Tif1gamma Is Essential for Macrophage Differentiation

Abstract Abstract 2370 TIF1gamma (or TRIM33) is an ubiquitous nuclear protein that belongs to the transcriptional intermediary factor 1 family. Human and mouse TIF1gamma are closely related to zebrafish moonshine (mon), a gene whose mutations disrupt embryonic and adult hematopoiesis with severe red blood cell aplasia. Targeted deletion of Tif1gamma is embryonic lethal in mice. In zebrafish and human CD34+ cells, TIF1gamma functionally links positive elongation factors such as p-TEFb and FACT to blood specific transcription complexes (e.g. the SCL/TAL1 complex) to regulate elongation of genes by antagonizing Pol II pausing. TIF1gamma also affects the human hematopoietic progenitor cell response to the cytokines of the transforming growth factor-beta superfamily through various mechanisms. Recently, we showed that the loss of Tif1gamma in mouse hematopoietic stem cells (cFES-Cre-Tif1gamma) favors the expansion of the granulo-monocytic progenitor compartment. The gene deletion induces the age-dependent appearance of a cell-autonomous myeloproliferative disorder with myelodysplastic features, monocytosis, and hepatosplenomegaly that recapitulates essential features of human chronic myelomonocytic leukemia (CMML). Interestingly, TIF1gamma is almost undetectable in leukemic cells of 35% of patients with CMML. This down-regulation is related to the hyper-methylation of CpG sequences in the gene promoter. Our results demonstrated that TIF1gamma is an epigenetically regulated tumor suppressor gene in hematopoietic cells. In addition, an altered production of peritoneal macrophages was observed in our mouse model. These macrophages did not adhere to the plastic and were morphologically abnormal in vitro. In bone marrow and in Lin- progenitor cells, Tif1gamma deletion leads to a significant decrease of cfms (Csf-1r) expression, required for the differentiation, proliferation, and survival of monocytic phagocytes. We also identified in CMML patients the association between low levels of TIF1gamma and cFMS (Aucagne et al., J. Clin. Invest., 121, 2361–2370, 2011). To gain insight into the possible mechanism accounting for diminished accumulation of macrophages, we examined the expression of c-Fms. We show that level of its expression is reduced significantly in blood monocytes isolated from Tif1gamma-deleted mice. When Tif1gamma-deleted sorted myeloid cells were induced to differentiate into macrophages in presence of CSF-1, a delayed production of few abnormal large macrophages was observed. Apoptosis was associated with this alteration of differentiation. This phenomenon was also characterized in young mice not developing the disease yet. The morphological abnormalities were correlated with very important alterations of specific macrophage differentiation markers. Expression of specific transcription factors involved in macrophage differentiation was deeply deregulated. Moreover, macrophage function such as migration, cytokine or chemokine secretion in response to LPS was altered. Likewise, in vitro differentiation of monocytes into dendritic cells was also abnormal. Altogether, our results suggest that monocyte plasticity is at least partially orchestrated by Tif1gamma. Disclosures: No relevant conflicts of interest to declare.

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HoxA10 Regulates Myeloid Progenitor Proliferation by Activating Transcription of the Gene Encoding Transforming Growth Factor Beta 2.

Blood ◽

10.1182/blood.v116.21.1572.1572 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1572-1572

Author(s):

Chirag Shah ◽

Hao Wang ◽

Elizabeth A. Eklund

Keyword(s):

Progenitor Cells ◽

Transforming Growth Factor Beta ◽

Target Genes ◽

Transforming Growth Factor ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Growth Factor Beta ◽

Differentiation Block

Abstract Abstract 1572 HoxA10 is a homeodomain transcription factor which functions as a myeloid leukemia promoter. Correlative clinical studies found that increased expression of a group of HoxA proteins, including HoxA10, in acute myeloid leukemia (AML) was associated with poor prognosis. In murine models, overexpression of HoxA10 in the bone marrow was associated with development of a myeloproliferative disease which progressed to AML with time. These results suggested that HoxA10-overexpression dysregulated cell proliferation and/or survival, and predisposed to acquisition of additional mutations which led to differentiation block and AML. Additional investigations, we and others demonstrated that HoxA10 overexpression in murine hematopoietic stem cells (HSC) expanded the granulocyte/monocyte progenitor (GMP) population in vitro and in vivo. Despite this information about the impact of HoxA10 overexpression on myeloid leukemogenesis, the mechanisms by which HoxA10 exerts this effect are largely unknown. To investigate such mechanisms, we have been identifying HoxA10 target genes. In previous studies, we identified a number of HoxA10 target genes that encode phagocyte effector proteins. HoxA10 represses transcription of these gene in myeloid progenitors, and decreased HoxA10 repression activity contributes to phenotypic differentiation as myelopoiesis proceeds. This provided a potential mechanism for HoxA10 involvement in differentiation block, but not progenitor survival or expansion. We used a chromatin immuno-precipitation based approach to identify additional HoxA10 target genes involved in these activities. Previously, we reported that HoxA10 activated the DUSP4 gene in myeloid progenitor cells. This gene encodes Mitogen Activated Protein Kinase Phosphatase 2 (Mkp2) which inhibits Jnk-induced apoptosis in myeloid progenitor cells. This provided a mechanism for increased cell survival in HoxA10-overexpressing cells. In the current studies, we identified TGFB2 as a HoxA10 target gene. This gene encodes Transforming Growth Factor Beta 2 (TgfB2) a member of the TgfB super family of cytokines. Similar to TgfB1 and 3, TgfB2 interacts with TgfB-receptors I and II. However, unlike these more classical family members, TgfB2 induces proliferation of hematopoietic stem and progenitor cells. We found that HoxA10 activated the TGFB2 promoter via tandem cis elements in the proximal promoter. This resulted in autocrine stimulation of proliferation in HoxA10-overexpressing GMP and leukemia cells in vitro. Increased proliferation in HoxA10-overexpressing cells involved activation of the MAP kinase pathway in a TgfB2 dependent manner. These studies identify autocrine production of pro-proliferative cytokines as a novel mechanism for the function of Hox proteins. These findings have implications for ex vivo expansion of HSC and myeloid progenitors for tissue engineering. These result also have implications for therapeutic approaches to poor prognosis AML characterized by increased Hox expression. Disclosures: No relevant conflicts of interest to declare.

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TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽

10.1182/blood.v118.21.4829.4829 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4829-4829

Author(s):

David C Dorn ◽

Wei He ◽

Joan Massague ◽

Malcolm A.S. Moore

Keyword(s):

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Transforming Growth Factor Β ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

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Temozolomide, irradiation and hypoxia promote homing of hematopoietic progenitor cells towards gliomas

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.20044 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 20044-20044

Author(s):

W. Wick ◽

G. Tabatabai ◽

B. Frank ◽

M. Weller

Keyword(s):

Progenitor Cells ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Tumor Hypoxia ◽

Glioma Cells ◽

Signaling Cascade ◽

Hematopoietic Stem ◽

The Impact

20044 Background: Temozolomide and irradiation are essential parts of the standard therapy and hypoxia is a critical aspect of the microenvironment of gliomas. IN the present study, we aimed at investigating the impact of these stimuli on the previously defined transforming growth factor beta (TGF-β)- and stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1α/CXCL12)-dependent migration of adult hematopoietic stem and progenitor cells (HPC) towards glioma cells in vitro and the homing to experimental gliomas in vivo. Hyperthermia served as control. Methods and Results: Cerebral irradiation of nude mice at 21 days after intracerebral implantation of LNT-229 glioma induces tumor satellite formation and enhances the glioma tropism of HPC in vivo. Supernatants of temozolomide-treated, irradiated or hypoxic LNT-229 glioma cells promote HPC migration in vitro. Reporter assays reveal that the CXCL12 promoter activity is enhanced in LNT-229 glioma cells at 24 h after irradiation at 8 Gy or after exposure to 1% oxygen for 12 h. The irradiation- and hypoxia-induced release of CXCL12 depends on hypoxia inducible factor-1 alpha (HIF-1α), but not on p53. Induction of transcriptional activity of HIF-1α by hypoxia and irradiation requires an intact signaling cascade of TGF-β. Conclusions: Thus, we delineate a novel stress signaling cascade in glioma cells involving TGF-β, HIF-1α and CXCL12. Stress stimuli can be temozolomide, irradiation and hypoxia but not hyperthermia. These data suggest that the use of HPC as cellular vectors in the treatment of glioblastoma may well be combined with anti-angiogenic therapies which induce tumor hypoxia. [Table: see text]

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Non-Cycling Cells From Acute Myeloid Leukemia Patients Harbor the FLT3-ITD Mutation and Are Insensitive to TKI258, a Potent FLT3-Directed Inhibitor, in Vitro.

Blood ◽

10.1182/blood.v114.22.479.479 ◽

2009 ◽

Vol 114 (22) ◽

pp. 479-479

Author(s):

Caroline L Alvares ◽

Tino Schenk ◽

Sanna Hulkki ◽

Toon Min ◽

Gowri Vijayaraghavan ◽

...

Keyword(s):

Cell Expansion ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Trisomy 13 ◽

Leukemic Cells ◽

Multi Drug Resistance ◽

Hematopoietic Stem ◽

Flt3 Inhibitors ◽

The Impact

Abstract Abstract 479 The identification of activating mutations in the FLT3 gene and their impact on prognosis has been crucial to the rationale behind the development of FLT3 inhibitors. While it has been shown that some leukemic cells with high tumorigenic potential exist mostly in a dormant state, it is unclear if this quiescent/non-cycling leukemia-initiating fraction carries the FLT3-ITD mutation and if it is successfully targeted by FLT3 inhibitors. As a paradigm, quiescent Ph+ stem cells in CML have been shown to exhibit resistance to bcr-abl targeted inhibitors. Additionally, results from clinical trials suggest that FLT3 inhibitors reduce the leukemic blast count in peripheral blood but are less successful in the bone marrow where factors regulating hematopoietic stem cell quiescence are active. In order to investigate the non-cycling and cycling human leukemic cell boundary, we devised a biological model that allowed us to distinguish non-cycling AML cells from cycling AML progenitors in human FLT3-ITD positive AML samples. CD34+ cells were isolated from AML samples using magnetic cell sorting, labeled with the cell membrane dye PKH26 to enable tracking of cell division, and cultured on murine stroma for 12 days. Non-cycling AML cells were then separated from cycling cells by FACS sorting and were found to retain a CD34+ primitive phenotype in contrast to expanding leukemic blasts. Fluorescence in situ hybridization analyses revealed that non-cycling cells carried leukemic gene rearrangements (trisomy 8, trisomy 13, t[3;21]and t[16;16] in our cases), and were therefore part of the original leukemic clone. PCR for the FLT3-ITD region showed that in four out of five cases, the FLT3-ITD mutation was present in the non-cycling fraction. To examine the distribution of FLT3-ITD to FLT3 wild type (WT) bearing cells, non-cycling AML cells were FACS sorted, DNA extracted and the PCR products subsequently cloned. Bacterial colonies were sequenced and colony-PCR used to determine the ratio of FLT3-ITD to WT bearing colonies for each patient. These data indicated that at least 25% of non-cycling cells (range 25%-100%) harbored the FLT3-ITD mutation. We then assessed the impact of a potent FLT3-directed inhibitor, TKI258 (Novartis), on leukemic cell expansion and the viability of non-cycling cells. TKI258 has been found to induce apoptosis of FLT3-ITD bearing cells of the human acute monocytic leukemia MV4;11 cell line. In our present study, CD34+ AML blasts from the same FLT3-ITD positive patient samples were grown in vitro in the presence of 0 μM, 0.3 μM (IC50 dose) and 1.25 μM TKI258. In stromal cultures, TKI258 significantly reduced leukemic cell expansion (range 2.13 to 20 fold for untreated cultures and 0.07 to 2.27 fold for 1.25 μM TKI258 treated cultures at day 12, p ≤ 0.05). In methylcellulose colony assays, TKI258 exposure resulted in dose-dependent suppression of colony formation of CD34+ FLT3-ITD positive leukemia cells (60% to 81% reduction in the mean plating efficiency of CD34+ AML cells at 0.3 μM TKI258). Despite this striking anti-proliferative effect, the majority of non-cycling cells from AML patients showed resistance to TKI258 (five out of six cases). In these samples, FLT3-ITD positive non-cycling cells could still be detected after treatment with the equivalent highest clinical dose (1.25 μM) of TKI258. Moreover, at a functional level, limiting-dilution experiments on non-cycling AML cells pre-treated with TKI258 showed no impairment in a modified leukemic cobblestone assay at four weeks compared to untreated non-cycling cells. These results suggest that the majority of non-cycling AML cells that harbor FLT3-ITD are unaffected by a FLT3 inhibitor and may constitute an as yet untargeted disease reservoir. Only one FLT3-ITD AML case showed exquisite sensitivity to TKI258 with elimination of the non-cycling fraction observed from 0.3 μM of TKI258 upwards. Possible explanations for this may include specific mutant receptor sensitivities or generic multi-drug resistance mechanisms operating in dormant cells. Interestingly, TKI258 selectively eradicated an ‘intermediate' dividing progenitor population in two of the insensitive cases, an indication that leukemic progenitors may be rendered sensitive to FLT3 inhibition on transiting the dormancy-cycling boundary. Further studies are needed to determine if these findings are representative of a generation of FLT3 inhibitors or specific for TKI258. Disclosures: No relevant conflicts of interest to declare.

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Impact of Cytokine Gene Polymorphisms on Risk and Treatment Outcomes of Aplastic Anemia

Blood ◽

10.1182/blood.v118.21.4369.4369 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4369-4369

Author(s):

Eunyoung Lee ◽

Yun-Gyoo Lee ◽

Inho Kim ◽

Ji-Hyun Kwon ◽

Dong-Yeop Shin ◽

...

Keyword(s):

Aplastic Anemia ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Treatment Modalities ◽

Dominant Model ◽

Necrosis Factor Alpha ◽

Hematopoietic Stem ◽

Cytokine Genes ◽

Ifn Γ

Abstract Abstract 4369 Immunosuppressive therapy (IST) is one of the main treatment modalities for acquired aplastic anemia (AA). Approximately 70% of AA patients have been known to achieve clinical improvements with IST consisting of antithymocyte globulin (ATG) and cyclosporine. This remarkably high response rate supports us to tell the immunogenic pathophysiology of AA. Autoreactive cytotoxic T cells play a key role in this immunogenic pathogenesis of AA by working with myelosuppressive cytokines like interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFβ), which induce apoptosis in hematopoietic stem cells, partially through the Fas-dependent pathway. The purpose of this study is to find out which single nucleotide polymorphisms (SNPs) in cytokine genes were relevant to the risk of AA and whether the relevant SNPs were associated with response to IST in AA patients. Between January 2000 and December 2008 84 patients were screened and 80 patients confirmed as having acquired AA by bone marrow biopsy, and 84 age- and sex-matched healthy controls were analyzed consecutively. We genotyped the polymorphisms in three cytokine genes (IFNG, TNF, and TGFB1) and FAS gene, which are known to be involved in T cell-mediated marrow destruction. We assessed the association between polymorphisms in those selected genes and risk for AA, and the association between those polymorphisms and response to IST in three genetic models (dominant, recessive, and additive). The IFNG -2353 T allele (dominant model, OR=0.43, p=.012) and TCA haplotype (dominant model, OR=0.50, p=.038) were significantly associated with the development of AA. In addition, this relevant IFNG -2353 T allele and TCA haplotype were related to the response of IST (dominant model, OR=0.076, p=.034). The presence of the minor T allele was protective and related to a 2.3-fold reduction in the risk for AA (p=.012), and the presence of the IFNG TCA haplotype was related to a 2-fold reduced risk for AA (p=.038). Concerning TGFB1, although its polymorphisms are not related to AA susceptibility, P10L T allele (recessive model, OR=0.18, p=.038) and CT haplotype (dominant model, OR=5.68, p=.038) were associated with response to IST. The T allele was related to a 4.3-fold reduced response to IST at 3 months (p=.038) and the presence of the CT haplotype was favorable to IST and was related to a 5.7-fold higher response at 3 months compared with the response in patients without the CT haplotype (p=.038). This exploratory study concurred with prior studies indicating that polymorphisms in IFNG are related to AA susceptibility. In addition, it was found that polymorphisms in IFNG and TGFB1 are associated with response to IST. AA patients with intracellular IFN-γ expression in peripheral lymphocytes showed almost 3-fold higher response to IST than patients without IFN-γ-expressing lymphocytes (p<0.0001) and this finding puts weight on the immunogenic association of responsiveness. Regarding TGFβ, the functionality of polymorphisms and in TGFB1 has rarely been reported. However, the significant relationship between TGFB1 polymorphisms and therapeutic response to IST in our study suggests their late effects on marrow suppression. Our results may help explain the variability of response to IST in AA and suggests that patients with a high probability of response might be treated first with IST rather than conventional marrow transplantation.Table 1.Factors relevant to response to ISTFactorCorrected for Patient CharacteristicsUncorrected DataGenotypeOR (95% CI)POR (95% CI)PResponse at 3-month after IST (n=43) Age1.00 (0.96–1.05).841.02 (0.98–1.06).46 Sex (male vs female)1.13 (0.26–4.84).871.26 (0.37–4.23).71 Severity (severe vs non-severe)1.03 (0.23–4.53).0971.63 (0.48–5.47).43 TGFB P10L C/TTT vs CT + CC0.18 (0.03–0.90).0380.23 (0.06–0.95).043 TGFB haplotypeCT-CT + CT-other vs Other-other5.68 (1.11–29.15).0384.28 (1.05–17.42).043Response at 6-month after IST (n=43) Age1.00 (0.95–1.05).911.02 (0.97–1.06).45 Sex (male vs female)0.38 (0.072–2.01).250.60 (0.16–2.21).44 Severity (severe vs non-severe)2.53 (0.47–13.62).282.22 (0.59–8.26).24 IFNG -2353 A/TTT+AT vs AA0.076 (0.007–0.82).0340.40 (0.10–1.56).19 IFNG haplotypeTCA-TCA + TCA-other vs Other-other0.076 (0.007–0.82).0340.40 (0.10–1.56).19 TGFB haplotypeTC-TC vs TC-other + Other-other0.22 (0.05–0.90).0360.19 (0.03–1.09).063 Disclosures: No relevant conflicts of interest to declare.

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Exhaustion in Myeloid Lineage and Very Early Defect in HSPC Pool: An Embryonic Origin of Fanconi Haematological Disorders

Blood ◽

10.1182/blood.v126.23.296.296 ◽

2015 ◽

Vol 126 (23) ◽

pp. 296-296 ◽

Cited By ~ 1

Author(s):

Carine Domenech ◽

Alix Rousseau ◽

Laurence Petit ◽

Sandra Sanfilippo ◽

Jean Soulier ◽

...

Keyword(s):

Bone Marrow ◽

Embryonic Development ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Hematopoietic Progenitor Cell ◽

Myeloid Lineage ◽

Hematopoietic Stem

Abstract Fanconi anemia (FA) is a genetic disorder due to mutations in one of the sixteen FANC genes involved in DNA repair. Many FA patients develop bone marrow failure (BMF) during childhood, and FA strongly predisposes to myelodysplasia syndrome and/or acute myeloid leukaemia. The pathogenesis of the BMF remains uncompletely understood. Low hematopoietic progenitor cell (HPCs) counts observed early in life and preceeding the onset of blood cytopenia in patients led we, and other, to hypothesize that the hematopoietic development might be abnormal in the FA embryo. Indeed, unlike adult hematopoietic stem cells (HSCs) which are quiescent in the BM niche, during embryonic life HSCs are in active proliferation in sites of expansion such as fetal liver and placenta, where they get amplified and acquire properties of adult HSC .We hypothesized that in FA, the FA defect in response to the replicative stress could impair the expension of the HSC pool.In order to investigate this hypothesis, we carried out studies in Fancg-/- knock out mice and in human FA fetuses obtained with informed consent from medical abortion. In Fancg-/- mice, FACS analysis revealed a 1,5- to 3-fold deficiency in hematopoietic stem and progenitor cells (HSPC) very early during embryonic development (i.e 11.5 days of gestation - E11.5) in fetal liver (FL) and placenta (Pl) (p <0.001). In both organs, this defect persists during the whole period of amplification (until E14.5 for FL and E12.5 for Pl). In vitro clonogenic assays also demonstrated a 2- fold defect in granulocyte, erythrocyte and macrophage (GEM) progenitors both in Fancg-/- FL or Pl compared to WT (p <0.001), and 4 to 5- fold defect in more immature mixed GEM progenitors in FL (p <0.001). LTC-IC frequency of the HSC-enriched Lineage- Sca1+ AA4.1+ population (LSA) of E14.5 Fancg-/- FL comforted this later result, since it was 5-fold lower than for WT. In vivo long-term hematopoietic reconstitution (LTR) assays confirmed a deficit of the HSC enriched LSA population of E14.5 Fancg-/- FL. Indeed, although the percentage of mice reconstituted was as good as that obtained with the same number of WT LSA, the CD45 Ly5.2 chimerism was reduced (49±20% vs 84±4% for 1000 LSA injected, and 56±12% vs 87±2% for 5000 LSA). Interestingly, bone marrow analysis of mice reconstituted with Fancg-/- LSA 22 weeks after injection showed a level of CD45 Ly5.2 chimerism 3-fold lower than that found in blood, spleen and thymus, as well as a very low chimerism for myeloid GEM lineages, contrasting with a high chimerism for B and T lymphoid lineages. Moreover, we were able to demonstrate that this deficit is already present at E12.5, both in Fancg-/- FL and Pl. Indeed, no mice reconstituted with 3.105 total Fancg-/- fetal liver cells, while 100% injected with the same number of WT FL cells got reconstituted with a chimerism of 59,5±5%. For Pl, when 500 000 cells were injected, reconstitution was observed in only 1 out of 3 mice for Fancg-/- (29% chimerism), and in 3 out of 3 mice for WT (88±4% chimerism). In human FA FL of 14 weeks of gestation, we also observed a 4-fold defect of HSPC with a total lack of in vitro amplification compared to control, in agreement with the mice data. Taken together, these data demonstrate that a profound deficit of HSCs and progenitors cells is present since the earlier stages of embryonic development in FA. In addition, using organotypic cultures of E11 aortas, we could show that this defect of amplification is already present in HSCs emerging from Fancg-/- aorta, which showed a 2-fold lower rate of amplification compared to WT. More importantly, our results show for the first time exhaustion in myeloid lineage of FA, in agreement with what is observed in children with FA disease. Altogether, our work suggests a role of the FA pathway during the development of the hematopoietic system leading to a deficit of amplification of HSC. Comparison of FA HSC transcriptome with that of control HSC in FL and Pl is in progress. It should allow to identify the key pathways involved in the embryonic HSC amplification that are deregulated in FA, and hopefully getting more insights in the pathogenesis of the BMF and leukemogenesis in FA patients. Disclosures No relevant conflicts of interest to declare.

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In Situ Microscopy Studies of Infused Megakaryocytes: Implications in Thrombopoiesis

Blood ◽

10.1182/blood-2021-150764 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4287-4287

Author(s):

Hyunjun Kim ◽

Danuta Jadwiga Jarocha ◽

Ian Johnston ◽

Hyunsook Ahn ◽

Deborah L French ◽

...

Keyword(s):

Progenitor Cell ◽

Large Scale ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cell ◽

Endothelial Lining ◽

Cross Sectional ◽

Hematopoietic Stem ◽

Nsg Mice

Abstract The questions of whether thrombopoiesis - the release of platelets from megakaryocytes - occurs both as megakaryocytes emerge from the intramedullar space or occurs as well in the pulmonary vascular bed remains unanswered. Studies by Lefrançais E, et al, (Nature, 2017) demonstrated by in situ microcopy that perhaps 50% of all platelet release in mice occurs from megakaryocytes released from the marrow and traveled to the lungs where they undergo thrombopoiesis over a 20- to 60-minute time-period. We examined whether CD34+-derived human megakaryocytes infused into immunocompromized NSG mice would also shed platelets in the lungs in a similar fashion. We differentiated CD34+-derived hematopoietic stem-progenitors for 12 days in culture using conditions previously described (Wang Y, et al., Blood 2015). We found that unlike platelet-like-particle (PLP) formation in in vitro cultures of CD34+ hematopoietic progenitor cell (HPC)-derived (CD34+) megakaryocytes, which undergo asynchronous shedding of the PLPs, that over 95% of infused CD34+ megakaryocytes shed their platelets within the first 40 minutes much as has been observed for endogenous murine megakaryocytes. The average number of cytoplasmic extensions per megakaryocytes was ~2.7, again very similar to what was seen with endogenous murine megakaryocytes. In contrast, CD34+ cells grown in culture into megakaryocytes for a shorter period of time of only 7 days, poorly shed any cytoplasmic fragments. We also studied human megakaryocytes grown from immortalized megakaryocyte progenitor cell lines (imMKCLs) from induced pluripotent stem cells (iPSCs) generated by the Eto laboratory and kindly provided by Dr. Koji Eto, Kyoto University). These cells were grown and differentiated into terminal megakaryocytes as described (Nakamura S, Cell Stem Cell, 2014) for 4 days in culture. These cells have been proposed to be useful for large-scale preparation of PLPs in vitro for clinical use in place of donor-derived platelets. The resultant infused human imMKCL-derived megakaryocytes also synchronously shed platelets, but only 50% of the infused cells shed their cytoplasm in contrast to >95% of CD34+ megakaryocytes. Moreover, cytoplasmic extensions were decreased to an average of ~1.1 per megakaryocyte. We had proposed that in vitro-generated megakaryocytes might be directly infused into patients in place of further manipulating the megakaryocytes to release functional platelets in vitro using a bioreactor. However, such megakaryocytes will likely be contaminated with a higher level of HPCs than anticipated from in vitro-prepared platelets, and concern exists that they may lead to unacceptable graft versus host complications. We, therefore, examined whether irradiating megakaryocytes as one strategy to eliminate this concern results in megakaryocytes that are still functional and found that megakaryocytes irradiated with up to 25 Gy retain platelet yield per infused megakaryocytes with the platelets having the same half-life. If irradiated and kept in culture, these megakaryocytes begin to shed platelets and undergo apoptosis notably by 24 hours. We also examined whether the pulmonary bed differs from other vascular beds, and infused CD34+ megakaryocytes both intravenously and intra-arterially in parallel studies and found that following intra-arterial infusion, megakaryocytes were mostly entrapped in various organs, but shed few platelets. Thus, our studies suggest that the pulmonary bed is unique for platelet shedding from entrapped megakaryocytes. Whether this is due to the structural organization of the pulmonary beds, its endothelial lining, its reverse exchange in oxygen, carbon dioxide and pH from other capillary beds or the mechanical forces of inhalation and exhalation that expand and contract the capillary cross-sectional area needs to be examined. Our studies show that infused human megakaryocytes synchronously release platelets over a 40-minute window and can do so even after being irradiated and that this occurs specifically in the lungs not only has potential clinical application, but also raises biological questions about what determines thrombopoiesis-readiness and what are the features of the pulmonary bed that allows this synchronous release. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Bone Marrow (BM) Stromal Expression Of Cytochrome P450 (CYP) Enzymes Protects Acute Myeloid Leukemia (AML) From All-Trans Retinoic Acid (atRA)

Blood ◽

10.1182/blood.v122.21.1449.1449 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1449-1449 ◽

Cited By ~ 1

Author(s):

Meng Su ◽

Richard J. Jones ◽

Gabriel Ghiaur

Keyword(s):

Bone Marrow ◽

Retinoic Acid ◽

Conflicts Of Interest ◽

Differentiation Markers ◽

Induced Differentiation ◽

Hematopoietic Stem ◽

Trans Retinoic Acid ◽

Cd15 Expression ◽

All Trans Retinoic Acid

Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved the outcome of acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of atRA resistance have been previously reported (i.e., over-expression of PRAME or up-regulation of Tal1 pathway), yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, although atRA induces complete remissions (CRs) in >80% of cases, most patients eventually develop disease recurrence. We have recently showed that BM stromal control of RA signaling via expression of CYP26 is important in determining normal hematopoietic stem cell fate. Thus, here we hypothesize that the BM microenvironment is responsible for the difference between the in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. It is well-recognized that pharmacological concentrations of atRA (10-7M x 3 days) differentiates NB-4 cells [a t(15;17) APL line] as demonstrated by decreased cellular expansion (32.9±1 vs. 3.5±1.3, mean±SD of cellular expansion folds of control vs. atRA-treated cultures, n=3, p<0.01) induction of cell cycle arrest (52.7% vs. 86.2% Go/G1, control vs. atRA), decreased clonogenic activity (61±12% of control in atRA cultures, n=3, p<0.01), increased expression of differentiation markers (47.4±7% vs. 84.5±13% CD11b positive cells, control vs. atRA, n=3, p=0.01), disappearance of blasts (44.5±7% vs 24.3±4% blasts, control vs. atRA, n=4, p<0.01) and emergence of morphological neutrophils. We observed similar pro-differentiation effects of atRA on other AML lines including HL-60 [non-t(15:17) APL line], KG-1 (primitive line arising from MDS), Kasumi-1 [t(8;21)] and OCI-AML3 (NPM1 mutated). When the incubations were repeated with the 5 AML lines in the presence of BM stroma, atRA activity (i.e., upregulation of differentiation markers, inhibition of clonogenic growth) was blocked. Inhibition of stromal CYP26 by the competitive inhibitor R115866, rescued the AML cell sensitivity to atRA: clonogenic recovery of NB4 cells was 92±17% on stroma with atRA compared to 56±15.9% when R115866 was added) (mean±SD of clonogenic activity of untreated stroma cultures, n=3, p=0.03) and 67.5±8.9% cells expressed the differentiation marker CD11b when treated with atRA on stroma compared to 95.1±4.9% with the combination of atRA+R115866 (n=3, p=0.03). Similar results were observed using HL-60, KG-1, Kasumi-1 and OCI-AML3 cells. CD34+ AML blasts from the BM of newly-diagnosed patients with t(8;21) AML were cultured as above. When isolated from the BM, the blasts showed only low-level expression of mature myelomonocytic markers (n=3). However, the expression of differentiation markers was significantly increased when cultured in serum (which contains about 10-9M atRA) and even further with the addition of 10-7M atRA (Figure). The presence of stroma during culture was associated with maintenance of an immature leukemic phenotype; moreover, as seen above, inhibition of CYP26 by R115866 restored AML sensitivity to atRA (Figure). CYP26 inhibitor had no effect on AML cells in stroma independent cultures. Our data suggest that CYP26 activity in the BM microenvironment creates retinoid low sanctuaries that protect AML cells from systemic atRA therapy. Thus, inhibition of CYP26 activity provides a new opportunity to expand the clinical activity of atRA in both APL and non-APL AML. It is also tempting to hypothesize that the P450-mediated detoxification of drugs by the stroma is not a retinoid-specific phenomenon but rather a more general, cell-extrinsic mechanism of drug resistance. Bone marrow stroma protects primary AML blasts from atRA induced differentiation Flow cytometry analysis of CD15 expression of CD34+ blasts isolated from the BM of a patient with t(8;21) AML. On day 0, these cells express no CD15. Culture in the presence of serum results in rapid acquisition of CD15 (liquid control). This is further enhanced by treatment with 10-7M atRA. Co-culture with BM stroma inhibits CD15 expression and atRA has only minimal effect in the presence of stroma. Inhibition of stromal CYP26 overcomes stroma’s inhibition of atRA-mediated up-regulation of CD15. Disclosures: No relevant conflicts of interest to declare.

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The VWRPY Domain Is Essential for RUNX1 Function in Hematopoietic Progenitor Cell Maturation and Megakaryocyte Differentiation

Blood ◽

10.1182/blood-2018-99-113400 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1319-1319

Author(s):

Halah Alkadi ◽

David McKellar ◽

Tao Zhen ◽

Tatiana Karpova ◽

Lisa J Garrett ◽

...

Keyword(s):

Genome Editing ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cell ◽

In Vivo Model ◽

Transactivation Domain ◽

Hematopoietic Stem ◽

Platelet Disorder ◽

The Impact ◽

Runx1 Mutation

Abstract RUNX1 is a transcription factor essential during definitive hematopoiesis. Germline mutations in RUNX1 results in a disorder called Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM). FPDMM patients have abnormal bleeding due to reduced platelet count and/or function. Importantly, 20-60% of the FPDMM patients develop hematological malignancies, which are mainly myeloid. Reported RUNX1 mutations in FPDMM families are mostly clustered in the N-terminal runt domain and the C-terminal transactivation domain. Recently, three mutations have been reported at or near the end of the C-terminal repression domain, the VWRPY motif. However, the mechanism behind the VWRPY motif involvement in the FPDMM pathogenesis has not been studied. Interestingly, these VWRPY-mutated RUNX1 proteins still have intact runt and transactivation domains, but patients still show FPDMM phenotype. Here, we evaluate the functional defects of a RUNX1 mutation, L472fsX, which was reported in a FPDMM family, using three different experimental models. Our study is aimed to unravel the significance of the VWRPY motif in FPDMM pathogenesis. The RUNX1 L472fsX mutation is caused by a GC insertion upstream of the VWRPY motif. The mutation results in a frameshift and a run on protein for an additional 123 amino acids. The frameshift abolishes the VWRPY motif, which is responsible for the binding between RUNX1 and a co-repressor protein, TLE1. As expected, from both FRET and co-IP assays, the mutated RUNX1 lost binding with TLE1. Interestingly, we observed increased binding between the mutated RUNX1 and its co-factor CBFβ in the FRET assay, as compared to the wildtype RUNX1. Furthermore, in reporter assays we found that TLE1 failed to repress the expression of a RUNX1 target, M-CSFR promoter, when co-transfected with the mutated RUNX1, which is contrary to what has been seen with wildtype RUNX1. Consistent with increased binding between the mutated RUNX1 and CBFβ in the FRET assay, co-transfecting mutant RUNX1 and CBFβ resulted in a significant increase of M-CSFR promoter expression as compared to wildtype RUNX1 with CBFβ. For another RUNX1 target, Hmga2, wildtype RUNX1 and CBFβ decreased Hmga2 expression, which could be restored by adding TLE1. Transfected mutant RUNX1 and CBFβ also decreased Hmga2 expression, but TLE1 could not restore Hmga2 expression when co-transfected with the mutant RUNX1. These findings suggest that the VWRPY-disrupting L472fsX mutation leads to the loss of binding between mutant RUNX1 and TLE1, which in turn resulted in defective repression of the RUNX1 activity by TLE1. To assess for hematopoietic defects in the FPDMM patients with the L472fsX mutation, blood cells from two family members were reprogrammed to induced pluripotent stem cells (iPSCs). Similar to previous studies, iPSCs from these patients gave rise to fewer megakaryocyte progenitors and mature megakaryocytes during in vitro differentiation. In addition, these FPDMM iPSCs showed decrease in hematopoietic stem cell (HSCs) maturation and differentiation to progenitors. The L472fsX mutation in the iPSCs was then corrected by genome editing using zinc finger nuclease. Importantly, the hematopoietic defects of the FPDMM iPSCs mentioned above were rescued after mutation correction. Overall, the findings in these iPSCs differentiation assays showed that the VWRPY motif is essential for RUNX1 activity in megakaryocytes differentiation. In addition, the VWRPY motif is important for HSCs maturation and differentiation to progenitors. To evaluate the impact of this VWRPY-deletion mutation on hematopoiesis in an in vivo model, CRISPR-mediated genome editing was used to generate mice with frameshift mutations that remove the VWRPY domain. Preliminary observations showed that the mutant mice have minor defects in the peripheral blood. More data on this mouse model will be presented at the meeting. In conclusion, we present a novel RUNX1 mutation (L472fsX) with unique hematopoietic defect that has not been reported previously in FPDMM. Our findings imply the significance of the VWRPY motif in megakaryopoiesis, as well as HSCs maturation and differentiation. Disclosures No relevant conflicts of interest to declare.

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Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood.v88.1.82.bloodjournal88182 ◽

1996 ◽

Vol 88 (1) ◽

pp. 82-88 ◽

Cited By ~ 8

Author(s):

E Sitnicka ◽

FW Ruscetti ◽

GV Priestley ◽

NS Wolf ◽

SH Bartelmez

Keyword(s):

Stem Cells ◽

Cell Division ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Proliferative Potential ◽

Tgf Beta ◽

Hematopoietic Stem ◽

Growth Factor Beta

Hematopoiesis appears to be regulated, in part, by a balance between extracellular positive and negative growth signals. Transforming growth factor beta-1 (TGF-beta 1) has been shown to be a negative regulator of primitive hematopoietic cells. This study examined the direct effect of TGF-beta 1 on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC) in vitro. We previously reported a cell fractionation approach that includes the selection of low Hoescht 33342/low Rhodamine 123 (low Ho/Rh) cell fractions that are highly enriched for long-term repopulating cells (LTR-HSC) and also clone to a very high efficiency in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6: 90% to 100% of individually cultured low Ho/Rh cells formed high proliferative potential clones. This high cloning efficiency of an LTR-HSC enriched cell population enabled proliferation inhibition studies to be more easily interpreted. In this report, we show that the continuous presence of TGF-beta 1 directly inhibits the cell division of essentially all low Ho/Rh cells (in a dose-dependent manner) during their 0 to 5th cell division in vitro. Therefore, it follows that TGF-beta 1 must directly inhibit the proliferation of LTR-HSC contained within these low Ho/Rh cells. The time required for some low Ho/Rh cells to undergo their first cell division in vitro was also prolonged in the presence of TGF-beta 1. Furthermore, when low Ho/Rh cells were exposed to TFG-beta 1 for varying lengths of time before neutralization of the TGF-beta 1 by monoclonal antibody, the ability to form macroclones was markedly decreased after approximately 4 days of TGF-beta 1 exposure. In addition, 1 to 10 ng/mL of TGF-beta 1 resulted in a maintenance of high proliferative potential-colony-forming cell (HPP-CFC) during 8 days of culture compared with loss of HPP-CFC in cultures with no added TGF- beta 1. In conclusion, this study shows that TGF-beta 1 directly inhibits the initial stages of proliferation of LTR-HSC and appears to slow the differentiation of daughter cells of low Ho/Rh cells.

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