Deletion of Irf5 protects hematopoietic stem cells from DNA damage-induced apoptosis and suppresses γ-irradiation-induced thymic lymphomagenesis

Abstract DNA double strand breaks (DSBs) are the most dangerous genomic lesions that can be induced by endogenous and exogenous sources. DNA damage response determines cellular fate decisions following DSBs and can lead to cell death or cell survival. Incorrect DSB repair via canonical Non-Homologous End Joining (cNHEJ) or Alternative NHEJ (Alt-NHEJ) is the main source of oncogenic aberrations, including leukemogenic translocations, DNA sequence deletions and insertions. The long life span of Hematopoietic Stem Cells (HSC) and their practically unlimited potential for self-renewal requires efficient strategies to cope with DNA damage to eliminate erroneous genetic information inheritance to daughter cells. Although the critical importance of maintaining genome integrity for normal hematopoiesis and prevention of leukemogenesis has been established, definitive analysis of DNA damage response and its mutagenic outcomes in human HSC and Progenitors in response to DSBs is missing. Here we repot that human cord blood purified HSC (defined as CD34+CD38-CD45RA-) are exquisitely sensitive to irradiation (IR)-induced apoptosis in contrast to committed progenitors (defined as CD34+CD38+) as validated by PARP cleavage induction. Interestingly, pan-caspase inhibitor Z-VAD-FMK prevented, whereas CHK2 inhibitor (PV1019) failed in altering apoptosis onset of irradiated HSC. Strikingly, CHK2 inhibitor blocked IR-induced apoptosis in cycling HSC, suggesting differential wiring of DNA damage induced apoptosis in quiescent versus mitogenically stimulated HSC. To characterize cNHEJ repair pathway and its mutagenic potential in live primitive hematopoietic cells we analyzed I-SceI endonuclease induced tandem DSBs joining capacity using DNA repair reporter assay. We found that HSC exhibit inferior cNHEJ capacity as compared with committed progenitors. By decreasing DSBs persistence we revealed that progenitors utilize to the higher degree than HSC the mutagenic component of cNHEJ pathway that results in DNA deletions. We identified HSC-specific contribution of CHK2 kinase activity in limiting incorrect DNA ends joining. Blockade of apoptosis induction also led to the selective increase in mutagenic NHEJ in HSC. On the other hand, inhibition of DNA-PK led to increased oncogenic repair in progenitors only. Importantly, we revealed that HSC utilized mutagenic Alt-NHEJ pathway that depends on microhomologies search and extensive DNA ends processing less efficiently than Progenitors. Thus, our results indicate that oncogenic consequences of DSBs repair in HSC are distinctly minimized by the non-redundant cell death and CHK2 dependent mechanisms. More broadly, these findings will help to elucidate additional repair modifiers and the mechanism by which HSC contend with genotoxic stress. Disclosures No relevant conflicts of interest to declare.

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¬¬Attenuated DNA Damage Responses and Increased Apoptosis Characterize Human Hematopoietic Stem Cells Exposed to Irradiation

Experimental Hematology ◽

10.1016/j.exphem.2018.06.097 ◽

2018 ◽

Vol 64 ◽

pp. S87

Author(s):

Michael Milyavsky ◽

Shahar Biechonski ◽

Leonid Olender ◽

Adi Zipin-Roitman ◽

Muhammad Yassin ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem ◽

Dna Damage Responses

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Genistein Protects Hematopoietic Stem Cells against G-CSF–Induced DNA Damage

Cancer Prevention Research ◽

10.1158/1940-6207.capr-13-0295 ◽

2014 ◽

Vol 7 (5) ◽

pp. 534-544 ◽

Cited By ~ 4

Author(s):

Liliana R. Souza ◽

Erica Silva ◽

Elissa Calloway ◽

Omer Kucuk ◽

Michael Rossi ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem

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NF-kB Family Proteins Participate in Multiple Steps of Hematopoiesis through Elimination of Reactive Oxygen Species (ROS).

Blood ◽

10.1182/blood.v104.11.1694.1694 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1694-1694

Author(s):

Soichi Nakata ◽

Itaru Matsumura ◽

Hirokazu Tanaka ◽

Yusuke Satoh ◽

Takumi Era ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Es Cells ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Growth And Survival ◽

Ros Accumulation ◽

Dependent Growth ◽

Induced Apoptosis

Abstract NF-kB family proteins have been reported to induce the expression of over 150 target genes, thereby crucially regulating immune responses, stress responses, and inflammation. These proteins also play important roles in cell growth and survival in various cell types. However, the precise roles of NF-kB in hematopoiesis and their mechanisms remain undetermined. To examine the roles for NF-kB family proteins in the growth and survival of hematopoietic cells, we expressed dominant negative NF-kB (IkBSR) in a murine IL-3-dependent cell line Ba/F3 using a Lac-inducible system, in which IkBSR was inducibly expressed by the IPTG treatment; this clone was designated Ba/F3/IkBSR. Furthermore, we introduced EPO receptor (R), TPOR, and G-CSFR/gp130 consisting of the extracelluar domain of G-CSFR and cytoplasmic domain of gp130 into this clone. At first, we confirmed that these clones could survive and proliferate under the cultures with IL-3, EPO, TPO, G-CSF, respectively. Although IPTG-induced IkBSR slightly suppressed IL-3- and EPO-dependent growth at low concentrations, it did not affect TPO- or gp130L-dependent growth, suggesting that NF-kB might not be so important for cytokine-dependent growth of hematopoietic cells. In contrast, IkBSR prominently enhanced factor-deprived apoptosis, which was accompanied by the ROS accumulation. To access the roles of ROS in IkBSR-enhanced apoptosis, we overexpressed ROS scavenger enzymes MnSOD and thioredoxin X (TRX) in Ba/F3/IkBSR, respectively. As a result, MnSOD and TRX significantly canceled IkB-SR-enhanced apoptosis, suggesting that ROS would be responsible for this apoptosis. We next analyzed the effects of IkBSR on the growth and survival of normal hematopoietic cells. When IkBSR was introduced into murine Lin−Sca-1+ hematopoietic stem/progenitor cells with the retrovirus system, it induced apoptosis even in the presence of appropriate cytokines. This apoptosis was also accompanied by the ROS accumulation due to the downregulated expression of anti-oxidants such as glutathione, MnSOD, glutathione peroxidase, and TRX. In addition, the expression of antiapoptotic BCl-2 family members, Bcl-XL, Bcl-2, and A1 was found to be repressed by IkBSR. However, since antioxidants such as MCI (3-methyl-1-phenyl-2-pyrazolin-5-one), N-acetylecysteine and TRX cancelled this apoptosis, ROS were supposed to be more important for IkBSR-induced apoptosis in normal hematopoietic stem/progenitor cells. To further analyze the roles for NF-kB proteins in the development of hematopoietic cells, we expressed IkBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from ES cells. When IkBSR was expressed at the stage of hemangioblasts, IkBSR induced apoptosis and inhibited the development of hematopoietic stem cells, which was also cancelled by MCI. Furthermore, when IkBSR was expressed after the development of hematopoietic stem cells, it also inhibited terminal differentiation towards granulocytes, erythrocytes, and megakaryocytes through ROS-mediated apoptosis; IkBSR inhibited granulopoiesis before the development of myeloblasts, erythropoiesis after the development of proerythroblasts, and megakaryopoiesis during polyploidization of megakaryocytes. These results indicate that NF-kB family proteins play essential roles to prevent apoptosis at multiple steps of hematopoiesis by eliminating ROS.

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A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs

Blood ◽

10.1182/blood-2018-99-114844 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1108-1108

Author(s):

Masoud Nasri ◽

Perihan Mir ◽

Benjamin Dannenmann ◽

Diana Amend ◽

Yun Xu ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Cell Line ◽

Gene Editing ◽

Jurkat Cells ◽

Cell Type ◽

Hematopoietic Stem ◽

Editing Efficiency ◽

Human Ipscs

Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.

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Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.2064oia143 ◽

2014 ◽

Vol 206 (4) ◽

pp. 2064OIA143

Author(s):

Cesare Lancini ◽

Paul C.M. van den Berk ◽

Joseph H.A. Vissers ◽

Gaetano Gargiulo ◽

Ji-Ying Song ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Dna Damage Response ◽

Hematopoietic Stem ◽

Functional Integrity ◽

Damage Response

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DNA damage and aging on hematopoietic stem cells: Impact of oxidative stress in ApoE−/− mice

Experimental Gerontology ◽

10.1016/j.exger.2021.111607 ◽

2021 ◽

pp. 111607

Author(s):

R. Aires ◽

M.L. Porto ◽

L.M. de Assis ◽

P.A.N. Pereira ◽

G.R. Carvalho ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem

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Inactivation of PUMA Protects Hematopoietic Stem Cells and Confers Long Term Survival in Response to High Dose γ-Irradiation

Blood ◽

10.1182/blood.v112.11.612.612 ◽

2008 ◽

Vol 112 (11) ◽

pp. 612-612 ◽

Cited By ~ 1

Author(s):

Hui Yu ◽

Hongmei Shen ◽

Feng Xu ◽

Xiaoxia Hu ◽

Yanxin Li ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Radiation Injury ◽

Γ Irradiation ◽

Hematopoietic Stem ◽

Cycle Arrest ◽

Stem And Progenitor Cells ◽

Radiation Induced

Abstract Radiation injury remains a significant health problem. New medical intervention to prevent or manage radiation damage is highly dependent on a deeper understanding of how radiation-induced cell death is accomplished in the irradiated tissue cells such as stem and progenitor cells. To date, relatively specific or untainted molecular mediators in apoptosis of tissue stem and progenitor cells upon radiation injury have not been clearly defined. The p53 pathway is known as a major molecular mechanism for cell apoptosis, upon the exposure of lethal radiation. Targeting p53 confers a radioprotective effect, but may increase tumorigenesis due to impaired cell cycle arrest for DNA repair. In our current study, we have examined the specific role of PUMA (p53 up-regulated mediator of apoptosis) in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). By quantitative RT PCR, we found that the level of PUMA mRNA was relatively low in the most primitive long-term repopulating hematopoietic stem cells (LT-HSC, isolated based on the immnunophenotype “CD34−LKS”) as compared to other hematopoietic cell populations from mice, but it was significantly elevated in response to γ-irradiation. In the mice lacking PUMA, while neither HSC number nor HSC function was altered under homeostatic conditions, the PUMA−/− HSCs appeared to be resistant to radiation damage in vivo as retrospectively quantified in a competitive HSC transplant model. Our further direct measurement with a single cell culture system for HSC growth in vitro, demonstrated that PUMA, but not p21 (the chief mediator of p53 in cell cycle arrest), is primarily responsible for the radiosensitivity of HSC in the p53 pathway (200 LT-HSCs analyzed for each cell type). Together, these data provide definitive evidence for PUMA as an essential mediator in radiation-induced apoptosis of tissue stem cells. We finally focused on the beneficial effects of targeting PUMA in HSCs and HPCs on the animal survival upon the exposure of lethal irradiation. Strikingly, the wild-type mice reconstituted with PUMA−/− hematopoietic cells exhibited a significant survival advantage after two rounds of 9-Gy γ-irradiation (18 Gy in total) as compared to the mice reconstituted with PUMA+/+ hematopoietic cells (95 % vs. 0 % survival in 20 days, n=21/each group; 50% vs. 0 % survival in 180 days, n=20 or 11/each group, respectively) as shown in the figure below. Moreover, unlike the p53−/− mice, those PUMA−/− reconstituted mice did not have an increased incidence of hematopoietic malignancies (n=20) within 180 days. Therefore, our current study establishes PUMA as an attractive molecular target for the development of therapeutic agents for the prevention and treatment of radiation injury.

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p53 Co-Activator Aspp1 Induces Apoptosis in Damaged Hematopoietic Stem Cells and Prevents Malignant Transformation

Blood ◽

10.1182/blood.v124.21.603.603 ◽

2014 ◽

Vol 124 (21) ◽

pp. 603-603 ◽

Cited By ~ 1

Author(s):

Masayuki Yamashita ◽

Eriko Nitta ◽

Toshio Suda

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Hematological Malignancies ◽

Aberrant Methylation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Apoptotic Genes

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Mutation accumulation in HSCs precedes the development of leukemia and lymphoma, and these “pre-leukemic HSCs” can survive after chemotherapy, contributing to the relapse of the disease. Thus, understanding for the DNA damage response at a HSC level is a matter of critical importance for lifelong hematopoiesis, yet the protection mechanism for HSCs from DNA damage accumulation remains to be elucidated. During our study on the response of HSCs to ionizing radiation (IR), we have detected higher responsiveness of HSCs to DNA damage compared with committed progenitor cells: higher p53 activation was observed in HSC-enriched LSK (Lin-Sca1+cKit+) cells and LT-HSCs (CD150+CD41-CD48-LSK) than in myeloid progenitor-enriched LKS- cells. Of note, when treated with 4 Gy IR, LSK cells exhibited stronger upregulation of pro-apoptotic genes Bax, Noxa and Puma compared with LKS- cells, whereas upregulation of survival-contributing p21 and Mdm2 genes was comparable between the two populations. Corresponding to such characteristic behavior, we have identified apoptosis-stimulating protein of p53 1 (Aspp1) as a novel specific regulator of HSCs that provides HSCs with high sensitivity to apoptosis. We found that mRNA and protein of Aspp1 were specifically detected in LSK cells and LT-HSCs. To uncover the roles of Aspp1 in the regulation of HSCs, we evaluated HSCs of adult Aspp1 knockout (KO) mice. These mutant mice exhibited a major increase in the absolute number of LSK cells (1.5-fold; P<0.05) and LT-HSCs (2-fold; P<0.0005). Furthermore, self-renewal capacity of Aspp1-null HSCs was significantly enhanced as measured by serial competitive bone marrow (BM) transplantation assays (P<0.01). To assess the cause of enhanced self-renewal of Aspp1-null HSCs, we examined gene expression profile of Aspp1-null LSK cells before and after BM transplantation using multiplex quantitative RT-PCR array. Aspp1-null LSK cells showed higher expression of multiple quiescence-related genes including Tek, Mpl and Ndn. In line with this, Ki67 staining revealed that Aspp1-null LSK cells showed resistance to the loss of quiescence after serial BM transplantation (P<0.01), and Aspp1 KO mice showed accelerated recovery of peripheral blood and BM when treated with a single dose of 5-FU (P<0.05). Moreover, when serially transplanted or subjected to 4 Gy IR in vivo, Aspp1-null LSK cells exhibited higher resistance to apoptosis which was detected as decreased proportion of Annexin V-positive cells (P<0.05). Gene expression analysis consistently revealed that the induction of pro-apoptotic genes Bax, Noxa and Puma was impaired in irradiated Aspp1-null LSK cells. As a result of the reduced apoptosis, Aspp1-null LSK cells exhibited the tendency to retain persistent DNA damage after genotoxic stress as assessed by γH2AX and 53BP1 foci (chi-square test, P<0.05). Importantly, by breeding Aspp1 KO mice with Mx1-Cre mice and p53flox/flox mice, we verified that Aspp1 synergized with p53 to regulate self-renewal and genomic integrity of HSCs beyond its canonical p53-dependent function. Aspp1 loss further enhanced self-renewal capacity of HSCs in a p53-null background when assayed by serial BM transplantation (P<0.05). Likewise, Aspp1 deficiency further accentuated the accumulation of DNA damage after IR exposure in the absence of p53 (P<0.05). Consequently, whereas approximately half of the recipients receiving p53-null LSK cells died of thymic lymphoma, the recipient mice transplanted with LSK cells deficient for both Aspp1 and p53 were 100% lethal within 6 months after BM transplantation (log-rank test, P<0.01). These mice succumbed to hematological malignancies, mostly T-cell acute lymphoblastic lymphoma and leukemia (ALL) (88%) but also B-cell (6%) and myeloid (6%) malignancies. Taken together, our study demonstrates that Aspp1 attenuates HSC quiescence and induces apoptosis in damaged HSCs, in both p53-dependent and -independent manners, thereby inhibiting the development of leukemia and lymphoma in conjunction with p53 in HSCs. As loss of Aspp1 expression due to aberrant methylation of its promoter has already been proven to be an independent poor prognosis factor in ALL patients, Aspp1 may be a potential target for stem cell-directed therapy of leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.

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