Counterpoint: In Some AMLs, Stem Cells are More Mature and Present at High Frequency.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-35-SCI-35
Author(s):  
Michael L. Cleary
Keyword(s):  
Stem Cells ◽  
Mouse Models ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Hierarchical Organization ◽  
Leukemia Stem Cells ◽  
Aberrant Expression ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Current Paradigm

Abstract Abstract SCI-35 Leukemia stem cells (LSCs) are responsible for sustaining and propagating malignant disease and, therefore, are promising targets for therapy. The current paradigm for LSC frequency, maturation and hierarchical organization is primarily based on transplantation studies in xenograft mouse models. To circumvent potential limitations of this experimental approach, investigators have recently employed syngeneic mouse models to study LSCs. In a mouse model of AML initiated by MLL oncogenes, which are associated with the FAB-M4 or M5 subtypes of human AML, LSCs are remarkably frequent, accounting for up to one-quarter of malignant myeloid cells at late-stage disease. Even in this syngeneic setting, however, transplant assays alone markedly underestimate LSC frequency due to poor engraftment efficiency. LSCs are organized in a phenotypic and functional hierarchy, and express myeloid lineage-specific antigens, placing them downstream of the known hematopoietic progenitor compartments. Thus, LSCs in this model are not synonymous with normal upstream progenitors that are targeted for leukemia initiation, but rather constitute myeloid lineage cells that have acquired an aberrant self-renewal program as well as other biologic features of hematopoietic stem cells. Gene expression profiling confirms the downstream myeloid character of LSCs in this model, and further demonstrates the aberrant expression of a stem cell associated transcriptional subprogram. However, LSC maintenance in the self-renewing compartment of AML employs a global transcriptional program more akin to embryonic rather than adult stem cells. Expression of LSC maintenance program genes is enriched in poor prognosis human malignancies, suggesting that the frequency of aberrantly self-renewing progenitor-like cancer stem cells may be linked to prognosis in human cancer. Consistent with this possibility, LSC frequencies in different syngeneic models of Hox-associated AML can vary over three orders of magnitude, depending on the particular initiating oncogene and expression levels of Hox pathway co-regulators, and correlate with leukemia biology. Studies in a human cord blood cell transduction/transplantation model of AML further support the downstream character of MLL LSCs. These findings prompt a revision of the current paradigm that AML leukemia stem cells are always rare and solely located within the most immature bone marrow progenitor compartments. The fact that LSCs can be more analogous to precursors and employ ESC-like genetic programs for their maintenance, may allow for their selective therapeutic targeting that spares HSCs required for hematopoiesis. Disclosures No relevant conflicts of interest to declare.

PU.1-Mediated Upregulation of M-CSFR Is Critical for MOZ-Leukemia Stem Cell Potential.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 216-216
Author(s):  
Yukiko Aikawa ◽  
Takuo Katsumoto ◽  
Daniel G. Tenen ◽  
Issay Kitabayashi
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinases ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Suicide Gene ◽  
Leukemia Stem Cells ◽  
Monocytic Leukemia ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Stem Cell Potential

Abstract Abstract 216 Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. The eradication of cancer stem cells is thought to be critical for successful anti-cancer therapy. However, there is little evidence for this. Using an acute myeloid leukemia (AML) model by introducing the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. Chromosomal translocations that involve the MOZ gene are typically associated with the FAB-M4 or -M5 subtype of human AML and often predict a poor prognosis. While MOZ is essential for the self-renewal of hematopoietic stem cells, MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells. The MOZ-fusion proteins interacted with PU.1 to stimulate the expression of macrophage-colony stimulating factor receptor (M-CSFR). Cells expressing high levels of M-CSFR (M-CSFR -high cells), but not those expressing low levels of M-CSFR, showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the M-CSFR promoter, AML was cured by ablation of the M-CSFR -high cells. Analysis of M-CSFR-deficient and PU.1-deficient mice showed that M-CSFR and PU.1 was essential to induce AML. Inhibitors for tyrosine kinases including M-CSFR slowed the progress of MOZ-TIF2-induced leukemia. Thus, M-CSFR -high cells contain leukemia stem cells, and the PU.1-mediated upregulation of M-CSFR is a useful therapeutic target for MOZ leukemia. Disclosures: No relevant conflicts of interest to declare.

CD34+/CD38- Leukemia Stem Cells Aberrantly Express CD82 Adhesion Molecule

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2168-2168
Author(s):  
Takayuki Ikezoe ◽  
Chie Nishioka ◽  
Jing Yang ◽  
Satoshi Serada ◽  
Tetsuji Naka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
Clinical Samples ◽  
Bone Marrow Niche ◽  
Absolute Quantitation ◽  
Aberrant Expression ◽  
Migration Capacity

Abstract Abstract 2168 To identify molecular targets in leukemia stem cells (LSCs), this study compared the protein expression profile of freshly isolated LSCs (CD34+/CD38- compartment) with that of non-LSC (CD34+/CD38+ compartment) counterparts from individuals with acute myelogenous leukemia (AML) using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 98 proteins were overexpressed, while six proteins were underexpressed in LSCs compared with their non-LSC counterparts. Proteins overexpressed in LSCs included a number of proteins involved in DNA repair, cell cycle arrest, gland differentiation, anti-apoptosis, adhesion, and drug resistance. Aberrant expression of CD82, a family of adhesion molecules, in LSCs was noted in additional clinical samples (n=6) by flow cytometry. In addition, we found that imatinib-resistant chronic eosinophilic leukemi EOL-1R cells expressed a greater amount of CD82 and remained in a dormant state compared to the parental EOL-1 cells. Interestingly, down-regulation of CD82 in EOL-1R cells by a small interfering RNA stimulated their migration capacity, as assessed by the transwell assay. These observations suggested that the aberrant expression of CD82 probably played a role in adhesion of hematopoietic cells to bone marrow microenvironment. Targeting CD82 could detach LSCs from bone marrow niche and sensitized these cells to anti-leukemia agents. Disclosures: No relevant conflicts of interest to declare.

Targeting on the Stem Cell Niche Can Protect Hematopoietic Stem Cell from Chemotherapy and G-CSF

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5790-5790
Author(s):  
Sidan Li ◽  
Qiongli Zhai ◽  
Dehui Zou ◽  
Changhong Li ◽  
Lugui Qiu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mouse Models ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Transplant Patients ◽  
Cell Engraftment ◽  
Cell Niche

Abstract The majority of hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow surrounded by specialized bone-shielded environment. The specialized microenvironment or niche not only provides a favorable habitat for HSPC maintenance and development but also governs stem cell function. Here we investigated the potential role of bone remodeling osteoblasts and osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors, then further tested the hypothesis that targeting the niche might improve stem cell–based therapies using six mouse models to mimic the multiple rounds of chemotherapy followed by autologous hematopoietic stem cells (HSCs) transplantation in a clinical setting. Herein, we show that multiple rounds treatment of cytotoxic drugs influence niche. Serum osteocalcin level declined obviously (22.19 ± 1.08 ng/mL, before treatment vs 16.08 ± 2.12 ng/mL, steady state, P=0.01) in autologous HSPCs transplant patients. In mouse models, the number of CD45- Ter119- OPN+ osteoblast was significantly reduced (untreated, 3993 ± 129 cells/femur; CTLs, 1937 ±196 cells/femur; Gs, 1055 ± 43 cells/femur; P<0.01). Pharmacologic use of parathyroid hormone (PTH) or receptor activator of nuclear factor kappa-B ligand (RANKL) increases the number of HSC mobilized into the peripheral blood for stem cell harvests and protects stem cells from repeated exposure to cytotoxic chemotherapy. Ttreatment with granulocyte colony stimulating factor (G-CSF) plus PTH led to relative preservation of the HSC pool (G vs PTH, P<0.01; CTL vs PTH, P<0.05). Recipient mice transplanted with circulation HSPCs of P+R and P+R+G groups also showed more robust myeloid and lymphatic cell engraftment than did HSCs from either CTL or G group. These data provide evidence that targeting the HSPC niche may improve the efficacy of HSPC mobilization. Disclosures No relevant conflicts of interest to declare.

A New Flowcytometry-Based Method to Discriminate Malignant From Normal Stem Cells in CML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3245-3245
Author(s):  
Jeroen J.W.M. Janssen ◽  
Wendy Deenik ◽  
Karlijn G.M. Smolders ◽  
Monique Terwijn ◽  
Angele Kelder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Risk Scores ◽  
Fish Analysis ◽  
Leukemic Stem Cells ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Culture Techniques

Abstract Abstract 3245 Poster Board III-182 Tyrosine kinase inhibitor (TKI) insensitivity of CML hematopoietic stem cells prevents eradication of the disease by these drugs and is presumably implicated in development of TKI resistance. Probably, improvement of treatment results will involve leukemic stem cell directed therapy. Therefore, more knowledge of stem cell specific targets would be instrumental. Previously, leukemic stem cells could only be identified indirectly by using culture techniques. We developed a new flowcytometric approach that enables to directly distinguish CML stem cells from their normal counterparts within single patient samples. In 24 newly diagnosed CML patients CML CD34+CD38- stem cells could be discriminated from normal stem cells by higher CD34 and CD45 expression and different forward/sideward light scatter properties, reflecting differences in size and granularity. In addition, aberrant expression of CD7, CD11b and CD56 was demonstrated on malignant stem cells, allowing clear discrimination from benign stem cells, that were always negative for these markers. Above all, in all tested CML patients we were able to demonstrate that high CD90 expression is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. FISH analysis on FACS sorted cells proved that populations were BCR-ABL positive (in case of high CD34 and CD45 expression and high CD90 expression) or negative (in case of low CD34 and CD45 expression and low CD90 expression), while long term liquid culture assays with subsequent CFU assays and FISH analysis proved their malignant/normal stem cell character. Patients with a large proportion of non-leukemic stem cells had significantly lower clinical risk scores (Sokal, Euro) than patients with few remaining normal stem cells. This new technique will expand our possibilities to identify new CML stem cell specific targets and may improve efficacy assessment of CML treatment as well. Disclosures No relevant conflicts of interest to declare.

Small-Molecule Inhibition of BRD4 Is a Novel Promising Approach to Therapeutically Target Leukemia Stem Cells in AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3484-3484 ◽  
Author(s):  
Harald Herrmann ◽  
Katharina Blatt ◽  
Junwei Shi ◽  
Amy R. Rappaport ◽  
Karoline V. Gleixner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Mouse Models ◽  
Small Molecule ◽  
Conflicts Of Interest ◽  
Leukemia Stem Cells ◽  
Clinical Value ◽  
In Vivo Evaluation ◽  
Small Molecule Inhibition

Abstract Abstract 3484 Acute myeloid leukemia (AML) is a stem cell-derived malignancy characterized by uncontrolled proliferation and accumulation of myeloblasts in hematopoietic tissues. The clinical course and prognosis in AML vary depending on deregulated genes, cell type(s) involved, and the biological properties of the clone. In most variants of AML, the complexity and heterogeneity of oncogenomes pose a challenge for the development of effective targeted therapeutics. However, diverse genetic aberrations in AML typically converge functionally to dysregulate the same cellular core processes. One key event is the corruption of myeloid cell-fate programs resulting in the generation of aberrantly self-renewing leukemia stem cells (LSC), which maintain and propagate the disease and are often resistant to conventional chemotherapy. Hence, strategies aimed at terminating aberrant self-renewal and eradicating LSC are considered as key for the development of more effective AML therapies. In an effort to systematically probe genes involved in chromatin regulation as potential therapeutic targets, we recently employed an unbiased screening approach combining AML mouse models and new in-vivo RNAi technologies, through which we identified the epigenetic ‘reader' BRD4 as new candidate drug target in AML (Zuber et al., Nature, in press). Inhibition of BRD4 using RNAi or a new small-molecule inhibitor (JQ1) blocking BRD4 binding to acetylated histones, showed profound antileukemic effects in AML mouse models, in all human AML cell lines tested (n=8) as well as in primary AML cells. In all models tested, BRD4 suppression was found to trigger apoptosis as well as terminal myeloid differentiation, and potently suppressed expression programs previously associated with LSC. As one key target, we observed a dramatic transcriptional repression of MYC, which recently has been discussed as core component of an LSC associated transcriptional module. To further evaluate suppression of BRD4 as a potential therapeutic approach to eradicate LSC in human AML, we analyzed the effects of JQ1 in primary AML cells obtained from 17 patients with freshly diagnosed or relapsed/refractory AML (females, n=5, males, n=12, median age: 54 years; range: 21–80 years). In unfractionated primary AML cells, submicromolar doses of JQ1 were found to induce major growth-inhibitory effects (IC50 between 0.05 and 0.5 μM) in a broad spectrum of AML subtypes. No differences in IC50 values were seen when comparing drug effects in AML cells kept in the presence or absence of growth-stimulating cytokines (G-CSF, IL-3, SCF). In addition, JQ1 treatment effectively triggered apoptosis in all patients tested, with similar anti-leukemic activities observed in newly diagnosed pts and refractory/relapsed AML. To further evaluate the clinical value of BRD4 as a clinically relevant target in AML, we analyzed the effect of JQ1 on AML LSC. In these experiments, JQ1 effectively induced apoptosis in CD34+/CD38+ progenitor cells as well as in CD34+/CD38− AML stem cells in all donors examined as evidenced by combined surface/Annexin-V staining. Furthermore, JQ1 was found to induce morphologic signs of maturation in 6 of 7 patients examined, thereby confirming our previous data obtained in mouse AML cells. Finally, we were able to show that JQ1 synergizes with Ara-C in inducing growth inhibition in HL60 cells and KG-1 cells. In summary, our data show that small-molecule inhibition of BRD4 has strong anti-leukemic effects in a broad range of AML subtypes. Furthermore, our results support the notion that JQ1's ability to suppress LSC specific transcriptional modules may translate into a therapeutic entry point for eradicating LSC in primary AML. While a more extensive in vivo evaluation of these effects, as well as the development of pharmacologically improved compounds will be required, all existing data unambiguously highlight small-molecule inhibition of BRD4 as a new promising concept in AML therapy. Disclosures: No relevant conflicts of interest to declare.

Roles of Ring1A/B on Stem Cell Potential of MOZ and Other Acute Myeloid Leukemias,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3998-3998
Author(s):  
Haruko Shima ◽  
Mika Shino ◽  
Kazutsune Yamagata ◽  
Yukiko Aikawa ◽  
Haruhiko Koseki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Acute Myeloid

Abstract Abstract 3998 Leukemia and other cancers possess self-renewing stem cells that help maintain cancer. Chromosomal translocations are often involved in the development of human acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene is one of the targets of such translocations. While MOZ is essential for the self-renewal of hematopoietic stem cells, the leukemia associated MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells. Ring1A and Ring1B are catalytic subunits of the polycomb-group repressive complex 1 (PRC1) complex containing Bmi1, and PRC1 complex plays an important role in the regulation of stem cell self-renewal. Using Ring1A-null and Ring1B-conditional deficient mice, we showed that Ring1A/B are required for continuous colony forming ability that is enabled by MOZ-TIF2 and other AML-associated fusions such as MLL-AF10, AML1-ETO, and PML-RARα. Furthermore, MOZ-TIF2- and MLL-AF10-induced AML development in mice were prevented by Ring 1A/B deficiency. To clarify the mechanism of stemness regulation in AML stem cells by PRC1 complex, we compared gene expression profiles of Ring1A/B deleted and non-deleted MOZ-TIF2-induced AML cells. As expected, Ink4a/Arf, a known major target of PRC1 complex involved in stem cell functions, was derepressed by deletion of Ring1A/B. Although deletion of Ink4a/Arf in Ring1A/B deficient AML cells partially restored colony formation ability, it was not substantial to initiate leukemia in recipient mice. Among several target genes which were derepressed by deletion of Ring1A/B, we focused on “Stemness inhibitory factor (SIF)”, known to be required for cell differentiation and morphogenesis in some specific organs. Enforced expression of SIF in MOZ-TIF2-induced AML cells stimulated differentiation of AML progenitors into macrophages. On the other hand, knock-down of SIF blocked cell differentiation block and restored the immortalizing ability of MOZ-TIF2-induced AML progenitors, despite of the absence of Ring1A/B. Collectively, our data demonstrate that Ring 1A/B play crucial roles in the maintenance of AML stem cells through repression of SIF, which strongly promote differentiation of leukemia stem cells. Disclosures: No relevant conflicts of interest to declare.

Inhibition of EZH2 Depletes MLL Fusion Leukemia Stem Cells Through Restoration of p16 Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3510-3510
Author(s):  
Koki Ueda ◽  
Akihide Yoshimi ◽  
Masahiro Nakagawa ◽  
Satoshi Nishikawa ◽  
Victor E Marquez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
High Specificity ◽  
Prolonged Survival ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Capacity

Abstract Abstract 3510 Leukemia stem cells (LSCs) are resistant to conventional chemotherapy and persistent LSCs after chemotherapy are supposed to be a major cause of disease relapse or refractoriness. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC-targeted drugs have scarcely been identified or used in clinical settings so far. Epigenetic regulators are associated with many cellular processes such as cell cycle, proliferation, and apoptosis. Of note are polycomb group proteins, because they potentially control stemness including activity of cancer stem cells, and can be pharmacologically targeted by a selective inhibitor of H3K27, 3-deazaneplanocin A (DZNep). We first administrated DZNep to MLL-related leukemia mouse model in order to test whether DZNep has potential to eradicate LSCs of the leukemic mice. Remarkably, the leukemic granulocyte-macrophage progenitors (LGMPs) in MLL/AF9 positive cells were significantly decreased in number by administration of DZNep while AraC did not affect the number of LGMPs, which implied that LSCs were targeted by DZNep. These data were reproduced by transplantation assays using short hairpin RNA (shRNA)-mediated knockdown of EZH2, a major component of polycomb repressive complex 2 (PRC2) which is responsible for H3K27 tri-methylation. Significantly, DZNep administration to wild-type mice led to only mild suppression of hematopoiesis, suggesting that this agent spares normal hematopoietic stem cells while eliminating LSCs, which is consistent with a previous report that genetic depletion of EZH2 did not compromise adult hematopoiesis in mice. Serial replating assay of MLL/AF9-induced leukemia cells showed that DZNep treatment in vivo diminished their colony forming capacity. Limiting dilution transplantation assays revealed that frequency of LSCs was markedly reduced by DZNep administration. DZNep treatment or EZH2 knockdown significantly prolonged survival of MLL/AF9 and MLL/ENL leukemic mice. To elucidate a molecular mechanism underlying the effects of DZNep on LSCs, we investigated transcriptional or epigenetic changes during DZNep treatment and EZH2 knockdown. Gene expression profiling revealed that p16 was significantly upregulated by EZH2 knockdown or DZNep administration. Knockdown of p16 completely canceled the survival advantage of the leukemia mice which received DZNep in vivo and restored the colony forming capacity of leukemia cells transduced with shRNA for EZH2 in vitro. These results supported the idea that p16 upregulation derived from EZH2 attenuation is central to the LSC reduction. Next, we investigated epigenetic status around p16 promoter and transcription start site (TSS) by chromatin immunoprecipitation (ChIP) assays. In MLL/ENL leukemia cells, both H3K4 and H3K27 methylation marks were highly enriched around the TSS of p16, together with EZH2 and Bmi1, a component of PRC1. Therefore removal of EZH2 is supposed to convert the promoter of p16 from a bivalent to an active state. The results of ChIP assays also indicated that MLL/ENL fusion protein binds to p16 coding region. In order to clarify whether dependency on EZH2 is specific for MLL fusion leukemia or can be applied for other types of leukemia, we evaluated the consequence of EZH2 inhibition in several types of leukemia. DZNep or shRNA for EZH2 strongly suppressed the proliferation of leukemia cell lines and immortalized cells harboring MLL fusion genes with high specificity. Administration of DZNep or transduction of shRNA targeting EZH2 significantly prolonged survival of MLL/AF9 and MLL/ENL-induced leukemia mice while TEL/PDGFRA-AML1/ETO-induced leukemia was not sensitive to DZNep, although bone marrow (BM) cells from either mice became globally hypo-methylated on H3K27 by exposure to this drug. Serial replating assay with DZNep or EZH2-shRNA demonstrated high sensitivity to EZH2 inhibition of MLL/AF9-transduced BM cells but not of AML1/ETO-transduced BM cells, E2A/HLF-transduced BM cells, or normal c-kit+ BM cells. Thus, the anti-leukemia effect of EZH2 inhibition is thought to be specific for MLL fusion leukemia. Collectively, our findings indicate that EZH2 is a potential therapeutic target of LSCs of MLL fusion leukemia to overcome the poor prognosis, encouraging the development of inhibitors against EZH2 with high specificity. Disclosures: No relevant conflicts of interest to declare.

Impact Of TET2 Deficiency On MPN Harboring JAK2V617F Mutation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 478-478
Author(s):  
Takuro Kameda ◽  
Kotaro Shide ◽  
Masaaki Sekine ◽  
Ayako Kamiunten ◽  
Tomonori Hidaka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Models ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Liver Weight ◽  
Mutant Mice ◽  
Spleen Weight ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Colony Forming Assay

Introduction JAK2V617F (JAK2VF) is the most frequent mutation in myeloproliferative neoplasms (MPN), and its role has been demonstrated in mouse models. Actually, JAK2VF transgenic (JAK2VF Tg) mice generated by us induce lethal MPN (Shide et al. Leukemia 2008). Recently, mutations of epigenetic regulator such as TET2 are also frequently identified in MPN, and several TET2 knock out or knock down (TET2KD) mouse models are generated. We previously analyzed TET2KD mice (Ayu17-449) (Shide et al. Leukemia 2012). TET2KD fetal liver (FL) or bone marrow (BM) cells showed a growth advantage over Wt BM cells, with increased self-renewal capacity of hematopoietic stem cells; however TET2KD mice didn’t develop MPN, and its role in MPN remained unclear. To explore the role of TET2 deficiency in MPN harboring JAK2VF, we examined the cooperative effect, using these mutant mice. Materials and methods (1) Mice and collection of test cells. JAK2VF Tg mice (C57BL/6, Ly5.2) and TET2KD mice (Ayu17-449, C57BL/6, Ly5.2) were used. We crossed them, and collected JAK2Wt-TET2Wt (Wt-Wt), JAK2Wt-TET2KD (Wt-KD), JAK2VF-TET2Wt (VF-Wt), and JAK2VF-TET2KD (VF-KD) FL cells. (2) Non-competitive repopulation assay (NCRA). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) without competitor cells. Recipients were analyzed by complete blood counts, flow cytometry, colony-forming assay, colony-replating assay, pathology at 20-28 weeks post-transplantation, and overall survival. (3) Competitive repopulation assay (CRA) and serial BM transplantation (sBMT). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) with competitor Wt BM cells (Ly5.1, 5x106 cells), and sBMT was performed by 1x106 BM cells of the recipients at every 12 weeks post-transplantation. Recipients which were not selected as the donors were analyzed. (4) Analyses of adult mutant mice. Mice were bred in BDF1 background and analyzed at 20 or more weeks of age, as well as the recipients in NCRA. (5) Statistical analysis. Results were presented as means±S.D. Two-tailed Student’s t-test and log-rank test were used. Result In NCRA, both recipients transplanted with VF-Wt cells and VF-KD cells developed MPN with increase in WBC and Plt, decrease in Hb, fibrosis in BM and spleen, and extramedullary hematopoiesis (EMH) of lung and liver; and the latter developed more severe MPN and died earlier: VF-Wt (n=10) vs. VF-KD (n=10); WBC (x104/µl), 4.2±1.6 vs. 7.3±3.3 (p<0.05); peripheral blood (PB) myeloid cells (%), 59.6±9.7 vs. 71.9±8.2 (p<0.05); liver weight (g), 1.15±0.22 vs. 1.48±0.22 (p<0.01); spleen weight (g), 0.26±0.11 vs. 0.52±0.19 (p<0.01): VF-Wt (n=36) vs. VF-KD (n=30); mean survival time (weeks), 36 vs. 39 (p<0.05). In colony-forming assay, number of CFU-GM was more increased in VF-KD cells than VF-Wt cells: VF-Wt (n=9) vs. VF-KD (n=9); colonies/2x104 BM cells, 107±37 vs. 157±46 (p<0.05). In colony-replating assay, VF-Wt BM cells lost replating capacity by 3rd to 5th passage; VF-KD BM cells retained replating capacity beyond 5th passage: VF-Wt (n=9) vs. VF-KD (n=6); number of colonies in 4th passage, 5.9±6.8 vs. 896±613 (p<0.01). In CRA, all recipients transplanted with VF-Wt cells (n=9) or VF-KD cells (n=9) showed ≥ 70% test cell-derived PB chimerism, and developed MPN with fibrosis and EMH at 12 weeks. In 2nd BMT, 4/9 recipients transplanted with VF-Wt cells showed ≥ 35% PB chimerism at 12 weeks. Six recipients were analyzed at 12-16weeks, and no one (0/6) showed pathological findings of MPN. Whereas, 7/9 recipients transplanted with VF-KD cells showed ≥ 35% PB chimerism. Five recipients were analyzed, and 3/5 developed MPN with fibrosis and EMH: VF-Wt (n=6) vs. VF-KD (n=5); liver weight (g), 1.00±0.12 vs. 1.39±0.15 (p<0.002); spleen weight (g), 0.069±0.019 vs. 0.20±0.097 (p<0.05). In analyses of adult mutant mice, both VF-Wt mice and VF-KD mice developed MPN, and disease severities or colony-replating capacities are similar tendencies as those in transplantation model. Conclusion TET2 deficiency increases severity of MPN harboring JAK2VF. TET2 deficiency enhances disease initiating potential of JAK2VF-MPN stem cells. TET2 deficiency is considered to be critical for both onset and progression of MPN harboring JAK2V617F. 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.

Remodeling Ca2+ Flux By ORP4L Is Essential for Leukemia Stem Cells (LSCs) Survival

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5257-5257
Author(s):  
Wenbin Zhong ◽  
Vesa Olkkonen ◽  
Xu Bing ◽  
Biying Zhu ◽  
Guoping Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Function ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
Ip3 Receptor ◽  
Hematopoietic Stem

Abstract Acute myelogenous leukemia (AML) is one of the deadliest hematological malignancies and there is at present no efficient strategy to defeat it. New detailed insight into AML leukemia stem cells (LSCs) survival will facilitate the identification of targets for the development of new therapeutic approaches. Recent work has provided evidence that LSCs are defective in their ability to employ glycolysis, but are highly reliant on oxidative phosphorylation, and the maintenance of mitochondrial function is essential for LSCs survival. It is increasingly clear that Ca2+ released constitutively from endoplasmic reticulum (ER) is taken up by mitochondria to sustain optimal bioenergetics and cell survival. Here we report three striking findings: 1) oxysterol-binding protein (OSBP)-related protein 4 (ORP4L) is expressed in LSCs but not in normal hematopoietic stem cells (HSCs). 2) ORP4L is essential for LSC bioenergetics; It forms a complex with PLCβ3 and IP3 receptor 1 (ITPR1) to control Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that sustain pyruvate dehydrogenase (PDH) activation and oxidative phosphorylation. 3) ORP4L inhibition eradicates LSCs in vitro and in vivo through impairment of Ca2+-dependent bioenergetics. These results suggest a novel role of ORP4L in governing Ca2+ release to sustain mitochondrial function and survival of LSCs and identify ORP4L as a putative new oncoprotein and therapeutic target for LSCs elimination. Disclosures No relevant conflicts of interest to declare.

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