scholarly journals MS4A3 Marks Early Myeloid Differentiation in Human Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4319-4319
Author(s):  
Tomohiko Ishibashi ◽  
Takafumi Yokota ◽  
Yusuke Satoh ◽  
Takao Sudo ◽  
Yukiko Doi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Myeloid Leukemia ◽  
Expression Level ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Lineage Differentiation ◽  
Hematopoietic Lineage ◽  
Acute Myeloid

Abstract Understanding lineage specific markers contributes to investigation into lineage commitment processes in hematopoiesis. Particularly in the human study, information about hematopoietic lineage divergence is essential to refine hematopoietic lineage tree. Lineage markers are also potentially useful for therapeutic target, such as CD20 in B-cell lymphoma, and CD33 in acute myeloid leukemia. We have recently reported that special AT-rich sequence-binding protein 1 (SATB1), a global chromatin organizer, promotes lymphocyte production from hematopoietic stem cells (HSCs) (Immunity 38;1105, 2013). Expression level of SATB1 increases with early lymphoid differentiation, whereas it is shut off in committed myeloid progenitors. To search a novel cell surface molecule that marks the point of branching lineage along early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSCs with mock-transduced HSCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (MS4A3). MS4A3, also called hematopoietic cell-specific transmembrane 4 (HTm4), is a member of the MS4A family. CD20, encoded by MS4A1 gene, belongs to the same family. We observed that expression level of MS4A3 in SATB1-overexpressed HSCs was decreased almost one tenth of that of mock HSCs. To confirm the relationship of SATB1 and MS4A3 in human hematopoietic cells, we first used chronic myeloid leukemia cell line K562, which was found to clearly express MS4A3 on their cell surface. While SATB1 expression was undetectable in original K562 cells, the exogenous expression of SATB1 significantly reduced their MS4A3 expression level, suggesting that SATB1 negatively regulates MS4A3 expression in human cells. Next, we analyzed MS4A3 expression pattern in primary human hematopoietic stem/progenitor cells. Bone marrow (BM) cells were obtained from healthy donors or patients with acute myeloid leukemia. The Institutional Review Board of Osaka University School of Medicine approved all of protocols, and written informed consents were obtained from all participants. Mononuclear cells were separated from the BM samples by density gradient centrifugation, and subsequently applied to cell sorting for Lineage marker-negative (Lin-) CD34+ CD38- HSCs, Lin- CD34+ CD38+ IL-3 receptor α (IL-3Rα)+ CD45RA- common myeloid progenitors (CMPs), Lin- CD34+ CD38+ IL3-Rα+ CD45RA+ granulocyte-macrophage progenitors (GMPs) and Lin- CD34+ CD38+ IL-3Rα- CD45RA-megakaryocyte-erythroid progenitors (MEPs). MS4A3 expression levels of the sorted cells were analyzed with real-time RT-PCR. We detected more than 10-fold amount of MS4A3 transcripts in CMPs than HSCs. Furthermore, its expression level continuously increased along myeloid lineage differentiation to GMP. On the other hand, megakaryocyte-erythroid lineage differentiation was not accompanied by MS4A3 expression and the amount of MS4A3 transcripts in MEPs was minimum as in HSCs. Flow cytometry analyses confirmed that HSCs and MEPs do not express MS4A3 on their cell surface whereas the MS4A3 expression on CMPs and GMPs is detectable. Further, the Lin- CD34+ CD38+ CD33+ cells could be fractionated according to the intensity of cell surface MS4A3 expression. To investigate the significance of cell surface MS4A3 expression for functional analyses of myeloid progenitor cells, we performed methylcellulose colony-forming assays. We found that MS4A3+ cells in Lin- CD34+ CD38+ CD33+ fraction only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. We also analyzed MS4A3 expression in BM cells obtained from patients with acute leukemia. Flow cytometry analyses revealed that leukemia cells of some patients expressed substantial amount of cell surface MS4A3. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis. In addition, our findings of MS4A3 expression on myeloid leukemia cells, while no expression on normal HSCs, imply that MS4A3 might be a therapeutic target molecule in myelogenous leukemia. Further studies would clarify the application of MS4A3 to anti-leukemia therapy. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia Cells Acquire Chemo-Resistance By Inducing Osteoblast Differentiation in Mesenchymal Stem Cells through up-Regulation of RUNX2

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2929-2929
Author(s):  
Venkata Lokesh Battula ◽  
Phuong M Le ◽  
Jeffrey Sun ◽  
Teresa McQueen ◽  
Anitha Somanchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Alkaline Phosphatase ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Cell Surface ◽  
Myeloid Leukemia ◽  
Osteoblast Differentiation ◽  
Leukemia Cells ◽  
Acute Myeloid

Abstract The leukemia bone marrow micro-environment (BME) is comprised of the endosteal and vascular niches, provides vital support for cellular growth and conveys drug resistance to leukemia cells. Several reports suggest that mesenchymal stem/stromal cells (MSCs) present in the bone marrow niche induce cell survival and anti-apoptotic proteins in acute myeloid leukemia (AML) cells and protect them from chemotherapy. The mechanisms underlying BME-mediated chemo-resistance however have not been fully elucidated. Here, we hypothesize that AML cells induce functional changes and prime MSCs to protect leukemia cells from chemotherapy. To test our hypothesis, we have compared age matched (between 40-60 years) bone marrow derived MSCs from AML patients (AML-MSC, n=10) and normal (N-MSC, n=10) individuals and analyzed their proliferation, cell surface phenotype, multi-lineage differentiation and chemo-protection potential. AML-MSCs are phenotypically different, with their polygonal morphology and larger cell size compared to N-MSCs which are elongated and spindle shaped appearance. The average cell doubling time of AML-MSCs is 52±8hrs compared to 34±6hours for N-MSCs during their exponential growth phase (p<0.01). Cell surface phenotyping by flow cytometry revealed that most of the markers known to be expressed on N-MSCs including CD105, CD90, CD73, CD51, CD44, SUSD2, CD106, CD140b, CD140a, CD106 and CD271 were also expressed on AML-MSCs at similar levels. Interestingly, tissue non-specific alkaline phosphatase (TNAP, clone W8B2), a cell surface protein highly expressed in naïve-MSCs and osteoblast progenitors (Battula VL et al., Haematologica, 2009) was 10-14 fold higher in AML- as compared to N-MSCs. Since TNAP is also a osteoblast specific marker, we compared osteoblast differentiation potential of N- vs AML-MSCs. Surprisingly, a dramatic increase in alkaline phosphatase activity (by BCIP/NBT substrate) was observed in AML-MSCs even without induction of osteoblast differentiation. mRNA analysis by qRT-PCR revealed that osteoblast specific genes including osteopontin, TNAP, osteocalcin, and osterix were 5-10 fold up-regulated in AML-MSCs compared to N-MSCs before induction. In N-MSCs, the expression of these markers was induced only under osteoblast differentiation conditions. These data indicate that AML-MSCs are primed to differentiate into-osteoblasts. Adipocyte differentiation was assessed by Oil-Red O staining for lipid droplets and revealed a > 95% reduction (p<0.0001) in the number mature adipocytes in AML-MSCs compared to N-MSCs suggesting that AML-MSCs lack the ability to differentiate into adipocytes. To understand the mechanism inducing osteogenic specific differentiation of AML-MSCs, we performed mRNA expression analysis of genes that regulate this process. We found RUNX2, a transcription factor that induces osteogenic but inhibits adipogenic differentiation, was 4-5 fold increased in AML-MSCs compared to N-MSCs. To validate these observations, we co-cultured N-MSCs in the presence or absence of OCI-AML3 cells for 3-5 days and FACS sorted the MSCs for gene expression analysis. We observed a 3-4 fold up-regulation of TNAP protein expression by flow cytometry and 4-6 fold up-regulation of osteoblast specific markers including osteopontin, alkaline phosphatase and osterix in MSCs co-cultured with OCI-AML3 cells. In addition, RUNX2 was up-regulated in MSCs when co-cultured with OCI-AML3 cells. These data suggest that AML cells induce osteogenic differentiation in BM-MSCs by up-regulation of RUNX2. To identify the clinical significance of these observations, we examined the ability of AML- and N-MSCs to protect AML cells from chemotherapy. Co-culture of OCI-AML3 cells with either AML- or N-MSCs and treatment with Cytarabine revealed a 15±4.5% increase in the number of live leukemia cells when co-cultured with AML-MSCs compared to N-MSCs. These data indicate that AML-MSCs protect leukemia cells better from chemotherapy than normal MSCs. In conclusion, AML cells induce osteogenic differentiation in MSCs through up-regulation of the RUNX2 transcription factor. Increased chemo-protection of AML cells by AML-MSCs suggests a prominent role of these cells in AML relapse. Targeting RUNX2 and thereby inhibition of osteoblast differentiation of MSCs may provide enhanced treatment options for AML therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2273-2285 ◽  
Author(s):  
James Ropa ◽  
Nirmalya Saha ◽  
Hsiangyu Hu ◽  
Luke F. Peterson ◽  
Moshe Talpaz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Critical Role ◽  
High Expression ◽  
Leukemia Cells ◽  
Biological Impact ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. Deregulation, including epigenetic deregulation, of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia. We recently showed that the Histone 3 Lysine 9 methyltransferase SETDB1 negatively regulates the expression of the pro-leukemic genes Hoxa9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation in MLL-AF9 leukemia cells. Here, we investigated the biological impact of altered SETDB1 expression and changes in H3K9 methylation on acute myeloid leukemia. We demonstrate that SETDB1 expression is correlated to disease status and overall survival in acute myeloid leukemia patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of leukemia cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While myeloid leukemia cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of cKit+ hematopoietic stem and progenitor cells. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of leukemia cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with acute myeloid leukemia, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in leukemia that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing acute myeloid leukemia.

Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3513-3513
Author(s):  
David B. Sykes ◽  
Mark K Haynes ◽  
Nicola Tolliday ◽  
Anna Waller ◽  
Julien M Cobert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Prostacyclin Receptor ◽  
Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

scholarly journals Prognostic Value of Minimal Residual Disease before Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5733-5733
Author(s):  
Olga Pérez-López ◽  
Teresa Caballero-Velázquez ◽  
Enrique Colado ◽  
Sara Alonso ◽  
José González-Campos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Prognostic Value ◽  
Residual Disease ◽  
Hematopoietic Stem ◽  
Multiparametric Flow Cytometry ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Introduction Several studies have shown that the minimal residual disease (MRD) in acute myeloid leukemia (AML) patients has a prognostic value after induction and consolidation therapy. Nevertheless the relapse is the most important cause of treatment failure in these patients, although they achieved a negative MRD, and even after an allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays, the value of the MRD before allogeneic BMT is still controversial. Method Multicentric study where we have studied correlative AML patients who went under an allo-HSCT in a situation of complete response, between 2012 and April'18. The MRD was analyzed by 8-coloured multiparametric flow cytometry, at least with 2 tubes per patient and 1,000,000 events per tube. We evaluated the prognostic value of the MRD before allo-HSCT. Results Between January'12 and April'18 we have gathered 90 allogeneic BMT in AML patients who were in CR, with a median age of 45 years old (17 - 66). The pre-HSCT situation was 1st complete remission (CR) in 75 patients and 2nd CR in 15. In 45 patients the conditioning regimen was myeoablative. In the group of patients (67) where we could know the risk group at diagnosis, the distribution was: low risk 18%, intermediate risk 59.7% and high risk 22.4%. The 46.7% of the donors were not related. In the last follow-up after allo-HSCT 24 patients have suffered a relapse (26.7%) and 41 (45.5%) have died (17 cases of mortality related to the transplant and 24 not related). In the global analysis the median follow-up of the overall survival (OS) was 37.5 months. Among the 90 patients, MRD was valuable in 86. Ten of 59 patients (16.9%) with negative MRD relapsed vs 12/27 (44.4%) with positive MRD, p= 0.016. If we consider only patients in 1st CR, 9/50 (18%) patients with negative MRD relapsed vs 10/22 (45.5%) with positive MRD, p= 0.02. This statistically significant difference does not exist if we consider only patients in 2nd CR. The median follow-up of OS and event free survival (EFS) was not reached in the negative MRD group and 571 days and 299 days in the positive MRD group. OS and EFS at 2 years after transplantation were 65% and 64% in the negative MRD group and 42% and 37% in the positive MRD group, p= 0.03 and p= 0.008 respectively (figure 1). Conclusions The detected MRD by 8-colour multiparametric flow cytometry previous an allo-HSCT in patients with AML in 1st CR is a prognostic factor in terms of relapse. Patients with a positive MRD before the allo-HSCT have a poorer OS and EFS than the patients with a negative MRD. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Grb10 Is a Tumor Suppressor in Human Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1344-1344
Author(s):  
Wanxing Chai-Ho ◽  
Martina M Roos ◽  
Michelle Li ◽  
Pang Amara ◽  
Yurun Zhang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Tumor Suppressor ◽  
Myeloid Leukemia ◽  
Negative Regulator ◽  
Leukemia Cells ◽  
Maternal Allele ◽  
Hematopoietic Stem ◽  
Colony Forming Capacity ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a common and potentially fatal hematologic malignancy. Allogeneic stem cell transplantation is the only curative therapy for most subtypes of AML, but carries a significant risk of transplant-related mortality. The development of novel therapies to eradicate AML remains a substantial area of unmet medical need. Growth factor receptor bound protein 10 (Grb10) is a member of the family of imprinted genes. Our laboratory demonstrated that in normal hematopoietic system, deletion of the maternal allele of Grb10 significantly increased hematopoietic stem cell long-term repopulating capacity (Yan et al. Cell Rep 2016). Grb10 has been shown to bind the intracellular domain of various tyrosine kinase receptors, e.g. KIT, FLT3 and RET, as well as low-density lipoprotein receptor-related protein 6, a negative regulator of the Wnt/β-catenin pathway. Analyzing RNAseq data from the Leucegene Project, we found that Grb10 is expressed on the majority of patient AML samples regardless of leukemia mutation profile. Silencing of Grb10 expression via Grb10 shRNA increased the proliferation and colony forming capacity of human AML cell lines, Kasumi-1, THP-1 and OCI-AML3 in vitro (p<0.0001 and p<0.01). Conversely, overexpression of Grb10 suppressed human AML cell growth (p<0.05). In order to determine the role of Grb10 in regulating AML growth in vivo, we transduced bone marrow lineage negative cells from mice with Grb10 maternal allele deletion (Grb10 m/+) and wild type (Grb10 +/+) mice with HoxA9-Meis1-neo-MSCV (gift from G. Savageau) and transplanted the progeny into congenic mice. Primary and secondary mice transplanted with Grb10m/+ HoxA9-Meis1 leukemia cells displayed significantly decreased survival compared to mice transplanted with Grb10+/+ HoxA9-Meis1 cells (p<0.001 and p<0.05). Furthermore, leukemia cells with Grb10 maternal allele deletion displayed an increase cell cycle progression and increased leukemia colony forming capacity. RNAseq analysis of Grb10 m/+ leukemia cells from diseased mice revealed significant dysregulation of the canonical Wnt/β catenin signaling pathway compared to Grb10 +/+ mice. RT-qPCR analysis confirmed that Wnt/β-catenin target genes, including MYC, CCND1, and SOX2 were significantly up-regulated in Grb10 knockdown human AML cells lines. Taken together, these data suggest that Grb10 is a powerful tumor suppressor in human AML, and represents a novel mechanistic target for the development of new therapies for human AML. Disclosures No relevant conflicts of interest to declare.

Prognostic Implications and Biological Roles of RhoH in Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4246-4246
Author(s):  
Toshihiro Iwasaki ◽  
Akira Katsumi ◽  
Hitoshi Kiyoi ◽  
Ryohei Tanizaki ◽  
Miki Kobayashi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Disease Free Survival ◽  
B Cell Lymphoma ◽  
Small Gtpases ◽  
Expression Level ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Rho Family ◽  
Acute Myeloid

Abstract The Rho family of small GTPases including Rho, Rac, and Cdc42 has been well characterized as molecular switches to transduce signals from plasma membrane to the downstream effectors. Rac1 and Rac2 are known to regulate engraftment and mobilization of hematopoietic stem cells. RhoH, a member of the Rho family, is specifically expressed in hematopoietic cells, and has been reported to inhibit the cell adhesion through regulating Rac and αLβ2 integrin. As RhoH is GTPase deficient and constitutively active, GTP-bound form, the activity of RhoH is directly related to the level of expression. Previous reports demonstrated the aberrant somatic hypermutation of RhoH gene as a novel mechanism of genetic lesion in diffuse large B-cell lymphoma, possibly through the deregulated expression although the role of the RhoH on leukemia is largely unknown. Here we have screened for the expression level of RhoH gene in the bone marrow samples from 90 previously untreated acute myeloid leukemia cases by using a real-time fluorescence detection method. The expression level of RhoH was neither related to the FAB classification, CR rate, nor WBC counts. In addition, the RhoH expression was not associated with the known gene mutations such as N-Ras, FLT3, and p53. However, the multivariate analysis demonstrated that low expression of RhoH was the independent unfavorable prognostic factor for overall and disease free survival (p=0.0028 and 0.003, respectively). RhoH did not affect the affinity modulation of α4β1 integrin, however, RhoH negatively regulate Rac activation in our system, suggesting that RhoH might work as a proapoptic molecule through Rac deactivation. Further investigations would be required to clarify the biological roles of RhoH on leukemic cells.

Impaired Hematopoietic Differentiation of iPSCs Derived From Patients with FPD/AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 767-767
Author(s):  
Masatoshi Sakurai ◽  
Hiroyoshi Kunimoto ◽  
Naohide Watanabe ◽  
Yumi Fukuchi ◽  
Ken Sadahira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Pluripotent Stem Cells ◽  
Myeloid Leukemia ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Differentiation Capacity ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Abstract 767 Somatic mutation of RUNX1 has been implicated in a variety of hematopoietic malignancies including myelodysplastic syndrome and acute myeloid leukemia, and previous studies using mouse models disclosed its critical roles in hematopoiesis. During embryonic development, Runx1 is absolutely essential in the emergence of hematopoietic stem and progenitor cells through hemogenic endothelium. In contrast, conditional disruption of Runx1 in adult hematopoietic system revealed that it was critical in the differentiation of megakaryocytes and lymphocytes as well as in the function of hematopoietic stem cells (HSCs). However, these results were derived from gene-disruption studies in mouse models, and the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder/ acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and the pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. These pedigrees have distinct heterozygous mutations in RUNX1 gene, two in the N-terminal RUNT domain affecting its DNA-binding activity and one in the C-terminal region affecting its transactivation capacity. After obtaining informed consent from the affected patients, we established iPSCs from their peripheral T cells by infecting Sendai viruses expressing four reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC). FPD-iPSCs could be established in comparable frequency as the one from normal individuals (WT-iPSCs). Initial characterization of FPD-iPSCs revealed that the established clones retained typical characteristics of pluripotent stem cells such as the expression of Nanog, Oct3/4, SSEA-3, SSEA-4, Tra-1-60 or Tra-1-81, and the teratoma formation in immunodeficient mice. Next we examined the hematopoietic differentiation capacity of FPD-iPSCs by co-culturing on AGMS-3 cells, a stromal cell line established from aorta-gonad-mesonephros (AGM) region. FPD-iPSCs and WT-iPSCs were dispersed and plated on inactivated AGM-S3 cells and were co-cultured in the presence of vascular endothelial growth factor. On day 10 through day 14 of co-culture, cells were collected and analyzed for the emergence of hematopoietic progenitors (HPCs) by flow cytometry. Interestingly, FPD-iPSCs generated CD34+ cells or CD45+ cells in significantly lower frequencies as compared to WT-iPSCs. To evaluate the differentiation capacity of HPCs generated from iPSCs, CD34+ cells were sorted by flow cytometry and subjected to colony forming assays. This revealed that CD34+ cells derived from FPD-iPSCs generated significantly fewer colonies as compared to those from WT-iPSCs in all colony types examined, showing that differentiation capacity of HPCs were impaired by RUNX1 mutation. Furthermore, CD34+ cells from FPD-iPSCs generated CD41a+CD42b+ megakaryocytes (MgK) in significantly lower frequencies as compared to WT in in vitro liquid culture with stem cell factor (SCF) and thrombopoietin (TPO). Of note, MgKs differentiated from FPD-iPSCs are smaller in size as evidenced by mean-FSC by flow cytometry. These results indicate that differentiation of MgKs is impaired both quantitatively and qualitatively. Importantly, all three FPD-iPSC lines share the same phenotype in the above-described assays, suggesting that N-terminal and C-terminal RUNX1 mutations impose similar defects in hematopoietic differentiation of FPD-iPSCs. Taken together, this study, for the first time, demonstrated that mutation of RUNX1 leads to the defective differentiation of hematopoietic cells in human settings. The phenotype observed in this study, at least in part, recapitulates the ones previously reported in Runx1-homozygously deficient mice, suggesting that the mutations of RUNX1 seen in FPD/AML indeed act in dominant negative manner. Disclosures: No relevant conflicts of interest to declare.

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