Discrimination Between Normal Hemopoietic Stem Cells and Myeloid Leukemia Cells Using Monoclonal Antibodies

Abstract Dendritic cell (DC) vaccines in leukemia show promise as a novel treatment modality however to date clinical evidence of efficacy has been limited. This is likely to be as a consequence of a combination of factors, which include insufficient immunogenicity of the DC vaccine and vaccination taking place in an environment adverse for generation of effective immune responses i.e. in patients with active disease. Our study aims to generate more efficient cytotoxic T cell (CTL) responses by improving DC uptake and presentation of leukemia cells in the remission state and will be applicable to both acute and chronic leukemias. Monoclonal antibodies (MoAbs) have been used to treat malignant cells prior to co-culture with DCs to enhance cross-presentation and generation of specific CTLs. We investigated whether this approach could improve DC induction of CTL responses in comparison to DCs loaded with UVB irradiated apoptotic leukemia cells. In this in vitro study we generated dendritic cells from adherent mononuclear cells (differentiation with GM-CSF and IL-4) of patients in remission following chemotherapy for acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). The immature DCs were loaded with autologous leukemia cells from the patients’ presentation samples. The presentation leukemia cells were treated with either UVB irradiation or appropriate monoclonal antibodies (the anti-CD33 MoAb Mylotarg in AML and CML; the anti CD20 MoAb Rituximab or the anti-CD 52 MoAb Alemtuzumab in CLL). Apoptosis was assessed by Annexin/Propidium iodide labelling. Treatment of the leukemia cells by different MoAbs induced varying degrees of apoptosis. DC uptake of antibody treated or apoptotic leukemia cells was assessed by dual colour staining. Leukemia cells were stained with PKH and DCs labelled with FITC-CD80 or CD86. DC uptake was more efficient with MoAb treated cells irrespective of the degree of apoptosis induced by the MoAb. DCs were matured with TNFa for two days then co-cultured with autologous T cells for one week. T cell subsets and Regulatory T cells were assessed on the presentation and remission samples.The T cells were harvested and their cytoxicity assessed in an Interferon Gamma (IFNg) ELISPOT assay where the unmodified blasts were used as stimulators. Initial results show enhanced anti-leukemia activity in the MoAb treated group as compared to the irradiated group. A similar set up using allogeneic DCs and T cells confirmed the augmentation of CTL responses with MoAb treatment of leukemia cells.The use of MoAb in this setting shows promise for improvement in the success and applicability of DC vaccine strategies in leukemia.

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Leukemia cells invading the liver express liver chemokine receptors and possess characteristics of leukemia stem cells in mice with MPD-like myeloid leukemia

Experimental Hematology ◽

10.1016/j.exphem.2010.11.005 ◽

2011 ◽

Vol 39 (2) ◽

pp. 187-194 ◽

Cited By ~ 3

Author(s):

Alexey E. Bigildeev ◽

Irina N. Shipounova ◽

Daria A. Svinareva ◽

Nina J. Drize

Keyword(s):

Stem Cells ◽

Chemokine Receptors ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Leukemia Stem Cells

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The Role of Hypoxia on ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Blood ◽

10.1182/blood-2019-129768 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5369-5369

Author(s):

Seiichi Okabe ◽

Yuko Tanaka ◽

Mitsuru Moriyama ◽

Akihiko Gotoh

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

K562 Cells ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Transfected Cells ◽

Resistant Cells

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Moreover, ABL TKIs cannot cure the Ph-positive leukemia patients because of leukemia stem cells in the bone marrow niche. Therefore, new approach against leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Hypoxia is an important component of the bone marrow microenvironment. Because oxygen tension plays a key role in driving normal hematopoiesis, leukemia stem cells may be maintained in hypoxic areas of the bone marrow. Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R and Ba/F3 T315I). We investigated gene expression profiles in cultured ABL TKI resistant cells and parental cell line, K562 in normoxia and hypoxia condition by DNA microarray. Results: We first investigated gene expression profiles in cultured K562 cells in hypoxia condition. We found gene expression of insulin-like growth factor 1 (IGF1) was increased K562 cells in hypoxia condition by DNA microarray. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. We could not detect the BCR-ABL point mutation in ABL TKI resistant cells. We found gene expression of insulin-like growth factor 1 (IGF1) receptor (IGF1R) was increased ABL TKI resistant K562 cells. IGF1R gene amplification was confirmed by RT-PCR analysis. IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). One of IGFBP, IGFBP5 is related to imatinib sensitivity and resistant in chronic myeloid leukemia (CML) patients (GSE12211). In hypoxia condition, several IGFBPs were also increased in ABL TKI resistant cells. IGF cause intracellular signaling that ultimately results in cellular growth and proliferation. Thus, we initially examined whether addition of IGF1R inhibition could enhance ABL TKIs sensitivity. One of IGF1R inhibitor, linsitinib was inhibited ABL TKI resistant cells and parental cell line, K562 in hypoxia condition. ABL TKI resistant cell lines were more sensitive against linsitinib. Combined treatment of ABL TKI resistant cells and K562 cells with ABL TKIs and linsitinib caused more cytotoxicity than each drug alone in hypoxia condition. Caspase 3/7 activity and cellular cytotoxicity was also increased after ABL TKIs and linsitnib treatment. In the colony formation method, the number of cell colonies were also reduced in hypoxia condition. Intracellular ATP levels have been implicated in vitro as a determinant of cell death by apoptosis. The concentrations of intracellular ATP were reduced after ABL TKIs and linsitinib. We next blocked IGF1R function by small interfering RNA (siRNA). SiRNA transfected cells were reduced cellular proliferation. We also found drug sensitivity of the cells to the imatinib was increased compared to mock-transfected cells. Apoptotic cells and caspase 3/7 activity were increased after imatinib treatment in siRNA transfected cells. Conclusion: The IGF1 pathway is involved in Ph-positive leukemia cells in hypoxia condition and ABL TKI resistant in CML cells. We also provide the promising clinical relevance as a candidate drug for treatment of residual leukemia cells in bone marrow niche which is in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

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KBP-1 Supports Acute Myeloid Leukemia Development

Blood ◽

10.1182/blood.v126.23.1378.1378 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1378-1378

Author(s):

Jingjing Xie ◽

Zhigang Lu ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Myeloid Leukemia ◽

Underlying Mechanism ◽

Leukemia Cells ◽

Human Leukemia ◽

Therapeutic Approaches ◽

Control Shrna ◽

Early Apoptosis ◽

Leukemia Development ◽

Acute Myeloid

Abstract Novel targets are needed to develop effective therapeutic approaches to treat acute myeloid leukemia (AML). We have developed a systematic strategy to identify factors important for leukemia development. We first use clinical databases to identify plasma membrane proteins that have correlations with the clinical outcomes of leukemia patients. We then validate the functions of candidate proteins in leukemia models and compare these functions to those in normal cells. The signaling pathways identified provide candidate targets for development of therapeutic approaches. Using this approach, we previously identified several ITIM-containing receptors that support AML development. Here we performed an in silico analysis of the relationship between gene expression and the overall survival of AML patients using data from three independent databases: the TCGA AML database (https://tcga-data.nci.nih.gov/tcga/; n = 187), the GSE6891 database (n = 520), and the GSE10358 database (n = 91). Expression of genes encoding several factors, including IL2RA, GPR56, ACDY7, and kappa-binding protein-1 (KBP-1), inversely correlated with the overall survival of AML patients. We focused on the potential function of KBP-1 in AML development in this study. KBP-1 is a transcriptional regulator that was known to inhibit NF-kB signaling and enhance TGF-beta signaling. Previous studies indicated that KBP-1 inhibits teratoma growth. We detected significantly higher kbp-1 mRNA levels in cells from human AML cells than other leukemia cells. To study the potential function of KBP-1 in human leukemia, we inhibited the expression of KBP-1 by introducing lentivirus-encoded shRNAs into MV4-11 and THP-1 cells. The KBP-1 deficiency resulted in significantly decreased in vitro growth of these leukemia cells over time. To determine the underlying mechanism by which KBP-1 supports the growth of leukemia cells, we compared the cell cycle status, migration, and apoptosis of AML cells treated with these shRNAs or scrambled control shRNA. KBP-1-deficient cells had significantly increased levels of apoptosis compared to cells treated with the control shRNA (for example, 68% early apoptosis in KBP-1 knockdown MV4-11 cells vs 15% early apoptosis in control MV4-11 cells at 3 days after shRNA infection). These results indicate that KBP-1 supports leukemia cell growth by suppressing apoptosis. We further studied the function of KBP-1 in AML development using the KBP-1 knockout mice and retrovirus transplantation mouse models. Consistent with the results of KBP-1 knockdown in human leukemia cells, the knockout of KBP-1 in MLL-AF9 AML cells dramatically delayed AML development in mice, as determined by survival, flow cytometry, immunohistochemistry, and colony forming analyses. Serial transplantation of wild-type and KBP-1 knockout AML cells indicates that KBP-1 deficiency impaired the self-renewal of AML stem cells. We are working on elucidating the underlying mechanism by which KBP-1 supports the activity of AML stem cells. Together, this is the first demonstration of KBP-1 function in hematopoietic maligancies. KBP-1 is highly expressed by AML cells and its expression correlates with AML development. KBP-1 supports the survival of human AML cells and the activity of AML stem cells. The tumor-supportive role of KBP-1 in AML is different from its tumor-suppressive function in teratoma. Disclosures No relevant conflicts of interest to declare.

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Inhibitory Effects of Homoharringtonine on Leukemic Stem Cells and BCR-ABL Induced Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia in Mice.

Blood ◽

10.1182/blood.v110.11.2912.2912 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2912-2912 ◽

Cited By ~ 1

Author(s):

Yaoyu Chen ◽

Yiguo Hu ◽

Shawnya Michaels ◽

Dennis Brown ◽

Shaoguang Li

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Acute Lymphoblastic Leukemia ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Leukemic Stem Cells ◽

Lymphoid Leukemia

Abstract The Abl tyrosine kinase inhibitors (TKIs) imatinib mesylate (IM) and dasatinib, targeting BCR-ABL for the treatment of Philadelphia-positive (Ph+) leukemia including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL), have produced impressive results in terms of therapeutic outcome and safety for patients. However, clinical resistance to these TKIs likely at the level of leukemic stem cell negates curative results in Ph+ leukemia. At present, an anti-stem cell strategy has not been developed for treating these leukemia patients. Homoharringtonine (HHT) (omacetaxine mepesuccinate - USAN/INN designation) has shown significant clinical activity in CML in combination with IM or alone for patients failing IM. However, little is known about whether HHT has an inhibitory effect on leukemic stem cells. The purpose of this study is to determine whether HHT inhibits BCR-ABL-expressing leukemic stem cells (Lin-c-Kit+Sca-1+) that we identified previously (Hu et al. Proc Natl Acad Sci USA 103(45):16870–16875, 2007) and to evaluate therapeutic effects of HHT on CML and B-ALL in mice. We find that in our in vitro stem cell assay, greater than 90% of leukemic stem cells were killed after being treating with HHT (12.5, 25, and 50 nM) for 6 days, and in contrast, greater than 75% or 92% of leukemic stem cells survived the treatment with dasatinib (100 nM) or imatinib (2 mM). We next treated CML mice with HHT (0.5 mg/kg, i.p., once a day). 4 days after the treatment, FACS analysis detected only 2% GFP+Gr–1+ myeloid leukemia cells in peripheral blood of HHT -treated CML mice and in contrast, 41% GFP+Gr–1+ myeloid leukemia cells in placebo-treated mice. We also treated mice with BCR-ABL induced B-ALL with HHT, and found that only 0.78% GFP+B220+ lymphoid leukemia cells were detected in peripheral blood compared to 34% GFP+B220+ lymphoid leukemia cells in placebo-treated mice. Furthermore, HHT significantly inhibited in vitro proliferation of K562 and B-lymphoid leukemic cells isolated from mice with B-ALL induced by BCR-ABL wild type and BCR-ABL-T315I resistant to both imatinib and dasatinib. In sum, HHT has an inhibitory activity against CML stem cells, and is highly effective in treating CML and B-ALL induced by BCR-ABL in mice.

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The Tumor Suppressor Role of the Msr1 Gene in Cancer Stem Cells of Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.188.188 ◽

2009 ◽

Vol 114 (22) ◽

pp. 188-188

Author(s):

Yaoyu Chen ◽

Con Sullivan ◽

Shaoguang Li

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Tumor Suppressor ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Wild Type ◽

Marrow Cells

Abstract Abstract 188 We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice (Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nature Genetics 41:783-792, 2009). We believe that the Alox5 pathway represents a major molecular network in LSCs. Therefore, we decided to further dissect this pathway by comparing gene expression profiles between wild type and Alox5−/− LSCs from CML mice using the DNA microarray analysis. We identified a small group of candidate genes that were changed in expression in the absence of Alox5. Among these genes, we have identified the Msr1 gene and chosen to test the function of this gene in regulating LSC function, because this gene was up-regulated, indicating that it might play a tumor suppressor role in LSCs. In our CML mouse model, we observed that recipients of BCR-ABL transduced Msr1−/− bone marrow cells developed CML much rapidly than recipients of BCR-ABL transduced wide type bone marrow cells. To test whether this accelerated CML is related to abnormal function of LSCs, we carried out a serial transplantation assay by transferring bone marrow cells from primary recipients of BCR-ABL-transduced wild type or Msr1−/− donor bone marrow cells into secondary and next-generation of recipient mice to biologically assess the effect of Msr1 on LSCs. BCR-ABL-expressing wild type leukemia cells from bone marrow of CML mice were only able to transfer CML once, whereas BCR-ABL-expressing Msr1−/− leukemia cells were able to transfer lethal CML for five genrations. This observation indicates that BCR-ABL-expressing Msr1−/− LSCs have markedly increased stem cell function. To further compare the stem cell function, we performed the leukemia stem cell competition assay by 1:1 mixing wild type (CD45.1) and Msr1−/− (CD45.2) bone marrow cells from CML mice. At day 25 or 30 after transplantation, more than 60% and 95% of GFP+Gr-1+ cells in peripheral blood of the mice were CD45.2+Msr1−/− myeloid leukemia cells, and all these mice developed CML and died of CML derived from Msr1−/− LSCs. To confirm the tumor suppressor role of Msr1 in CML development, we co-expressed BCR-ABL and Msr1 in MSR1−/− bone marrow cells by retroviral transduction, followed by transplantation of these cells into recipient mice. The ectopically-expressed Msr1 in MSR1−/− bone marrow cells rescued the accelerated CML phenotype, and some recipient mice did not even develop the CML. Together, these results demonstrate that Msr1 plays a tumor suppressor role in LSCs. The Msr1 pathway is a novel molecular network in LSCs, and it will be important to fully study this pathway for developing curative therapeutic strategies for CML. Disclosures: No relevant conflicts of interest to declare.

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