Conditional TRF1 Knockout in Haematopoietic Progenitor Cells Causes Telomere Shortening and Bone Marrow Failure by Induction of Cellular Senescence

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 49-49
Author(s):  
Fabian Beier ◽  
Miguel Foronda ◽  
Jose A Palacios ◽  
Paula Martinez ◽  
Maria A Blasco
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Conditional Knockout ◽  
Telomere Maintenance ◽  
Loss Of Function ◽  
Telomere Shortening ◽  
Exon 1

Abstract Abstract 49 Introduction: Mutations in the telomerase complex may cause bone marrow failure syndromes due to loss-of-function and consecutive telomere shortening. In addition to the telomerase complex, the six “shelterin” proteins (TRF1, TRF2, TIN2, RAP1, POT1 and TPP1) are required for telomere maintenance. TRF1 has a prominent role in chromosome capping function and prevents the recognition of telomeres by DNA repair mechanisms. At the moment, only TIN2 mutations have been linked to bone marrow failure. Here we aimed to identify other shelterin proteins might cause bone marrow failures. A previous study reported an clinical association between TRF1 mutations and acquired aplastic anemia, however the proof-of-principle that TRF1 can cause bone marrow failure is still missing (Savage SA Exp Hematol 2006). Material and Methods: To address this issue, we used the Mx1-Cre system in combination with the recently generated TRF1 allele in which the exon 1 of TRF1 is flanked by floxP (Martinez P Gen Dev 2009). The bone marrow of the bitransgenic mice was transplanted into B6 wildtype mice and poly (P:I) injections allowed the conditional knockout of TRF1. Results: Initiation of poly (P:I) injections 4 weeks after transplantation resulted in a failure of all three haematopoietic lineages after 17 days and histopathology revealed massive hypocellular bone marrow consistent with a bone marrow failure. Transplanted control animals showed normal histopathology and even increased neutrophil and thrombocyte counts. Further detailed FACS analysis 7 days after initiation of poly (P:I) injections showed a significant depletion of common myeloid, megakaryocte-erythocyte and common lymphoid progenitor cells, but only a slight decrease of lin-, c-kit+,Sca-1+ haematopoietic stem cells. Interesting, we found no increased rate of apoptosis for the decrease of the progenitor cells, but ß-galactosidase staining showed significant higher amounts of senescent cells in the bone marrow. Further detailed analysis of FACS sorted bone marrow cells showed that especially the c-kit positive progenitor fraction underwent senescence and cell cycle analysis showed an increased G2-M phase indicating a G2-M arrest. In line with these findings RT-PCR of FACS sorted BM revealed increased levels of p21 in the c-kit positive fraction. In addition BrdU injections into the mice on day 7 after poly (P:I) initiation showed increased incorporation and telomere length analysis of transplanted animals with and without poly (P:I) injections revealed massive telomere shortening on day 17. Conclusions: Our data indicates that TRF1 knockout especially affects haematopoietic progenitor cells by inducing G2-M arrest, induction of p21, and subsequent senescence. Further, compensation of the progenitor cell depletion leads to higher cell turnover and consecutively massive telomere shortening. Taken together this is the first report proving that TRF1 can cause a bone marrow failure and is accompanied with significant telomere shortening. Disclosures: No relevant conflicts of interest to declare.

Defective Hematopoiesis and Erythroid Differentiation Resulting From Telomerase Deficiency

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2410-2410
Author(s):  
Aparna Raval ◽  
Brenda Kusler ◽  
Steven Artandi ◽  
Beverly S. Mitchell
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Failure ◽  
Telomere Maintenance ◽  
P Value ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Telomere Shortening ◽  
Cell Numbers ◽  
Bone Marrow Failure Syndromes

Abstract Abstract 2410 Telomere shortening and anemia are common in bone marrow failure syndromes (BMF) such as dyskeratosis congenita (DKC), acquired aplastic anemia and myelodysplastic syndromes (MDS). Components of the telomerase complex including TERT, TERC and Dyskerin are mutated in multiple BMF syndromes, strongly suggesting a link between aberrant telomere maintenance and BMF. However, the specific defects in hematopoiesis that lead to BMF have not been completely defined. To better understand the effects of telomere shortening on hematopoiesis, we crossed Tert−/− mice for five successive generations to derive 5th generation Tert−/− (G5 Tert−/−) mice with short telomeres. The G5 Tert−/− mice were smaller in size and their peripheral blood cell counts had significantly reduced numbers of red blood cells and hemoglobin content (p-value < 0.01). There was also a significant reduction in immature erythroid (CD71+; p-value < 0.03) and mature erythroid (CD71-Ter119+; p-value < 0.05) cells in the bone marrows of these mice as compared to the G0 Tert+/− controls, while myeloid (Mac-1, Gr1+) cell numbers were not decreased. Annexin V and PI staining showed increased apoptosis in immature erythroid cells from G5 Tert−/− mice. In order to determine if the reduction in erythrocytes was due to defective hematopoiesis, we studied hematopoietic stem cell (HSC), committed erythroid progenitor (MEP) and myeloid progenitor (GMP) cell numbers in these mice. We observed a marked decrease in MEP (Lin-cKit+Sca1-CD34-CD16/CD32lo; p-value < 0.02) and HSC (Lin-cKit+Sca1+CD34-CD150+; p-value < 0.03) populations in G5 Tert−/− mice marrow, while GMP (Lin-cKit+Sca1-CD34+CD16/CD34hi; p-value < 0.6) cells were not affected. In contrast, there was an increase in the number of immature erythroid cells (CD71+), hematopoietic progenitor cells (Lin-cKit+Sca1+) and HSC in the spleens of G5 Tert−/− mice, strongly suggesting extramedullary hematopoiesis. These data demonstrate that there is a defect in the regulation of hematopoiesis, starting at the level of HSC, in the bone marrow of G5 Tert−/− mice. MEPs from G5 Tert−/− mice showed increased DNA damage, as demonstrated by phospho-H2Ax staining by flow cytometry, although enhanced apoptosis was not present. In contrast, GMPs did not show any difference in phospho-H2Ax staining, suggesting that MEPs may be selectively sensitive to telomere shortening or that telomeres shorten more rapidly in these cells. Total bone marrow cells from G0 Tert+/− and G5 Tert−/− mice were plated in Methocult supporting erythroid colony formation; we observed that the G5 Tert−/− cells had significantly reduced numbers of CFU-E and BFU-E colonies (p-value < 0.01) compared to cells from the G0 mice, suggesting that the ability of erythroid progenitor cells to differentiate is compromised by dysfunctional telomeres. These data show for the first time a direct and distinctive link between the loss of telomerase function and erythropoiesis. These results, in conjunction with single-cell “mass cytometry” experiments to explore the molecular pathways that are selectively deregulated in the HSC and erythroid precursors of G5 Tert−/− mice, will elucidate the mechanisms underlying the cell-specific effects of telomere shortening on erythropoiesis and help to elucidate the relationship between shortened telomeres and the anemia of bone marrow failure syndromes. Disclosures: No relevant conflicts of interest to declare.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 513-513
Author(s):  
Pekka Jaako ◽  
Shubhranshu Debnath ◽  
Karin Olsson ◽  
Axel Schambach ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Stem And Progenitor Cells

Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.

Calr Mutants Retroviral Mouse Models Lead to a Myeloproliferative Neoplasm Mimicking an Essential Thrombocythemia Progressing to a Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 157-157 ◽  
Author(s):  
Caroline Marty ◽  
Nivarthi Harini ◽  
Christian Pecquet ◽  
Ilyas Chachoua ◽  
Vitalina Gryshkova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
In Vivo Model ◽  
Common Mechanism ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Platelet Counts

Abstract Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are malignant homeopathies resulting from the transformation of a multipotent hematopoietic stem cell (HSC). The common mechanism of transformation is the constitutive activation of the cytokine receptor/JAK2 pathway that leads to the myeloproliferation. The acquired point mutation JAK2V617F is the most prevalent (95% of PV and 60% of ET or PMF). In addition, other mutations affecting the same signaling pathway have been described such as JAK2 exon 12 mutations, mutations of MPL affecting W515, and loss-of-function mutations of LNK and also mutations of c-Cbl in 3% of PMF. Recently, whole exome sequencing allowed identifying a new recurrent genetic abnormalities in the exon 9 of the calreticulin gene (CALR) in about 30% of ET and PMF patients. All CALR mutants induce a frameshift of the same alternative reading frame and generate a novel C-terminus tail. To address the role of these new mutants in the pathophysiology of MPN, the goal of this study was to investigate the effect of the CALR mutant (del52 and ins5) expression by a retroviral mouse modeling. For that purpose, we transduced bone marrow cells with retrovirus expressing either CALRdel52, CALRins5, CALRWT or CALRDexon9 and performed a transplantation in lethally irradiated recipient mice (10 mice / group), which were then followed over one year. CALRdel52 expressing mice showed a rapid and strong increased in platelet counts (over 5 x106/mL) without any other changes in blood parameters during 6 months. In contrast, CALRins5 expressing mice presented platelet counts much lower than CALRdel52 but significantly higher than CALRWT or CALRDexon9 expressing mice. After 6 months, CALRdel52 expressing mice showed a decreased in platelets count associated with anemia and development of splenomegaly suggesting the progression to a myelofibrosis. Importantly, the disease was transplantable to secondary recipient for both CALRdel52 and CALRins5 mutants. The bone marrow and spleen were also analyzed over time. We observed a progressive increased in immature progenitors (SLAM cells) as well as a hypersensitivity of the megakaryocytic progenitors (CFU-MK) to thrombopoietin. Altogether, these results demonstrate that CALR mutants are able and sufficient to induce a thrombocytosis progressing to myelofibrosis in retroviral mouse model, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPN. Disclosures No relevant conflicts of interest to declare.

Benzene-Induced Leukemogenesis: Irreversible Inhibition of PTPN2 and Subsequent STAT1 Signaling Alteration By the Hematotoxic Metabolite Benzoquinone

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3671-3671
Author(s):  
Romain Duval ◽  
Linh-Chi Bui ◽  
Cécile Mathieu ◽  
Emile Petit ◽  
Jean-Marie Dupret ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Hematopoietic Cells ◽  
Loss Of Function ◽  
Irreversible Inhibitor ◽  
Irreversible Inhibition ◽  
Stat Signaling

Abstract Benzene (BZ) is a chemical compound of industrial and toxicological interest classified as a class I human carcinogen. Environmental and occupational exposure to BZ lead to bone marrow malignancies such as leukemia. The leukemogenic effects of BZ relies on its metabolization in bone marrow cells into reactive metabolites, in particular benzoquinone (BQ) that can react with macromolecules (arylation) and/or induce oxidative stress. Although BZ is well recognized as a leukemogenic chemical, most of the key molecular and cellular mechanisms underlying its hematotoxicity are not fully understood. PTPN2 is a protein tyrosine phosphatase (PTP) mainly expressed in hematopoietic cells and playing a key role in the homeostasis of the hematopoietic system. In particular, this PTP is an important modulator of growth factors and JAK/STAT signaling pathways. Loss of function analyses in patients with mutation/deletion of the PTPN2 gene and knock-out mouse models indicate that PTPN2 acts as a tumor suppressor in haematologic disorders such as leukemia. We found that BQ, the prime hematotoxic metabolite of BZ, is an irreversible inhibitor of human PTPN2. Kinetic and biochemical analyses using purified PTPN2 indicated that the irreversible inhibition of the enzyme by BQ is mainly due to arylation of its active site cysteine. Exposure of immortalized human hematopoietic cells (Jurkat T and THP-1 lines) to BQ leads to the irreversible inhibition of endogenous PTPN2 activity with a concomitant over activation of JAK/STAT signaling pathway. Irreversible BQ-dependent inhibition of PTPN2 in cells was found to be mainly due to overoxydation of its catalytic cysteine into sulfinic and/or sulfonic forms. In Vivo experiments conducted in mice confirmed that exposure to BZ leads to irreversible inhibition of PTPN2 in bone marrow and spleen cells. Our data provide the first mecanistic evidence that irreversible inhibition of PTPN2, a tumor suppressor tyrosine phosphatase, may contribute to benzene-dependent leukemogenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential Effects of TLR1/2 and TLR2/6 Signaling on Normal and Premalignant Hematopoietic Stem and Progenitor Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1806-1806
Author(s):  
Darlene A. Monlish ◽  
Zev J. Greenberg ◽  
Sima T. Bhatt ◽  
Dagmar Ralphs ◽  
John L. Keller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
White Blood Cells ◽  
Hematopoietic Stem ◽  
Specific Effects ◽  
Stem And Progenitor Cells ◽  
Cell Cycling

Abstract Prior studies from our lab and others have demonstrated a role for Toll-like receptor 2 (TLR2) in regulating both normal and premalignant hematopoietic stem and progenitor cells (HSPCs), however the contributions of its binding partners, TLR1 and TLR6, remain unknown. In CD34+ bone marrow cells of patients with myelodysplastic syndrome (MDS), increased TLR2 was associated with lower-risk disease, elevated rates of apoptosis associated with improved prognosis, and enhanced survival. Conversely, increased levels of TLR6, but not TLR1, was associated with higher-risk disease and an increased percentage of bone marrow blasts (Zeng et al., Exp Cell Res 2016 and Wei et al., Leukemia 2013). These data suggest that there may be heterodimer-specific effects of TLR2 signaling on HSPCs influencing disease progression. To elucidate the unique contributions of the heterodimer pairs in MDS pathogenesis and leukemogenesis, we utilized a well-established mouse model of MDS that expresses the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter. The "NHD13" mice recapitulate many of the salient features of human MDS and succumb to cytopenias or leukemia by 14 months of age (Lin et al., Blood 2005). Importantly, we observed significantly increased expression of TLRs 1, 2, and 6 on the c-Kit+, Sca-1+, Lineage- ("KSL") HSPCs of the NHD13 mice, similar to the increased expression of these TLRs on CD34+ cells of MDS patients. To begin to delineate the heterodimeric differences, NHD13 mice were treated chronically with either PAM2CSK4 (PAM2), a TLR2/6-specific agonist, or PAM3CSK4 (PAM3), a TLR1/2-specific agonist, to assess the effects on cytopenias and survival. After five months of treatment, a significant increase was observed in the total number of white blood cells in NHD13 mice treated with PAM2 (p=0.007), but not PAM3 (vs. vehicle (water)-treated controls), a finding that was not recapitulated in wild-type (WT) controls. On the contrary, a significant decrease in the total number of platelets in both NHD13 and WT mice treated with PAM3 was observed as compared to vehicle-treated controls (p=0.024 and p=0.011, respectively). Further supporting the existence of heterodimer-specific differences, death was expedited in NHD13 mice treated with PAM2 as compared to those treated with PAM3 (p=0.019), with a median survival of 243 days vs. 338 for the PAM3-treated cohort. The cause of death, as determined by a hematopathologist based on cytology and blast percentage, was most often due to leukemia. To investigate the potential mechanism through which enhanced TLR2/6 signaling accelerates leukemogenesis and death in NHD13 mice, the HSPCs of premalignant NHD13 mice treated with PAM2 or PAM3 were characterized by flow cytometry and evaluated for cell cycling and cell death. Both the total number and frequency of KSL cells were significantly increased in NHD13 mice treated with PAM2 (p=0.007 and p<0.0001, respectively), but not PAM3, vs. water-treated controls. No significant changes were noted in either cell cycling or apoptosis following agonist treatment. A microarray of bone marrow KSL cells revealed that stimulation of the TLR2/6 pathway is associated with an activated c-Myc signature, suggesting that enhanced signaling through this pathway, but not TLR1/2, may enhance leukemogenesis via Myc activation. Further, the expression levels of six downstream targets of c-Myc, including BAX, APEX1, ODC1, FKBP4, NCL, and HSPD1, were significantly increased in both WT and NHD13 mice following PAM2 treatment. Evaluation of serum cytokines also revealed heterodimer-specific alterations, including increased IL-6 levels in NHD13 mice treated with PAM2, but not PAM3. These data corroborate numerous previous reports linking IL-6 to MDS pathogenesis and transformation to acute myeloid leukemia. Ongoing studies involving mass cytometry, IL-6knockout mice, and pharmacological inhibitors of both IL-6 and c-Myc aim to further elucidate the mechanism through which TLR2/6-specific activation accelerates leukemogenesis and death in the NHD13 mouse model of MDS. These studies hope to inform more targeted therapeutics that could potentially delay MDS progression and reduce off-target effects. Disclosures No relevant conflicts of interest to declare.

scholarly journals NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1155-1155
Author(s):  
Stefanie Kreutmair ◽  
Rouzanna Istvanffy ◽  
Cathrin Klingeberg ◽  
Christine Dierks ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stress Response ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated. NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days). To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response. To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population. Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. Disclosures No relevant conflicts of interest to declare.

Identification of Cytogenetically Normal Human CD34+CD38+ Hematopoietic Stem/Progenitor Cells from Inv(16)+ Leukemic Bone Marrow

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1059-1059
Author(s):  
Jason N. LeGrand ◽  
Stephanie C. Heidemann ◽  
C. Scott Swindle ◽  
Christopher A. Klug
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Cell Subset ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Heterogeneous Expression

Abstract For many subtypes of AML including cases with the inv(16), mutations that give rise to the leukemic phenotype occur, at least in part, in the hematopoietic stem/progenitor (HSPC) cell subset, as suggested by studies showing that primitive CD34+ CD38- bone marrow cells can function as leukemia-initiating cells (LIC) when transferred into immunodeficient mice. A significant challenge has been that LIC share many of the same cell-surface markers as their normal HSPC counterparts, thus making it difficult to purify and functionally characterize either subset from the bulk bone marrow of leukemia patients. Here we report the FACS analysis of several previously reported human LIC markers on bone marrow samples from inv(16) AML patients and show that a combination of TIM3, CLL1, and CD33 can significantly enrich for a rare population of CD34+ CD38- cells that lack the inv(16) fusion mRNA when tested by nested RT-PCR. Heterogeneous expression of these markers among different patient samples often causes incomplete elimination of the fusion mRNA when FACS-sorting the CD34+ CD38- population as single TIM3-, CLL1-, or CD33- subsets. The combination of TIM3 with CLL1 and/or CD33 leads to a more consistent elimination of the fusion mRNA from the FACS-sorted CD34+ CD38- subsets. Results from methylcellulose assays showed that the TIM3- CLL1- CD33- subset of CD34+CD38- cells could form multiple colony types, including CFU-GEMM, that were all negative for the fusion mRNA by RT-PCR. In contrast, colonies derived from bulk bone marrow were all positive for the fusion mRNA. The TIM3- CLL1- CD33- subset of CD34+CD38- cells displayed greater than 600-fold enrichment for progenitor activity compared to bulk bone marrow but did not form additional colonies upon serial re-plating. These results have important implications for the therapeutic targeting of inv(16)+ hematopoietic stem/progenitor cells in patients with relapsed and refractory disease and for purification of normal HSPC from leukemic bone marrow samples. Disclosures No relevant conflicts of interest to declare.

Id2 Is Required for the Self-Renewal and Proliferation of Hematopoietic Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1173-1173 ◽  
Author(s):  
Lei Sun
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Conditional Knockout ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The production of mammalian blood cells is sustained throughout life by the self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation in this system leads to different pathologies including anemia, bone marrow failure and hematopoietic malignancies. The Helix-Loop-Helix transcriptional regulator Id2 plays essential roles in regulating proliferation and cell fate of hematopoietic progenitors; however, its role in regulating HSC development remains largely unknown. To assess the function of Id2 in HSCs, we developed two mouse models, including an Id2 conditional knockout model and an Id2-EYFP model, in which EYFP expression is driven by endogenous Id2 promoter. When we examined HSC function by serial transplantation, we found that mice transplanted with Id2F/F Mx1-Cre+ conditionally deleted bone marrow cells became moribund more rapidly after primary and secondary transplantation, compared to those transplanted with Id2+/F Mx1-Cre+ bone marrow, suggesting that HSC self-renewal is impaired when Id2 is deleted. To further determine if self-renewal and maintenance of HSCs depends on the expression level of Id2, we purified HSCs with different levels of Id2 expression using Id2-EYFP mice to specifically address the role of Id2 in HSCs. First, we confirmed Id2 is highly expressed in HSCs in this model. Second, when HSCs with either low or high levels of Id2-EYFP were transplanted into irradiated mice, cells with high levels of Id2 reconstituted transplanted recipients faster than those with low levels of Id2 at 3 weeks and longer, suggesting that Id2 expression is associated with repopulation advantage. Furthermore, Ki-67 staining showed that HSCs with high levels of Id2 have 15-fold more cells in G2/M phase, and fewer cells in G0. BrdU staining also suggested that there are 5-fold more BrdU+ cells in HSCs with high levels of Id2, indicating that Id2 expression correlates with cell cycle progression in HSCs. In addition, p57 has been reported to be required for quiescence of HSCs. Our preliminary data showed that p57 is downregulated in HSCs with high levels of Id2, and p57 is correspondingly upregulated in Id2-null HSCs. Altogether, our data demonstrate that Id2 is required for the self-renewal and proliferation of HSCs, and suggest a link between Id2 and the transcriptional regulatory networks that regulate the functional hematopoietic system. Since Id2 is also expressed in other adult stem cells including muscle and neuronal stem cells, as well as cancer cells, we believe our results can improve our understanding of stem cell biology and cancer development, and contribute to the identification of novel molecules that may be targeted to eliminate cancer stem cells. Disclosures No relevant conflicts of interest to declare.

SETD2, a H3K36 Lysine Methyltransferase, Is Essential for Adult Normal Hematopoiesis and Leukemia Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1055-1055
Author(s):  
Yile Zhou ◽  
Yunzhu Dong ◽  
Jiachen Bu ◽  
Xiaomei Yan ◽  
Yoshihiro Hayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Conditional Knockout ◽  
The Other ◽  
Leukemia Stem Cells ◽  
Transcriptional Networks ◽  
Loss Of Function ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are characterized by their capability for self-renewal and multi-potency. Hematopoiesis is dynamically controlled by the interplay between epigenetic and transcriptional networks. Dysregulation of these networks can lead to unfitness of hematopoiesis, cell transformation, and hematological diseases. The human SETD2 gene was originally isolated from HSCs and progenitors. SETD2 is a histone methyltransferase, which specifically catalyzes tri-methylation of histone 3 lysine 36 (H3K36me3). SETD2 functions as a tumor suppressor, as loss-of-function mutations have been identified in many cancers. However, the role of SETD2 in hematopoiesis has not been fully understood. To assess the function of Setd2 in hematopoiesis, we generated three Setd2 mouse alleles with Crispr/CAS9 technology; Setd2F2478/WT knock-in, Setd2Exon6-Δ/WT, and Setd2-Exon6flox/flox/Mx1-Cre conditional knockout alleles, as homozygous Setd2 mutation showed embryonic lethality. Setd2-F2478 point mutation, which is located in the SRI domain, can express SETD2 mutant protein but completely lose the interaction with RNA pol II. Setd2Exon6-Δ/WT allele results in a frame shift and nonsense mediated decay of Setd2 mRNA and protein. After induction of excision with pIpC injection, Setd2-exon6flox/flox/Mx1-Cre+ (Setd2Exon6-Δ/Δ) mice showed severe anemia, increased platelet count, and a reduction in bone marrow (BM) cellularity compared to wild-type (WT) mice, while Setd2F2478/WT and Setd2Exon6-Δ/WT mice did not show any obvious hematological changes. The Lin- Sca-1+ c-Kit+ (LSK) population in Setd2Exon6-Δ/Δ mice was 2.5-fold decreased compared to those in WT, while the LSK populations in Setd2F2478/WT and Setd2Exon6-Δ/WT mice were comparable with those in WT. Interestingly, all three of these Setd2 mutant alleles showed a higher frequency of Lin- Sca-1- c-Kit+ (LK) cells in the BM. In the LK populations, we found an increased CMP population in Setd2F2478/WT and Setd2Exon6-Δ/WT mice; of note, the CMP population in the Setd2Exon6-Δ/Δ mice had disappeared while the MEP population expanded with higher expression of CD16/32. Next, to assess the function of the HSPCs, we performed CFU assays and competitive bone marrow transplantations (CBMT). Consistent with our phenotypic findings, the number of colonies derived from Setd2F2478/WT and Setd2Exon6-Δ/WT BM cells was increased in the first two passages, while the number of colonies derived from Setd2Exon6-Δ/Δ mice was significantly decreased. In CBMT, we found that mice transplanted with Setd2Exon6-Δ/Δ BM cells showed anemia and an impaired BM reconstitution, compared to the control (p = 0.0002). On the other hand, the Setd2F2478/WT and Setd2Exon6-Δ/WT models showed comparable capabilities of BM reconstitution. Taken together, these results suggest that Setd2 has an essential role in the maintenance of adult hematopoiesis. SETD2 mutations (mainly one allele mutation) have been frequently identified in acute leukemia, especially in about 22% of MLL leukemia. To understand the role of SETD2 in leukemic stem cells, Setd2 mutant mice were bred with the Mll-AF9 knock-in mouse. The Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice showed higher frequencies of LK and LSK populations compared to Mll-AF9 mice, indicating that Setd2 mutations may increase the stemness of leukemia stem cells (LSCs). The cells derived from Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice resulted in a significantly higher yield of colonies and growth advantage in serial replating CFU assay compared to the cells derived from Mll-AF9 mice. After BMT of equal numbers of cells from Mll-Af9 or Mll-AF9/ Setd2F2478/WT mice into recipient mice, the Mll-AF9/ Setd2F2478/WTBMT mice developed leukemia with significantly shortened latencies compared with MLL-Af9 BMT mice. In conclusion, our data suggests that Setd2 plays an important role in maintaining normal HSPCs. Half the doses of Setd2 can still maintain the normal hematopoiesis while a total loss of Setd2 leads to a failure of hematopoiesis. In leukemia, heterozygous mutants of Setd2 can accelerate leukemogenesis by expanding LSCs. Whether the remaining WT allele is required for leukemia maintenance is unclear. Further reduction of Setd2 levels, or complete deletion of the other WT allele, may diminish SETD2-mutated leukemia. Such tumor vulnerability can be explored as a therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document