scholarly journals Role of Signal Transducing Adaptor Protein-1 (STAP-1) in Chronic Myelogenous Leukemia Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4245-4245
Author(s):  
Jun Toda ◽  
Michiko Ichii ◽  
Hirohiko Shibayama ◽  
Hideaki Saito ◽  
Yuichi Kitai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Z Score ◽  
Rna Seq ◽  
Interacting Protein ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Apoptotic Genes ◽  
Induced Apoptosis

Abstract Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder caused by hematopoietic stem cells expressing the BCR-ABL fusion oncoprotein, which constitutively activates multiple signal transduction pathways such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and Janus kinase/signal transducer and activator of transcription (JAK/STAT). Although tyrosine kinase inhibitor (TKI) therapy results in dramatic clinical success, studies have shown that TKIs are unable to eradicate leukemic stem cells (LSCs). Several key signaling molecules and pathways have been proposed to regulate the survival of CML LSCs in the presence of TKI; however, the details remain unclear. It is necessary to elucidate the mechanisms that maintain LSCs to better understand the pathogenesis of CML and develop new treatment approaches. The family of signal-transducing adaptor proteins (STAPs), which includes STAP-1 and STAP-2, has been implicated in various intracellular signaling pathways. In 2003, we cloned STAP-2 as a c-fms interacting protein and reported that STAP-2 binds to BCR-ABL and enhances activity, leading to the activation of downstream molecules such as ERK, STAT5, BCL-xL, and BCL2. STAP-1 was cloned as a c-kit interacting protein from a hematopoietic stem cell library, but it is unknown whether STAP-1 plays a role in CML. Given the structural homology between STAP-1 and STAP-2 and the hematopoietic expression of STAP-1, we hypothesized that STAP-1 might contribute to the leukemogenesis of CML. A STAP-1-deficient (KO) CML mouse model was developed. To generate this model, lineage (Lin)− Sca-1+ c-Kithigh (LSK) fraction isolated from bone marrow (BM) cells was infected with a retrovirus carrying BCR-ABL1 and GFP and subsequently transplanted into congeneric recipients. STAP-1 KO CML mice showed significantly longer survival than WT CML mice and displayed less severe splenomegaly and lung hemorrhages compared with WT mice. In recipient BM, absolute numbers of STAP-1 KO LSCs (GFP+ LSK cells) were significantly lower than WT LSCs. In the colony-forming assay, STAP-1 KO LSCs generated fewer colonies compared to WT LSCs. Using flow cytometric analysis, we found that STAP-1 KO LSCs had a higher apoptotic rate than WT LSCs. These findings suggest that the suppression of apoptosis induced by STAP-1 mediates longer survival of LSCs. To further understand the effects of STAP-1, we performed a gene expression analysis using RNA-sequence (RNA-seq) and compared WT and STAP-1 KO CML LSCs. When canonical pathways were analyzed with Ingenuity Pathway Analysis, various pathways associated with inflammatory cytokines were observed to be regulated in STAP-1 KO CML LSCs. Changes in mRNA expression, including that of SOS1, SOS2, FOXO3, FASLG, NFKB2, and BCL-xL, indicated that the PTEN signaling pathway, known to play a tumor suppressive role in CML, was significantly activated by STAP-1 KO (p=1.096E-3, activation Z-score=2.611). The pathway related to JAK/STAT signaling was also affected (p=2.04E-5, activation Z-score=-3.286). Downstream genes in the JAK/STAT signaling pathway, such as STAT5B and BCL-xL, were downregulated more than 2-fold in STAP-1 KO LSCs, suggesting that the deletion of STAP-1 inhibits the expression of STAT5-targeted anti-apoptotic protein and induced apoptosis of CML LSCs. To confirm the results of the RNA-seq experiment, an intracellular flow cytometric assay with CML Lin− cells was conducted. The frequency of cells positive for phosphorylated STAT5 was reduced for STAP-1 KO compared with that for WT. Quantitative PCR with CML LSCs confirmed the downregulation of BCL2 and BCL-xL, which are STAT5-targeted anti-apoptotic genes, in STAP-1 KO CML LSCs. In conclusion, we show that STAP-1 plays a crucial role in the maintenance of CML LSCs using a murine model of CML. STAP-1 deficiency results in the reduction of phosphorylated STAT5, downregulation of anti-apoptotic genes BCL-2 and BCL-xL, and induced apoptosis of CML LSCs. These findings suggest that STAP-1 and related signaling pathways could be potential therapeutic targets for CML LSCs. Disclosures Ichii: Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Fujimoto Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

scholarly journals Monitoring ESAM Expression Levels Reveals Autonomous Fluctuation of Leukemia Stem Cells By Autocrine/Paracrine Cytokine Signaling

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3950-3950
Author(s):  
Yasuhiro Shingai ◽  
Takafumi Yokota ◽  
Takayuki Ozawa ◽  
Tomoaki Ueda ◽  
Yukiko Doi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Signaling Pathways ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Induced Apoptosis

Abstract Recently , we showed that hematopoietic stem cells (HSCs) fluctuate in a dynamic trajectory for self-renewal and lineage commitment, which regulates HSC heterogeneity (Cell Rep 2018). Although the significance of heterogeneity is difficult to interpret, it might enable HSCs to maintain a dynamic equilibrium of homeostatic hematopoiesis and to adapt promptly to stress-induced hematopoiesis based on physiological demands. We hypothesized that similar to HSCs, leukemia stem cells (LSCs) are heterogeneous with active fluctuation, which contributes to treatment resistance. Hence, this study aimed to show the fluctuation of LSCs and determine the underlying molecular mechanisms. We previously reported that the endothelial-cell selective adhesion molecule (ESAM) is useful as a life-long marker for HSCs in humans and mice (Blood 2009, Exp Hematol 2013). ESAM expression levels precisely indicated the activation status of HSCs (J Immunol 2012). Furthermore, approximately two-thirds of patients with acute myelogenous leukemia (AML) were positive for ESAM expression on leukemia cells in a heterogeneous manner. (Exp Hematol 2013). We exploited human AML lines as LSC-like cells and monitored expression levels of ESAM. Flow cytometry analyses revealed wide-ranging ESAM expression levels on KG1a and CMK lines. In KG1a cells, ESAM-high cells were concentrated in the CD34+CD38- fraction with high growth capability, whereas ESAM-low cells were less proliferative. Notably, when sorted into ESAM-high or ESAM-low subgroups, both groups reconstituted the parental heterogeneous distribution of ESAM levels in culture. Furthermore, single self-renewing LSC-like clones also reproduced the same heterogeneous population regardless of their original ESAM levels. These results suggested that LSC-like clones developed the heterogeneous AML cell population by fluctuating, which could be monitored with ESAM levels. The transcriptomes of ESAM-high and ESAM-low LSC-like cells in KG1a line were compared to obtain comprehensive information of key molecules involved in fluctuation and heterogeneity. We found that expression of T-cell immunoglobulin and mucin domain 3, reported as a reliable LSC marker for human AML (Cell Stem Cell 2010), was variable in parallel with ESAM. In addition, network analysis demonstrated that "genes related to hematopoietic progenitor cells" were activated whereas "genes related to blood cell maturation" were downregulated in ESAM-high cells. These results suggested that ESAM-high LSC-like cells were more identical to the conventional concept of LSCs than ESAM-low cells. However, expression levels of BAALC, SPP1, and ITGB7 were elevated in ESAM-low cells. BAALC encodes "brain and acute leukemia cytoplasmic (BAALC)" protein, which inhibits drug-induced apoptosis in KG1a cells (Hematology 2012). SPP1 encodes "secreted phosphoprotein 1 (SPP-1), also known as osteopontin, which induces chemo-resistance in AML cells (Life sci 2017). Furthermore, a recent paper reported that the active form of integrin □7 can be a therapeutic target of myeloma stem cells (Nat Med 2018). Thus, we infer that fluctuating to the ESAM-low stage may contribute to the refractory nature of LSCs in AML. We performed an up-streaming analysis on the transcriptome data to determine the signaling pathways involved in the fluctuation. The tumor necrosis factor alpha (TNFα) and the transforming growth factor beta (TGFβ) signaling pathways were found to affect ESAM expression levels. When TNFα or TGFβ1 was added to culture medium, the proliferation of KG1a cells was suppressed in a dose-dependent manner. Furthermore, addition of TNFα or TGFβ1 significantly suppressed ESAM expression levels on a subset of KG1a cells, which enhanced their heterogeneity. When cultured in serum-free cytokine-free medium, KG1a cells became uniform in terms of ESAM expression levels, with the ESAM-low population disappearing . Intriguingly, we found that KG1a cells produced TNFα and TGFβ1 autonomously, which induced the cell heterogeneity in an autocrine/paracrine manner. Surprisingly, blocking these signaling pathways by receptor inhibitors induced apoptosis in KG1a cells. These results suggest that autocrine/paracrine cytokine signaling plays important roles in promoting the variable and heterogeneous nature of LSCs, the inhibition of which might be a strategy for therapeutic intervention in refractory AML. Disclosures Yokota: Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding; Celgene: Research Funding. Ichii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Jansen Pharmaceutical K.K: Honoraria; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Bone Marrow (BM) Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated with Nilotinib

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1570-1570
Author(s):  
Santa Errichiello ◽  
Simona Caruso ◽  
Concetta Quintarelli ◽  
Biagio De Angelis ◽  
Novella Pugliese ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Hematopoietic Progenitor Cell ◽  
Newly Diagnosed ◽  
Hematopoietic Stem ◽  
Optimal Response

Abstract Introduction Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Deletion of Rac2 inhibits Proliferation of Chronic Myelogenous Leukemia (CML) Stems Cells and Progenitors (HSC/P) In Vivo and Promotes Survival of Scl/p210-BCR-ABL Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3253-3253
Author(s):  
Amitava Sengupta ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
Jose Cancelas
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myelogenous Leukemia ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Rac Gtpases ◽  
And Migration

Abstract Abstract 3253 Poster Board III-1 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy induced by p210-BCR-ABL and is characterized by myeloproliferation in the bone marrow (BM) and egress of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSCs in patients under imatinib suggests that inhibition of ABL-kinase alone is not sufficient to completely eliminate the LSC/P population. Rac GTPases represent integrative molecular switches for p210-BCR-ABL-induced HSC transformation and combined pharmacological and genetic attenuation of Rac GTPases significantly prolong survival in vivo, as reported in a retroviral transduction/transplantation model (Thomas EK & Cancelas JA et al, Cancer Cell 2008). Here, we analyzed the role of Rac2 GTPase in the leukemic maintenance and in the interaction of LSC/P with the leukemic microenvironment in vivo. We used a stem cell leukemia (Scl) promoter-driven, tet-off, Scl-tTA x TRE-BCR-ABL (Scl/p210-BCR-ABL) binary transgenic mouse model (Koschmieder S et al., Blood 2005), where expression of BCR-ABL is restricted to the HSC/P compartment, allowing the study of the intrinsic molecular changes in LSC/P during leukemogenesis. In these mice, Scl-driven expression of BCR-ABL is active in HSC (Lin-/Sca1+/c-kit+; LSK) and progenitors (Lin-/c-kit+/Sca-1-; LK), and CML development is associated with the activation of downstream signaling effectors CrkL, p38-MAPK and JNK. Additionally, Scl/p210-BCR-ABL mice had increased cycling of LSK cells and expansion of circulating and splenic, but not BM, LSC/P, suggesting egress of LSC/Ps from the marrow. These mice share all the characteristics of HSC/P transformation in CML, including increased HSC/P proliferation and survival, severely reduced adhesion to fibronectin, increased migration towards CXCL12, increased cell surface expression of CD44 and decreased expression of L-selectin. Myeloproliferative disease (MPD) in these mice is transplantable into recipient mice, and CML splenocytes have a 10-fold increase in homing to the spleen than towards BM (P<0.05). Leukemic splenocytes are also enriched in endosteal lodging progenitors, compared to the BM-derived progenitors (1.9-fold, P≤0.05). In order to determine the contribution of Rac2 GTPase in the transformation phenotype of leukemic stem cells and progenitors, Scl/p210 mice were intercrossed with Rac2-/- mice. Interestingly loss of Rac2 GTPase alone significantly prolongs survival of the leukemic mice (P≤0.001). Prolonged survival, as observed in Scl/p210 x Rac2-/-, is associated with significantly reduced proliferation of leukemic LK (3-fold, P<0.05) and LSK (6-fold P<0.005) cells, both in BM as well as in spleen, in vivo. Scl/p210 x Rac2-/- mice are also characterized by increased apoptosis (1.7-fold) and lower frequency of LSK cells (2-fold) compared to the Scl/p210 mice in vivo. However, deletion of Rac2 does not significantly reverse the adhesion and migration transformation phenotype of LSC/P. In summary, Rac2 deficiency induces a significant survival of CML mice in a HSC-initiated model of disease through decrease proliferation and survival but does not reverse the transformation phenotype affecting adhesion and migration. This murine model may represent an adequate in vivo system to dissect out the specific signaling pathways involved in p210-BCR-ABL-induced stem cell transformation. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

scholarly journals Immunoglobulin Superfamily Member 8 Is Indispensable for Myeloid Leukemia Via Wnt/β-Catenin Signaling Pathway

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-24
Author(s):  
Koji Jimbo ◽  
Takaaki Konuma ◽  
Takahiro Ito ◽  
Yaeko Nakajima-Takagi ◽  
Atsushi Iwama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Immunoglobulin Superfamily ◽  
Hematopoietic Stem

Immunoglobulin superfamily member 8 (IGSF8, also known as EWI-2, PGRL, and CD316), is a cell surface protein containing 4 immunoglobulin domains. IGSF8 directly binds to the tetraspanin molecules, CD9 and CD81, and modulates cell adhesion, migration, and growth. Previous studies demonstrated that IGSF8 was associated with prognosis and metastasis in several solid tumors. However, the role of IGSF8 in normal hematopoiesis and myeloid leukemia is still unclear. First, we examined the expression levels of Igsf8 in various hematopoietic fraction of wild-type murine bone marrow cells, and found that Igsf8 is expressed in all hematopoietic lineages. To investigate hematopoietic functions of Igsf8, we generated hematopoietic cells specific Igsf8 deleted mice (Igsf8fl/fl; Vav-Cre) and tamoxifen induced Igsf8 deleted mice (Igsf8fl/fl; Rosa26-CreERT). Igsf8fl/fl, Vav-Cre (denoted as Igsf8-/-) mice represented normal maturation. Deletion of Igsf8 did not significantly affect adult hematopoiesis in peripheral blood and bone marrow. Igsf8-/- long-term hematopoietic stem cells (LT-HSCs: CD34- Flk2- c-Kit+ Sca-1+ Lineage- cells) reduced colony forming ability in vitro, and serial competitive transplantation assay showed comparable donor chimerism by 3 months, but led to decrease Igsf8-/- donor chimerism at 4 months and those after second transplantation in vivo. These results suggest that Igsf8 does not affect the adult hematopoiesis, but it can affect their proliferative and reconstitutive capacity of HSCs. To investigate the effects of Igsf8 on myeloid leukemia, we generated MLL-AF9 and NRASG12V-driven acute myelogenous leukemia (AML), or BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) mice models. Igsf8-/- led to a dramatic reduction in the number of leukemic colonies formed in vitro (Figure 1A). Igsf8-/- leukemia mice showed significantly longer survival in vivo (Figure 1B). This effect was also observed by eliminating Igsf8 expression after leukemia establishment using conditionally deletion. Igsf8-/- AML cells showed decreased S phase fraction. Igsf8-/- leukemia stem cells (LSCs: c-Kit+ Lineage- cells) triggered an increment of the apoptosis, which contribute to significantly lower proportion of LSCs in spleen of Igsf8-/- leukemic mice. Given that Igsf8-/- did not affect homing ability of leukemia cells, these results indicate that Igsf8 is required for propagation of myeloid leukemia and maintenance of LSC. To understand the Igsf8-mediated regulation of myeloid leukemia, we conducted RNA sequencing analysis of LT-HSCs, and LSCs of AML and CML-BC. Gene set enrichment analysis exhibited increase apoptosis related genes and decrease Wnt/β-catenin related genes in Igsf8-/- leukemic cells, but not in LT-HSCs (Figure 1C). Increment of pro-apoptosis genes, and decrement of anti-apoptosis genes and Wnt/β-catenin target genes in Igsf8-/- AML stem cells were validated in quantitative polymerase chain reaction analysis. Further, expression levels of β-catenin protein in Igsf8-/- leukemic cells were significantly lower compared to Igsf8+/+ leukemic cells, but not in normal hematopoietic stem and progenitor cells (Figure 1D). These results suggest that Igsf8 might be critical for myeloid leukemia maintenance via Wnt/β-catenin signaling pathway. Then, we investigated the effects of IGSF8 on human myeloid leukemia. We confirmed IGSF8 expression in several human myeloid leukemia cell line and primary patient-derived leukemia cells. Knockdown of IGSF8 by small hairpin RNA in myeloid leukemia cell lines (THP-1, MV4-11, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro. Knockdown of IGSF8 also caused decrease expression of β-CATENIN in AML cell lines. These results indicate that IGSF8 is also required for propagation of human myeloid leukemia cells. Taken together, our present study reveals that Igsf8 is indispensable for myeloid leukemia, but not adult hematopoiesis, suggesting that IGSF8 inhibition should be considered for targeting myeloid leukemia. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Konuma:SGH Foundation: Research Funding; The Japanese Society of Hematology: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Ito:Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

scholarly journals Polyclonal hematopoiesis in interferon-induced cytogenetic remissions of chronic myelogenous leukemia

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 997-1002 ◽  
Author(s):  
D Claxton ◽  
A Deisseroth ◽  
M Talpaz ◽  
C Reading ◽  
H Kantarjian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Polymorphism ◽  
Chronic Myelogenous Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Clonality Analysis ◽  
Ifn Therapy ◽  
Cytogenetic Remission ◽  
The One

Interferon (IFN) therapy of early chronic myelogenous leukemia (CML) frequently produces partial or complete cytogenetic remission of the disease. Patients with complete cytogenetic remission often continue on therapy for several years with bone marrow showing only diploid (normal) metaphases. We studied hematopoiesis in five female patients with major cytogenetic remissions from CML during IFN therapy. Clonality analysis using the BstXI PGK gene polymorphism showed that granulocytes were nonclonal in all patients during cytogenetic remission. BCR region studies showed rearrangement only in the one patient whose remission was incomplete at the time of sampling. Granulopoiesis is nonclonal in IFN-induced remissions of CML and may be derived from normal hematopoietic stem cells.

scholarly journals Absence of Evidence Implicating Hematopoietic Stem Cells As Common Progenitors for DLBCL Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4107-4107
Author(s):  
Max Jan ◽  
Florian Scherer ◽  
David M. Kurtz ◽  
Aaron M Newman ◽  
Henning Stehr ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
B Cells ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Phylogenetic Trees ◽  
Somatic Mutations ◽  
Hematopoietic Stem ◽  
Tumor Biopsies ◽  
Myd88 L265p

Abstract Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs. Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs. Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs. Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

scholarly journals Single-cell RNA-seq reveals a concomitant delay in differentiation and cell cycle of aged hematopoietic stem cells

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Léonard Hérault ◽  
Mathilde Poplineau ◽  
Adrien Mazuel ◽  
Nadine Platet ◽  
Élisabeth Remy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Cycle Regulators ◽  
Potential Mechanism ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Undifferentiated State ◽  
Reference Map

Abstract Background Hematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging, heterogeneity of HSCs changes, contributing to the deterioration of the immune system. In this study, we revisited mouse HSC compartment and its transcriptional plasticity during aging at unicellular scale. Results Through the analysis of 15,000 young and aged transcriptomes, we identified 15 groups of HSCs revealing rare and new specific HSC abilities that change with age. The implantation of new trajectories complemented with the analysis of transcription factor activities pointed consecutive states of HSC differentiation that were delayed by aging and explained the bias in differentiation of older HSCs. Moreover, reassigning cell cycle phases for each HSC clearly highlighted an imbalance of the cell cycle regulators of very immature aged HSCs that may contribute to their accumulation in an undifferentiated state. Conclusions Our results establish a new reference map of HSC differentiation in young and aged mice and reveal a potential mechanism that delays the differentiation of aged HSCs and could promote the emergence of age-related hematologic diseases.

scholarly journals Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4640-4640
Author(s):  
Heng-Yi Liu ◽  
Nezia Rahman ◽  
Tzu-Ting Chiou ◽  
Satiro N. De Oliveira
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Lines ◽  
Immune Cells ◽  
Research Funding ◽  
Humanized Mice ◽  
Tumor Challenge ◽  
Hematopoietic Stem

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

scholarly journals Mouse acute leukemia develops independent of self-renewal and differentiation potentials in hematopoietic stem and progenitor cells

2019 ◽  
Vol 3 (3) ◽  
pp. 419-431 ◽  
Author(s):  
Fang Dong ◽  
Haitao Bai ◽  
Xiaofang Wang ◽  
Shanshan Zhang ◽  
Zhao Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Leukemia ◽  
Progenitor Cells ◽  
Lymphoblastic Leukemia ◽  
The Self ◽  
Myelogenous Leukemia ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract The cell of origin, defined as the normal cell in which the transformation event first occurs, is poorly identified in leukemia, despite its importance in understanding of leukemogenesis and improving leukemia therapy. Although hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were used for leukemia models, whether their self-renewal and differentiation potentials influence the initiation and development of leukemia is largely unknown. In this study, the self-renewal and differentiation potentials in 2 distinct types of HSCs (HSC1 [CD150+CD41−CD34−Lineage−Sca-1+c-Kit+ cells] and HSC2 [CD150−CD41−CD34−Lineage−Sca-1+c-Kit+ cells]) and 3 distinct types of HPCs (HPC1 [CD150+CD41+CD34−Lineage−Sca-1+c-Kit+ cells], HPC2 [CD150+CD41+CD34+Lineage−Sca-1+c-Kit+ cells], and HPC3 [CD150−CD41−CD34+Lineage−Sca-1+c-Kit+ cells]) were isolated from adult mouse bone marrow, and examined by competitive repopulation assay. Then, cells from each population were retrovirally transduced to initiate MLL-AF9 acute myelogenous leukemia (AML) and the intracellular domain of NOTCH-1 T-cell acute lymphoblastic leukemia (T-ALL). AML and T-ALL similarly developed from all HSC and HPC populations, suggesting multiple cellular origins of leukemia. New leukemic stem cells (LSCs) were also identified in these AML and T-ALL models. Notably, switching between immunophenotypical immature and mature LSCs was observed, suggesting that heterogeneous LSCs play a role in the expansion and maintenance of leukemia. Based on this mouse model study, we propose that acute leukemia arises from multiple cells of origin independent of the self-renewal and differentiation potentials in hematopoietic stem and progenitor cells and is amplified by LSC switchover.

Differential expression of a novel C-terminally truncated splice form of SMAD5 in hematopoietic stem cells and leukemia

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3945-3950 ◽  
Author(s):  
Yunfang Jiang ◽  
Hong Liang ◽  
Wei Guo ◽  
Lazar V. Kottickal ◽  
Lalitha Nagarajan
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Receptor Activation ◽  
Myelogenous Leukemia ◽  
Blood Leukocytes ◽  
Hematopoietic Stem ◽  
Acid Protein ◽  
Acute Myelogenous ◽  
Inactivating Mutations

Abstract SMADs are evolutionarily conserved transducers of the differentiation and growth arrest signals from the transforming growth factor/BMP (TGF/BMP) family of ligands. Upon receptor activation, the ligand-restricted SMADs1–35 are phosphorylated in the C-terminal MH2 domain and recruit the common subunit SMAD4/DPC-4 gene to the nucleus to mediate target gene expression. Frequent inactivating mutations of SMAD4, or less common somatic mutations ofSMAD2 seen in solid tumors, suggest that these genes have a suppressor function. However, there have been no identified mutations of SMAD5, although the gene localizes to the critical region of loss in chromosome 5q31.1 (chromosome 5, long arm, region 3, band 1, subband 1) in myelodysplasia (MDS) and acute myelogenous leukemia (AML). A ubiquitously expressed novel isoform,SMAD5β, encodes a 351 amino acid protein with a truncated MH2 domain and a unique C-terminal tail of 18 amino acids, which may be the functional equivalent of inactivating mutations. The levels of SMAD5β transcripts are higher in the undifferentiated CD34+ hematopoietic stem cells than in the terminally differentiated peripheral blood leukocytes, thereby implicating the β form in stem cell homeostasis. Yeast 2-hybrid interaction assays reveal the lack of physical interactions between SMAD5β and SMAD5 or SMAD4. The expression ofSMAD5β may represent a novel mechanism to protect pluripotent stem cells and malignant cells from the growth inhibitory and differentiation signals of BMPs.

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