A Murine Model of Human Primary Leukemia for Studying Leukemia-Stromal Interactions and Prediction of Clinical Outcome.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1192-1192
Author(s):  
John Yu ◽  
Li-en Shao ◽  
Chia-lin Huang ◽  
Alice L. Yu
Keyword(s):  
Stem Cells ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Scid Mice ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Similar Treatment ◽  
High Level ◽  
Primary Leukemia

Abstract Acute or chronic leukemias resist apoptosis in vitro when co-cultured with marrow stromal cells, suggesting that the growth/survival of leukemia cells relies in part on interactions with stromal cells in the microenvironment. We have recently demonstrated that consistent and high-level engraftment of human primary leukemia obtained from patients can be achieved in NOD/scid mice by preconditioning with either adherent cord blood or marrow mesenchymal stem cells. High success rate of engraftment (84.9 ± 2.9 % leukemia blasts in mouse marrow) was obtained with many lineages of leukemia including acute T- or B-cell lymphoblastic and myeloid leukemia. In general, leukemia blasts were detectable in peripheral blood of mice by week 4–5, leading to fatal outcome by week 6 (42 ± 4 days). Furthermore, cells from the marrow of these preconditioned mice were found to secrete leukemia-promoting activities, suggesting that the mouse marrow had been altered to favor the proliferation/survival of leukemic cells in vivo. We also showed that the human leukemia cells harvested from mice could be serially transferred to other mice for many generations with ~100 fold increase in the number of leukemic and clonogenic cells in mice, while retaining properties similar to primary leukemia samples obtained from patients. Several lines of evidence in our studies including the appearance of leukemia blasts in marrow and blood, the dissemination to other tissues, and gene expression profiling in microarray analysis confirm that this xenograft model recapitulates key features of human leukemia. Furthermore, weekly i.p. injections of NOD/scid mice with vincristine at 0.5 mg/kg for three weeks, starting at second week after inoculation of patients primary leukemia, resulted in a significant delay in the appearance of human leukemia blasts in blood, doubly the length of mouse survival. In addition, pairs of primary leukemia samples collected at diagnosis and at relapse from the same patients were engrafted into NOD/scid mice. The NOD/scid mice transplanted with either samples developed leukemia in mouse peripheral blood at week 4–5 with similar kinetics after inoculation. However, weekly vincristine treatment x 3 of the mice transplanted with diagnosis leukemia samples, prevented the appearance of leukemia blast cells in the circulating peripheral blood for at least 5 weeks. In contrast, similar treatment of mice engrafted with relapse leukemia samples had 50% leukemia blasts in blood at week 5 and subsequently developed fatal leukemia dissemination at week 7. These findings indicate that this robust high level engraftment model of human primary leukemia may be useful in predicting clinical response to chemotherapy. Evidence has further suggested that these human leukemia in NOD/scid mice may be derived from a small subset of immature stem cells in the samples that give rise to leukemia expansion and phenotypic diversity in these mice. Therefore, we had developed a robust and predictive animal model of human primary leukemia, which is valuable for studying leukemia stem cells and for testing or prioritizing new agents/regimens in vivo.

In Vivo Treatment with TG-02 Results in Increased Mobilization and Sensitization of Leukemia Stem Cells to Chemotherapeutic Agents

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3765-3765
Author(s):  
Zeena Salman ◽  
Jeanne P. De Leon ◽  
Eric J. Feldman ◽  
Francis Burrows ◽  
Gail J. Roboz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Human Leukemia ◽  
Human Leukemia Cells

Abstract TG02 is a potent cyclin-dependent kinase 9 (CDK9) inhibitor. It also inhibits CDK1, CDK2, ERK5 and JAK2 at clinically relevant doses. In vitro studies of TG02 have shown robust induction of apoptosis in both acute myeloid leukemia (AML) cell lines and primary cells (Goh et al Leukemia 2011). A phase I dose escalation trial enrolled relapsed/refractory AML patients >18 years of age or patients >65 years with newly diagnosed AML unable to undergo standard induction therapy. Leukemia stem cells (LSCs) comprise a largely quiescent, highly chemotherapy-resistant cell population that contributes to the initiation, propagation and relapse of disease. Thus, the effect of in vivo treatment with TG02 in LSCs was investigated. Peripheral blood (PB) and bone marrow (BM) samples were evaluated (n=16) for LSC percentages and cell cycle status using flow cytometry. Colony forming assays were also performed. TG02 was not found to have an effect on AML tumor burden; however, 8 patients were found to have an increase in immunophenotypically-defined LSCs in both BM and PB with increased colony formation, suggestive of LSC mobilization from marrow into the circulation (Guzman et al Blood 2013). Thus, we hypothesized that exposure to TG02 in vivomay result in mobilization of LSCs from marrow into the periphery, potentially allowing their sensitization to chemotherapeutic agents, such as cytarabine. We tested this hypothesis in vivo by xenotransplanting NOD/SCID mice with primary human AML samples. Mice were divided randomly into one of four groups which received either TG02, cytarabine, both drugs, or saline (control). TG02 was dosed orally at 50mg/kg twice weekly, and the combination group received two doses of TG02 prior to initiation of intraperitoneal cytarabine 10mg/kg days 1-5/week, and for its duration. The total treatment time for all groups was three weeks. Flow cytometry was used to assess the effects of these agents, individually and in combination, on LSCs. BM examination revealed significantly fewer human leukemia cells in mice receiving the combination of TG02 and cytarabine than in those receiving TG02 alone (p=0.027), and both groups had significantly fewer human leukemia cells compared to controls (p=0.018). Mice receiving TG02 alone had significantly higher numbers of leukemic cells in the peripheral blood than untreated controls (p=0.005), suggesting that the agent resulted in mobilization of leukemic cells from marrow. In the group of mice treated with TG02 combined with cytarabine, there were significantly fewer peripheral leukemia cells (p<0.001), suggesting that cytarabine successfully eliminated the circulating cells mobilized with TG02 treatment. Our data suggest that TG02 induces an effect on LSCs or their niche, resulting in mobilization of these cells to the periphery. Furthermore, the addition of cytarabine to TG02 was associated with a significant decrease in both marrow and peripheral blood leukemia cells, suggesting that treatment with TG02 may sensitize these typically chemotherapy-resistant cells to cytarabine. Further investigation of the LSC-mobilizing and chemo-sensitization effects of TG02 is warranted in patients with AML. Disclosures Burrows: Tragara Pharmaceuticals: Employment.

Activity of the Stemness Inhibitor, BBI608, on the Self-Renewal of BCR-ABL1 Positive Leukemia Cells: Molecular Mechanisms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 905-905 ◽  
Author(s):  
Tetsuzo Tauchi ◽  
Seiichiro Katagiri ◽  
Seiichi Okabe ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Scid Mice ◽  
The Self ◽  
Leukemia Cells ◽  
Proliferative Potential ◽  
Human Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Background: Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and β-catenine show differential activation in normal versus leukemia stem cells. BBI608 is an oral first-in-class stemness inhibitor which inhibits the Stat3, β-catenine and Nanog patheways. In a phase I study, BBI608 demonstrated telerability as well as signs of anti-cancer activity in patients with solid tumors. In the present study, we investigated the molecular mechanisms by which BBI608 regulates the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. Methods: To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or ponatinib-resistant Ph-positive acute lymphoblastic leukemia (n=2, Y253H/E255K/T315I BCR-ABL1 and T315I BCR-ABL1) into NOD/SCID/IL-2γc-/- mice. The cell fractions with CD34+CD38- CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of BBI608 on self-renewal and the relevance as a therapeutic target in ABL-tyrosine kinase-resistant BCR-ABL1 positive leukemia, we examined the activity of BBI608 against CD34+CD38-CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with BBI608 (20 mg/kg; p.o.) for 28 days. Results: All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with BBI608 reduced the population of CD34+CD38- positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from BBI608-treated donors. These results demonstrate the persistent effects of BBI608 on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of Stat3 / Nanog pathway modulation on in vitro clonogenic growth. CD34+CD38-CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 2 μM of BBI608 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. BBI608 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by BBI608 (p<0.001). To identify the mechanisms that limit the self-renewal of BCR-ABL1-positive cells by BBI608, NOD/SCID mice engrafted with T315I -BCR-ABL1-positive CD34+ CD19+ fractions were treated with BBI608 (20 mg/kg; p.o.) for 14 days. BBI608 induced the expressions of p21Cip1 , cleaved PARP and reduced the expression of BMI-1, phospho-Stat3, c-Myc, Sox-2, and Bcl-XL. Coclusion: Our preclinical results indicate that BBI608 have potential as an important option for controlling the drug-resistant leukemia initiating cells in BCR-ABL1 positive leukemia. It is expected that the BBI608 may become extremely useful therapeutic interventions in a number of hematological neoplasms, including BCR-ABL1 positive leukemia, where the persistence of cancer stem cells. Disclosures No relevant conflicts of interest to declare.

Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2271-2286 ◽  
Author(s):  
M. Rosenzweig ◽  
T.J. MacVittie ◽  
D. Harper ◽  
D. Hempel ◽  
R.L. Glickman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
High Level

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

Distinct in Vivo Engraftment and Growth Patterns of t(1;19)+/E2A-PBX1+and t(9;22)+/BCR-ABL+Human Leukemia Cells in SCID Mice

1998 ◽  
Vol 32 (1-2) ◽  
pp. 77-87 ◽  
Author(s):  
Barabara J. Waurzyniak ◽  
Nyla Heerema ◽  
Martha G. Sensel ◽  
Paul S. Gaynon ◽  
Peter Kraft ◽  
...  
Keyword(s):  
Growth Patterns ◽  
Scid Mice ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Human Leukemia Cells

Targeting the Hedgehog Signaling Pathway Limits the Self-Renewal of BCR-ABL1 Positive Leukemia Cells: Molecular Mechanisms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 531-531 ◽  
Author(s):  
Tetsuzo Tauchi ◽  
Seiichiro Katagiri ◽  
Seiichi Okabe ◽  
Yosuke Minami ◽  
Tomoki Naoe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hedgehog Signaling ◽  
Molecular Mechanisms ◽  
Scid Mice ◽  
The Self ◽  
Hedgehog Pathway ◽  
Leukemia Cells ◽  
Hedgehog Signaling Pathway ◽  
Self Renewal

Abstract Abstract 531 Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and BMI-1 show differential activation in normal versus leukemia stem cells. Hedgehog signaling is increased in BCR-ABL1 positive stem and progenitor cells becoming more active with disease progression. We have previously shown that the combination with the hedgehog signaling pathway inhibitor, vismodegib and ABL tyrosine kinase inhibitor (ABL TKIs) inhibits the minimal residual cells in BCR-ABL1-positive leukemia cells (Blood: ASH Annual Abstracts, Nov 2011; 118: 63). In the present study, we investigated the molecular mechanisms by which vismodegib and LDE225 regulate the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or Ph-positive acute lymphoblastic leukemia (n=2, T315I BCR-ABL1 and WT-BCR-ABL1) into NOD/SCID/IL-2γc−/− mice. The cell fractions with CD34+CD38−CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of hedgehog inhibition on self-renewal and the relevance of the hedgehog pathway as a therapeutic target in BCR-ABL1 positive leukemia, we examined the activity of vismodegib and LDE225 against CD34+CD38−CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with vismodegib (20 mg/kg; p.o.) or LDE225 (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with vismodegib or LDE225 reduced the population of CD34+CD38− positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients, subsequently treated with vismodegib or LDE225 for 28 days. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from vismodegib or LDE225 treated donors. These results demonstrate the persistent effects of hedgehog inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of hedgehog pathway modulation on in vitro clonogenic growth. CD34+CD38−CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 1 μM of vismodegib or 200 nM of LDE225 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. Hedgehog pathway inhibition by vismodegib and LDE225 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by vismodegib and LDE225 (p<0.001). To identify the mechanisms that limit the self-renewal of BCR-ABL1-positive cells by vismodegib and LDE225, NOD/SCID mice engrafted with WT-BCR-ABL1-positive CD34+ CD19+ fractions were treated with vismodegib (20 mg/kg; p.o.) or LDE225 (20 mg/kg; p.o.) for 14 days. Both vismodegib and LDE225 induced the expressions of p21Cip1, pATM, pChk2 and γH2AX related with DNA damage response, and reduced the expression of Gli-1, Gli-2, Bcl-2, and cyclin D2. Our preclinical results indicate that vismodegib and LDE225 have potential as an important option for controlling the drug-resistant leukemia initiating cells in BCR-ABL1 positive leukemia. Although several hedgehog inhibitors have now entered clinical evaluation, it is expected that hedgehog inhibitors may become extremely useful therapeutic interventions in a number of hematological neoplasms, including BCR-ABL1 positive leukemia, where the persistence of cancer stem cells. Disclosures: No relevant conflicts of interest to declare.

Anti-PR1/HLA-A2 Antibody (8F4, A TCR-Mimic) Mediates Specific Lysis of AML Stem Cells but Not Normal Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 15-15
Author(s):  
Anna Sergeeva ◽  
Gheath Alatrash ◽  
Karen Clise-Dwyer ◽  
Kathryn E Quintanilla ◽  
Sijie Lu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Healthy Donor ◽  
Mononuclear Cells ◽  
Scid Mice ◽  
Specific Lysis ◽  
Isotype Control

Abstract Abstract 15 PR1 (VLQELNVTV) is an HLA-A2-restricted peptide derived from endogenous myeloid leukemia-associated antigens (LAA) proteinase 3 and neutrophil elastase. PR1/HLA-A2 is targeted by PR1-specific cytotoxic T lymphocytes (PR1-CTL) that contribute to cytogenetic remission in patients with CML after allogeneic stem cell transplantation or interferon therapy, and PR1 peptide vaccination induces specific CD8 immunity in AML, MDS, and CML patients. Because clinical effects after PR1 vaccination are associated with a low amount of leukemia and because the effects on leukemia stem cells (LSCs) are unknown, we sought to develop a PR1/HLA-A2-specific MAB that could be more effective in high disease burden cases and that might also target LSC. We previously reported a mouse IgG2A antibody (8F4) with PR1/HLA-A2 specificity. We now used 8F4 to study PR1 expression by FACS and confocal mircroscopy on leukemia subsets and normal peripheral blood mononuclear cells (PBMC). Circulating blasts from peripheral blood (PB) or leukapheresis products (LP) from AML patients (5 HLA-A2+, 2 control HLA-A2-,) were studied (see Table 1). PR1 expression (MFI±SEM) was higher in AML (23.7±5.18) compared to control (4.0±2.42) and normal PBMC (13.6±0.23; p = 0.046). Confocal microscopy confirmed bright heterogeneous surface expression of PR1 on AML, but was nearly absent on healthy donor PB. Interestingly, PR1 expression was higher on LSC (CD34+CD38-lin-) (76±44) compared to healthy donor HSC (CD34+CD38-lin-) (13.4), which suggests that only differentiated myeloid cells might be more susceptible to 8F4 toxicity. To study whether 8F4 mediated specific lysis of PR1+ cells, we determined cell-mediated cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of 8F4 compared to isotype control. 8F4 induced 51% specific lysis by CDC and 15% specific lysis by ADCC of PR1-pulsed T2 cells, which was dose-dependent on both 8F4 and PR1, with no lysis of target cells by isotype control. Moreover, 8F4 induced CDC-mediated lysis of blasts from 5/5 HLA-A2+ AML patients, but not blasts from an HLA-A2- AML patient (see Table 1), or mononuclear cells from HLA-A2+ healthy donors. To study the in vivo effects of 8F4 on AML, we used an established xenogeneic model of human AML in NOD/SCID mice. 1 × 106 blasts from 4 HLA-A2+ AML patients (including the HLA-A2+ patients susceptible to 8F4-mediated lysis in vitro) and 1 HLA-A2- patient were mixed for one hour under serum-free conditions with 8F4 or isotype control antibody and adoptively transferred to NOD/SCID mice. Engraftment of AML was compared on day 14 and AML growth was confirmed on day 28 by comparing FACS analysis and histopathology of BM from sacrificed animals. 8F4 treatment of AML cells, but not isotype treatment, abrogated engraftment of 4/4 HLA-A2+ AML patients, but not of 1 HLA-A2- AML patient on d14. By day 28, AML expanded by 150% in one surviving isotype-treated animal. Thus, 8F4-mediated specific lysis of AML and LSC by CDC and ADCC mechanisms in vitro were confirmed by in vivo results of AML engraftment. Taken together, these results show that 8F4, a novel TCR-mimic antibody that targets only PR1/HLA-A2+ cells, mediates lysis of AML blasts and LSC, but not total normal BM cells or HSC. Therefore, 8F4 may be useful as an immunotherapeutic agent in the treatment of HLA-A2+ myeloid leukemia, and as the first antibody therapy that may target and eliminate LSC. Disclosures: No relevant conflicts of interest to declare.

Donor stromal cells from human blood engraft in NOD/SCID mice

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3971-3978 ◽  
Author(s):  
Silvia-Renate Goan ◽  
Ilse Junghahn ◽  
Manuela Wissler ◽  
Michael Becker ◽  
Jutta Aumann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Immunohistochemical Staining ◽  
Scid Mice ◽  
Rt Pcr ◽  
Specific Antibodies ◽  
Human Specific

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34+ peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34+ PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-α (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice.

Aurora-a Kinase: A Novel Target of Cellular Immunotherapy for Human Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2904-2904 ◽  
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Koichiro Suemori ◽  
Taichi Azuma ◽  
Kiyotaka Kuzushima ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cellular Immunotherapy ◽  
Human Leukemia ◽  
Normal Cells ◽  
Specific Ctls

Abstract [Purpose] Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family, and the Aur-A gene is located at chromosome 20q13. Aur-A is mainly expressed in the G2/M phase of the cell cycle and regulates mitotic cell division in normal cells. Aur-A is expressed exclusively in testis in normal tissues, but is abundantly expressed in various kinds of cancer including hematological malignancies. Its overexpression usually accompanies chromosomal abnormality related with the poor prognosis. Furthermore, the silencing of Aur-A gene has disrupted the proliferation of cancer cells and augmented their susceptibility to anti-cancer agents. These data strongly suggest that Aur-A is one of the crucial oncognes and a rational target for cancer treatment. Thus, we postulated that Aur-A might be an ideal target for cancer immunotherapy, and attempted to verify the feasibility of cellular immunotherapy for leukemia targeting Aur-A. [Methods] Nine-mer peptides derived from Aur-A which were algorithmically predicted to bind to HLA-A*0201 molecule were synthesized and assessed their binding affinities by the stabilization assay with T2 (HLA-A*0201) cells. Aur-A-specific cytotoxic T lymphocytes (CTLs) were generated by stimulation of CD8+ T cells with Aur-A peptide-pulsed autologous dendritic cells. Cytotoxicity of Aur-A-specific CTLs was defined by standard 51Cr-release assay. HLA class I restriction was examined with anti-HLA class I antibody and target-specificity of Aur-A-specific CTLs was examined by cold target inhibition assay with 51Cr-labeled HLA-A*0201-positive leukemia cell lines in combination with Aur-A peptide-loaded 51Cr-unlabeled HLA-A*0201-positive lymphoblastoid cell lines. The expressions of Aur-A mRNA and Aur-A protein in leukemia cells and normal cells were assessed by semi-quantitative real-time PCR and Western blotting. The frequencies of Aur-A-specific CTL precursors in the peripheral blood of healthy individuals and patients with leukemia were measured by tetramer staining. [Results] We established an HLAA* 0201-restricted and Aur-A207-215 peptide (YLILEYAPL)-specific CTL line (designated as AUR-1) by repeated stimulations of CD8+ T cells with Aur-A207-215 peptide-pulsed peripheral blood mononuclear cells from an HLA-A*0201-positive healthy individual. AUR-1 exerted cytotoxicity to various kinds of leukemia cell lines in an HLA-A*0201- restricted fashion. AUR-1 could discriminate freshly isolated leukemia cells from normal cells and normal mitotic cells, according to the expression levels of Aur-A mRNA. By using freshly isolated leukemia cells from CML patients’ bone marrow mononuclear cells (BMMCs), we further investigated whether AUR-1 could lyse the leukemic progenitor cells. The CD34+CD38low fraction of CML BMMCs which include leukemic stem cells but not the normal counter part cells overexpressed Aur-A mRNA. Additionally AUR-1 could lyse CD34+ CML BMMCs, but not normal CD34+ cells. Finally, by using tetramer assay, Aur-A207-215-specific CTL precursors were detected more frequently in the peripheral blood of HLA-A*0201-positive patients with leukemia than that of healthy individuals. [Conclusion] This is the first report that Aur-A-specific CTLs can exert the cytotoxicity against leukemic stem cells but not normal hematopoietic stem cells. Results in this study suggests that the cellular immunotherapy targeting Aur-A is a promising strategy for the treatment of human leukemia.

Targeting The Dopamine Receptor Signaling Limits The Self-Renewal Of BCR-ABL1 Positive Leukemia Cells: Molecular Mechanisms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1298-1298
Author(s):  
Tetsuzo Tauchi ◽  
Seiichiro Katagiri ◽  
Seiichi Okabe ◽  
Yosuke Minami ◽  
Tomoki Naoe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Receptor Antagonist ◽  
Dopamine Receptor ◽  
Molecular Mechanisms ◽  
Scid Mice ◽  
The Self ◽  
Leukemia Cells ◽  
Receptor Signaling ◽  
Self Renewal

Abstract Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and Dopamine Receptor show differential activation in normal versus leukemia stem cells. Using a high-throughput discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells, the norvel dopamine receptor antagonist, thioridazine, was identified from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal (Cell 149, 1284, 2012). In the present study, we investigated the molecular mechanisms by which dompamine receptor antagonist, thioridizine, regulates the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or ponatinib-resistant Ph-positive acute lymphoblastic leukemia (n=2, Y253H/E255K/T315I BCR-ABL1 and T315I BCR-ABL1) into NOD/SCID/IL-2γc-/- mice. The cell fractions with CD34+CD38- CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of the domamine receptor antagonist on self-renewal and the relevance as a therapeutic target in ABL-tyrosine kinase-resistant BCR-ABL1 positive leukemia, we examined the activity of thioridazine against CD34+CD38-CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with thioridazine (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with thioridazine reduced the population of CD34+CD38- positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from thioridazine-treated donors. These results demonstrate the persistent effects of domapine receptor signaling inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of dompamine receptor pathway modulation on in vitro clonogenic growth. CD34+CD38-CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 5 μM of thiorizaxine for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. Dopamine receptor pathway inhibition by thioridazine had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by thioridazine (p<0.001). To identify the mechanisms that limit the self-renewal of BCR-ABL1-positive cells by thioridazine, NOD/SCID mice engrafted with T315I -BCR-ABL1-positive CD34+ CD19+ fractions were treated with thoridazine (20 mg/kg; p.o.) for 14 days. Thioridazine induced the expressions of p21Cip1 and reduced the expression of c-Myc, Oct-3/4, Sox-2, and Bcl-2. Our preclinical results indicate that thioridazine have potential as an important option for controlling the drug-resistant leukemia initiating cells in BCR-ABL1 positive leukemia. It is expected that the domapine receptor antagonist may become extremely useful therapeutic interventions in a number of hematological neoplasms, including BCR-ABL1 positive leukemia, where the persistence of cancer stem cells. Disclosures: Ohyashiki: Novartis: Honoraria, Research Funding.

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