Mediated Apoptotic Pathways.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 31-31
Author(s):  
Wen-Shu Wu ◽  
Dong Xu ◽  
Stefan Heinrichs ◽  
A. Thomas Look
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Γ Irradiation ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

Abstract An antiapoptotic role for Slug/Snail in mammals was suggested by studies in C. elegans, where CES-1/Scratch, a member of the Slug/Snail superfamily, was found to control the apoptotic death of NSM sister neurons by acting as a transcriptional repressor of EGL-1, a BH3-only proapoptotic protein. Identification of Slug as the target gene of the E2A-HLF oncoprotein in human pro-B leukemia cells led us to demonstrate its antiapoptotic function in IL-3-dependent murine pro-B cells. In contrast to its aberrant expression in pro-B leukemia cells, endogenous Slug is normally expressed in both LT-HSC and ST-HSC, as well as committed progenitors of the myeloid series, but not in pro-B and pro-T cells, implying its function in myelopoiesis. Using Slug−/− mice produced in our laboratory, we showed that these knockouts are much more radiosensitive than Slug+/− and wild-type mice, and that apoptotic cells increase significantly in the hematopoietic progenitor cells of Slug−/− mice as compared to wild-type mice following γ-irradiation, indicating a radioprotective function in vivo. We showed here that although the development of myeloid progenitors is not impaired under steady-state conditions, their repopulation is incomplete γ-irradiated in in Slug−/− mice. We demonstrate further the radiation-induced death of Slug−/− mice is exclusively a result of bone marrow failure with no apparent contribution from systemic injures to other tissues. By two-way bone marrow transplantation, we provide firm evidence that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. Interestingly, regenerative capacity of hematopoietic stem cells (HSC) was retained in irradiated Slug−/− mice, which could be rescued by wild-type bone marrow cells after irradiation, indicating that Slug exerts its radioprotective function in myeloid progenitors rather than HSCs. Furthermore, we establish that Slug radioprotects mice by antagonizing downstream of the p53-mediated apoptotic signaling through inhibition of the p53-resposive proapoptotic gene Puma, leading in turn to inhibition of the mitochondria-dependent apoptotic pathway activated by γ-irradiation in myeloid progenitors. More interestingly, we observed that Slug is inducible by γ-irradiation in a p53-dependent manner. Together, our findings implicate a novel Slug-mediated feedback mechanism by which p53 control programmed cell death in myeloid progenitor cells in vivo in response to γ-irradiation.

scholarly journals Comparative Analysis of Gene Expression Identifies Distinct Molecular Signatures of Bone Marrow- and Periosteal-Skeletal Stem/Progenitor Cells

2017 ◽  
Author(s):  
Lorenzo Deveza ◽  
Laura Ortinau ◽  
Kevin Lei ◽  
Dongsu Park
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Statistical Significance ◽  
Fracture Repair ◽  
Functional Difference ◽  
Molecular Signatures ◽  
Reporter Mice ◽  
Selection Of

AbstractPeriosteum and bone marrow (BM) both contain skeletal stem/progenitor cells (SSCs) that participate in fracture repair. However, the functional difference and selective regulatory mechanisms of SSCs in different location are unknown due to the lack of specific markers. Here, we report a comprehensive gene expression analysis of bone marrow SSCs (BM-SSCs), periosteal SSCs (P-SSCs), and more differentiated osteoprogenitors by using reporter mice expressing Interferon-inducible Mx1 and NestinGFP, previously known SSC markers. We first defined that the BM-SSCs can be enriched by the combination of Mx1 and NestinGFP expression, while endogenous P-SSCs can be isolated by positive selection of Mx1, CD105 and CD140a (known SSC markers) combined with the negative selection of CD45, CD31, and osteocalcinGFP (a mature osteolineage marker). Comparative gene experession analysis with FACS-sorted BM-SSCs, P-SSCs, Osterix+ (OSX) preosteoblasts, CD51+ stroma cells and CD45+ hematopoietic cells as controls revealed that BM-SSCs and P-SSCs have high similarity with few potential differences without statistical significance. We also found that CD51+ cells are highly heterogeneous and little overlap with SSCs. This was further supported by the microarray cluster analysis, and the two populations clustered together. However, when comparing SSC population to controls, we found several genes that were uniquely upregulated in endogenous SSCs. Amongst these genes, we found KDR (aka Flk1 or VEGFR2) to be most interesting and discovered that it is highly and selectively expressed in P-SSCs. This finding suggests that endogenous P-SSCs are functionally very similar to BM-SSCs with undetectable significant differences in gene expression but there are distinct molecular signatures in P-SSCs, which can be useful to specify P-SSC subset in vivo.

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

scholarly journals DNA Damage-Response Pathway in Lymphoma Determines Interactions with Macrophages By Altered PD-L1 Expression and Exosome Formation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 275-275
Author(s):  
Daniela Vorholt ◽  
Elena Izquierdo-Alvarez ◽  
Benedict Sackey ◽  
Jan Schmitz ◽  
Nadine Nickel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Research Funding ◽  
Therapeutic Response ◽  
Wild Type ◽  
Lymphoma Cells ◽  
Damage Response ◽  
Dna Damage Response Pathway ◽  
Support Research

Abstract The tumor microenvironment is characterized by multiple interactions of transformed malignant cells with non-transformed stroma or immune cells. Particularly macrophages play a pivotal role in this network determining disease progression and therapeutic response. In previous work we could show that macrophages are an essential mediator of therapeutic response in the synergistic response to the administration of the chemoimmunotherapy. The combination treatment strongly increases tumor clearance by repolarization of tumor-associated macrophages from a suppressive to an activated phenotypic state. Here, se analyzed the functional implications of the DNA damage response pathway for the generation of the ASAP and synergy in chemoimmunotherapy. We attempted to disrupt DNA damage response pathway in lymphoma cells generated from the hMB humanized Double-Hit-Lymphoma model by knock-down of key elements like ATM, DNA-PK or p53. We could prevent the formation of the stimulatory cytokine release effect on macrophage phagocytic capacity. Here, p53 status displays a key regulatory role on macrophage mediated malignant cell depletion. TP53 activation via Nutlin-3A treatment of lymphoma cell enhances ADCP in in p53 wild-type cells, while not displaying enhancement in p53-deficient lymphoma cells. Addressing the treatment in vivo using the hMB model for modeling of Double-Hit Lymphoma bearing mice we could demonstrate diminished ASAP and ADCP for p53-deficient lymphoma treated with cyclophosphamide in vivo. Using primary human CLL patient cells comparing both wild-type and p53-deficient status, the p53-deficient CLL cells failed to induce the stimulatory, cytokine-mediated effect on macrophage phagocytosis in response to combination treatment as seen with the p53 proficient CLL cells. Using a CLL mouse model by treating Eµ-TCL1/p53wt/wt as well as Eµ-TCL1p53-/- mice we could show that low-dose cyclophosphamide treated Eµ-TCL1p53-/- mice failed to induce an antibody mediated stimulatory effect on macrophage phagocytosis capacity as seen with Eµ-TCL1/p53wt/wt mice. A similar effect was seen for primary multiple myeloma cells in response to daratumumab displaying significantly less ADCP of p53-deficient multiple myeloma cells. As for the mechanism of p53-defined interaction within the tumor microenvironment we subjected p53-wild-type and p53-deficient lymphoma cells for proteomic analysis. Here we could identify a significantly deregulated protein expression profile for exosome release in p53 deficient lymphoma cells. Verifying this finding by assessing size and frequency exosomes released by respective cell populations we reveal profound changes induced by p53 loss. Furthermore we could identify up-regulation of PD-L1 in p53-deficient cells. Blocking this checkpoint in the ADCP assay could significantly restore phagocytic capacity of macrophages and overall therapeutic response. In this work, we indicate that p53 functional status determines phagocytic function and therapeutic response to monoclonal antibodies. We can verify this finding in independent models in vitro and in vivo as in primary CLL and myeloma patient cells. We furthermore identify altered exosome profiles and checkpoint inhibitor expression in lymphoma cells as underlying mechanism of macrophage modulation. Finally our ongoing research offers possibility to reveal and tailor new combinatorial treatment approaches for chemo-refractory patients. Disclosures Wendtner: Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding. Hallek:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Pallasch:Gilead: Research Funding.

Negative regulation of activated α2 integrins during thrombopoiesis

Blood ◽  
2009 ◽  
Vol 113 (25) ◽  
pp. 6428-6439 ◽  
Author(s):  
Zhiying Zou ◽  
Alec A. Schmaier ◽  
Lan Cheng ◽  
Patricia Mericko ◽  
S. Kent Dickeson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Surface Expression ◽  
Wild Type ◽  
Vessel Injury ◽  
Rapid Signaling ◽  
Α2 Receptors

Abstract Circulating platelets exhibit rapid signaling and adhesive responses to collagen that facilitate hemostasis at sites of vessel injury. Because platelets are anuclear, their collagen receptors must be expressed by megakaryocytes, platelet precursors that arise in the collagen-rich environment of the bone marrow. Whether and how megakaryocytes regulate collagen adhesion during their development in the bone marrow are unknown. We find that surface expression of activated, but not wild-type, α2 integrins in hematopoietic cells in vivo results in the generation of platelets that lack surface α2 receptors. Culture of hematopoietic progenitor cells ex vivo reveals that surface levels of activated, but not wild-type, α2 integrin receptors are rapidly down-regulated during cell growth on collagen but reach wild-type levels when cells are grown in the absence of collagen. Progenitor cells that express activated α2 integrins are normally distributed in the bone marrow in vivo and exhibit normal migration across a collagen-coated membrane ex vivo. This migration is accompanied by rapid down-regulation of activated surface integrins. These studies identify ligand-dependent removal of activated α2 receptors from the cell surface as a mechanism by which integrin function can be negatively regulated in hematopoietic cells during migration between the adhesive environment of the bone marrow and the nonadhesive environment of the circulating blood.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

Tet2-Deficient Bone Marrow Progenitors Have a Proliferative Advantage in the Presence of TNF-Alpha and IFN-Gamma: Implications for Clonal Dominance in Inflammaging and MDS

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2850-2850
Author(s):  
Samuel Ojo Abegunde ◽  
Michael J. Rauh
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Liquid Culture ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Proliferative Advantage

Abstract Background: TET2 is a hematopoietic tumor suppressor gene that has been implicated in DNA demethylation and the epigenetic regulation of gene expression. Inactivating TET2 mutations are common in aging-associated clonal hematopoiesis of indeterminate potential (CHIP) and myelodysplastic syndromes (MDS). TET2 mutations may contribute to early clonal dominance and myeloid transformation, although the exact mechanisms remain to be elucidated. Emerging evidence suggests that the MDS bone marrow niche may be abnormal and this abnormal niche may act as fertile ground for expansion of neoplastic cells in vivo. Common to the environment of MDS and “inflammaging” are elevations in cytokines, such as TNFa and IFNg. We hypothesized that TET2 mutant clones may thrive in an inflammatory environment and further condition this environment to promote their own survival. Methods: Adult (10-14 weeks-old) Tet2 wild type and Tet2 mutant C57BL/6 mice strains (JAX) were chosen as a model system. The floxed Tet2 allele was deleted by targeting exon 3 with Vav1-cre mediated, hematopoietic-specific excision. We isolated lineage negative cells (Lin-), enriched for hematopoietic stem and progenitor cells (HSPC), from Tet2 wild type and -/- and bone marrow (BM) (EasySep; StemCell Technologies) and cultured these in the absence or presence of TNFα (0.1, 1, or 10 ng/ml) and IFN-γ (0.01, 0.1 or 1 ng/ml) in a methylcellulose colony formation assay (MethoCult; StemCell) or liquid culture media, and then examined their colony growth, cell count and phenotypic characterization over a period of 12 days. Where indicated, serial re-plating was performed. Results: We found an increased proportion of Lin- cells in Tet2 -/- BM compared to wild type, suggesting in vivo HSPC expansion. In triplicate experiments starting with equal numbers of wild type and Tet2 -/- Lin- cells (104 cells/MethoCult well), we found no significant difference in colony counts on days 3, 6, 9, or 12, when cultured in the absence or presence of increasing TNFα concentrations. As expected, TNFα dose-dependently reduced colony counts in both genotypes (up to 3 to 4-fold at 10 ng/ml). However, Tet2 -/- Lin- cells displayed a proliferative advantage over wild type in serial re-plating assays. In the presence of TNFα, this Tet2 -/- re-plating advantage was striking. As exemplified by day 12 colony counts at first re-plating (Fig. 1), while wild type colonies declined with increasing TNFα, Tet2 -/- colony counts increased with TNFα concentration (i.e. average 20-fold higher than wild type at 10 ng/ml TNFα; p<0.05). We next shifted our analysis to IFNg, and found significantly increased day 6, 9 and 12 Tet2 -/- methylcellulose colonies at first plating. Upon re-plating in IFNg, Tet2 -/- cells demonstrated significantly increased (1.5 to 2-fold; p<0.05) mean day 12 colony counts at 0.01, 0.1 and 1 ng/ml IFNg. To gain some insight into the nature of the cells emerging under IFNg stress, we performed flow cytometry upon re-plating. Preliminary experiments revealed increases (1.5 to 2-fold) in Mac1+Gr1+ and Sca1+Kit1+ populations in Tet2 -/-, as compared to wild type, in the presence of IFNg. We are currently comparing apoptosis in wild type and Tet2 -/- cells in the MethoCult system +/- TNFα and IFNg (and a more amenable liquid culture system), using Annexin V/Propidium Iodide-based flow cytometry. These results will be reported. Future directions include the characterization of differential: a) gene expression signatures in Tet2 wild type and -/- Lin- cells under TNFα and IFNg stress, and b) TNFα and IFNg signatures in TET2 -mutant and non-mutant human MDS. Conclusion: Tet2 -deficient murine bone marrow progenitors demonstrate a proliferative advantage, as compared to their wild type counterparts, under TNFα and IFNg stress. Given that these inflammatory cytokines have been associated with inflammaging and myelodysplasia, it is worth exploring whether TET2 -mutant human clones may emerge under inflammatory stress, leading to CHIP and/or MDS, and presenting a novel therapeutic target for clone eradication. Disclosures No relevant conflicts of interest to declare.

scholarly journals Protein Kinase C-β Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B-Cells in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 314-314
Author(s):  
Gloria Lutzny ◽  
Thomas Kocher ◽  
Martina Rudelius ◽  
Marc Schmidt-Supprian ◽  
Ludger Klein-Hitpass ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
B Cells ◽  
Stromal Cells ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Kinase C ◽  
Pkc Β ◽  
Nf Kappab

Abstract Abstract 314 Defects in the apoptosis program are a hallmark of chronic lymphocytic leukemia (CLL), characterized by high expression levels of bcl2 and Mcl1. Notably, this de-regulation of anti-apoptotic proteins is not sufficient to maintain long-term survival of CLL cells, which remain highly dependent on pro-survival factors provided by the leukemia-microenvironment. Bone marrow stromal cells (BMSCs) play an important role for microenvironment mediated survival of CLL cells, based on the provision of soluble and membrane-bound factors. The stroma-CLL interactions not only protect CLL cells from spontaneous, but also from drug-induced apoptosis, clinically recognized as minimal-residual disease. Therefore, understanding the molecular mechanisms of CLL-stroma interactions may offer new therapeutic options and help in eradicating CLL cells from the bone-marrow niche. Here we describe that monoclonal B-cells from CLL patients impose morphological and genetic changes in stromal cells, which become reminiscent of cancer-associated fibroblast (CAF). Comparative gene expression profiles indicate that contact with primary CLL cells induce the expression of pro-inflammatory genes in stromal cells. Further characterization of the underlying signaling pathways activated in stromal cells revealed that CLL cells induce the expression of protein-kinase C-β in BMSCs. Blocking the up-regulation of PKC-β by siRNA abrogated the pro-survival effects of stromal cells on CLL cells. Furthermore, following induction of PKC-β, BMSCs activate NF-kappaB through a Bcl10-independent, but NEMO/IKKgamma-dependent pathway. Gene expression profiling of NEMO-proficient and deficient BMSCs indicated that NF-kappaB regulates the expression of pro-inflammatory cytokines and adhesion molecules by stromal cells, required to promote survival of CLL. Interference with the NF-kappaB activation in BMSCs abrogated the pro-survival effects of stromal cells on CLL, similar to PKC-β deficient stromal cells. To demonstrate that this pathway is also important in vivo, Tcl1-CLL was transplanted into syngeneic PKC-β knock-out and wild-type mice. Notably, all PKC-β wild-type mice died of a CLL-like disease, whereas PKC-β kock-out animals were entirely resistant to CLL transplants. Immunofluorescence staining of PKC-β in bone marrow trephine biopsies indicated that this pathway is also activated in mesenchymal stromal cells of CLL patients. Importantly, our data provide further evidence that the PKC-β – NF-kappaB pathway is also activated in stomal cells by monoclonal B-cells from ALL and mantle-cell lymphoma (MCL) patients. Conclusively, we describe a novel survival signaling pathway activated by monoclonal B-cells in BMSCs. Interference with the PKC-β-NF-kappaB pathway activated in the leukemia/lymphoma microenvironment may offer new therapeutic options to fully eradicate malignant B-cells from bone-marrow niches. Disclosures: No relevant conflicts of interest to declare.

HSC Exit From Dormancy Provokes De Novo DNA Damage, Leading To Bone Marrow Failure If Unresolved By The Fanconi Anemia Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 799-799
Author(s):  
Dagmar Walter ◽  
Amelie Lier ◽  
Anja Geiselhart ◽  
Sina Huntscha ◽  
David Brocks ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Aplastic Anemia ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Damage Response ◽  
Fold Induction

Abstract Long-term quiescence has been proposed to preserve the genomic stability of hematopoietic stem cells (HSCs) during aging. The current models of HSC aging are limited in their ability to observe both DNA damage in vivo and the consequences of this damage upon hematopoiesis. Fanconi Anemia (FA) is a hereditary multisystem disorder, characterized by defective DNA damage response and progressive bone marrow failure in most patients. However, the existing genetic models of FA do not develop aplastic anemia, suggesting that cell-extrinsic factors may play a causal role. We sought to identify whether physiologic mediators of HSC activation could be used as agonists to provoke DNA damage and HSC attrition in vivo. Mice were treated with a range of agonists that promote the in vivo exit of HSC from a dormant state into active cycling (polyI:polyC; Interferon-α; G-CSF; TPO; and serial bleeding). Highly purified HSC demonstrated a rapid 3-5-fold induction of DNA damage after treatment with all agonists (p<0.01), as assessed by both enumerating γ-H2AX foci and by alkaline comet assay. Mechanistically, stress-induced exit from quiescence correlated with increased mitochondrial metabolism in HSC, as evaluated by elevated mitochondrial membrane potential (2-fold increased, p<0.01) and superoxide levels (1.5-fold increased, p<0.05). Critically, we could directly implicate these reactive oxygen species in DNA damage as we observed a 1.4-fold increase in 8-Oxo-dG lesions in HSC that had been activated into cycle in vivo(p<0.05). At 48 h post-treatment, γ-H2AX levels began to decrease and this repair was concomitant with an induction of the FA signaling pathway in HSC, as demonstrated by both increased levels of FA gene expression and elevated FANCD2 foci (4-fold induction, p<0.01). Treatment of Fanca-/- mice with polyI:polyC led to a HSC proliferative response comparable to wild type (WT) mice but resulted in a 2-fold higher level of activation-induced DNA damage (p<0.05), demonstrating that this repair pathway is involved in resolving activation-induced DNA damage. Four rounds of serial in vivo activation led to a permanent depletion of the most primitive label-retaining Fanca-/- HSC and this correlated with a 4-fold depletion of functional HSC (p<0.01) as defined by competitive repopulation assays. Subsequent rounds of HSC activation with polyI:polyC resulted in the onset of a severe aplastic anemia (SAA) in 33% of treated Fanca-/- mice but not in any of the WT controls. SSA was characterized by a dramatic reduction in bone marrow (BM) cellularity, profound thrombocytopenia (21-246x106 platelets/ml), leukocytopenia (0.4-0.5x106 WBC/ml), neutropenia (0.03-0.1x106/ml) and anemia (1.5-2.3 g/dL Hb). Examination of BM HSC/progenitors demonstrated nearly complete loss of HSC, MPP, CMP and CLP (depletion of ≥33x, 8x, 4x and 12x respectively compared to PBS-treated Fanca-/-controls). Taken together, these data demonstrates that enforced exit from dormancy in vivo leads to de novo DNA damage in HSC, which is repaired by activation of a FA-dependent DNA damage response. Furthermore, the highly penetrant bone marrow failure observed in Fanconi anemia patients can be recapitulated by the serial application of a physiologic HSC activating signal to Fanca-/- mice. This suggests that the BM failure in FA may be caused by an aberrant response to HSC activation, most likely during exposure to infection or other physiologic stressors. These data provides a novel link between pro-inflammatory cytokines, DNA damage and HSC dysfunction and may have important clinical implications relevant to both prevention of BM failure in FA and in the study of age-related hematopoietic defects in non-FA patients. Moreover, these data provide the first evidence that FA knockout mouse models accurately recapitulate and provide novel insights into the etiology of BM failure in patients with FA. Disclosures: No relevant conflicts of interest to declare.

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