Increase of Sensitivity to Chemotherapy and NK-Mediated Cytotoxicity of Leukemic Stem Cell by a Farnesyltransferase Inhibitor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4161-4161
Author(s):  
Miaorong She ◽  
Jinggao Li ◽  
Xilin Chen ◽  
Xingqing Niu ◽  
Kunyuan Guo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Malignant Disease ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Nkg2d Ligands ◽  
Enhanced Sensitivity ◽  
Cell Immunotherapy ◽  
Bcl2 Protein ◽  
Mitochondria Pathway ◽  
Induced Apoptosis

Abstract Abstract 4161 Acute myeloid leukemia (AML) is a serious and often lethal hematopoietic malignant disease arising from stem cells. Leukemic stem cells (LSCs) play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population. We previous found that LSC were resistant to chemotherapy and NK-mediated cytotoxicity, which were resulted from the apoptosis defect and NKG2D ligands change. Our previous data also showed that Manumycin induced apoptosis in leukemia cells through mitochondria pathway. Here, we demonstrated that manumycin increased the sensitivity of LSC to chemotherapy and NK cell-mediated cytotoxicity. Manumycin enhanced sensitivity to chemotherapy in LSCs via induced apoptosis. Analysis of signaling pathways revealed that manumycin enhanced mitoxantrone activation of caspase 3 and significantly decreasing Bcl2 protein. These finding indicated that manumycin sensitized mitoxantrone to chemotherapy via down-regulating Bcl2. Importantly, manumycin enhanced the sensitivities of LSCs to NK cells cytotoxicity through up-regulating the expressions of NKG2D ligands MICA/B and ULBP3. Thus, Manumycin present a promising novel therapeutic approach for AML therapy especially when resistant to chemotherapy and NK cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Role of the SIRT1 Deacetylase in Survival and Imatinib Resistance of CML CD34+ Progenitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 189-189
Author(s):  
Ling Li ◽  
Liang Li ◽  
Hongfeng Yuan ◽  
Wenyong Chen ◽  
Ravi Bhatia
Keyword(s):  
Stress Responses ◽  
Conflicts Of Interest ◽  
Erythroid Cell ◽  
Imatinib Resistance ◽  
Sirt1 Expression ◽  
Knock Down ◽  
Enhanced Sensitivity ◽  
Cd34 Cells ◽  
Lentivirus Vectors ◽  
Induced Apoptosis

Abstract Abstract 189 Imatinib mesylate (IM) treatment has primarily anti-proliferative effects on CML progenitors, and only modest induction of apoptosis is observed. Quiescent, primitive progenitors are especially insensitive to IM induced apoptosis. Identification and targeting of mechanisms of resistance to IM is required to allow enhanced elimination of residual CML progenitors in IM treated patients. SIRT1 is a NAD+ dependent deacetylase that regulates activity of several proteins involved in stress responses. We have observed significantly increased expression of SIRT1 mRNA and protein in CML compared to normal CD34+ progenitors. Here we investigated the effect of inhibition of SIRT1 expression on growth, survival and IM sensitivity of CML progenitors. CML and normal CD34+ cells were transduced with lentivirus vectors expressing SIRT1 shRNAs (Si-1 or Si-2) or control shRNA (Ctrl). Inhibition of SIRT1 expression in Si-1 (95% inhibition) and Si-2 (80% inhibition) transduced CD34+ cells was confirmed on Western blotting. SIRT1 inhibition resulted in modest increase in apoptosis of CML progenitors (Si-1 16±9%, Si-2 10±5% and Ctrl 8±4% apoptosis), and significantly enhanced sensitivity of CML progenitors to IM (2.5μM) induced apoptosis (Si-1, 27±12%, Si-2, 15±4% and Ctrl, 14±5%, Si-1 versus Ctrl, p=0.04, n=4). SIRT1 inhibition did not induce apoptosis in normal progenitors or increase their sensitivity to IM (Si-1 14±3%, Si-2, 13±4% and Ctrl, 11±2% without IM; and Si-1, 14±2%, Si-2, 12±3% and Ctrl, 12±2% with IM). Importantly SIRT1 inhibition significantly enhanced apoptosis of non-dividing (CFSE bright) CML progenitors treated with IM (Si-1, 49±16% and Ctrl, 33±4%, p=0.02, n=4). SIRT1 knock down inhibited proliferation of CML progenitors measured by CFSE labeling (Si-1, 46±5% and Si-2, 14±3% inhibition versus Ctrl, p<0.01, n=4) and enhanced inhibition of CML progenitors in combination with IM (Si-1, 69±11% and Si-2, 53±12% inhibition versus Ctrl, 44±9%, p<0.05, n=4). Inhibition of normal progenitor proliferation was also seen, but was significantly less than that of CML progenitors (Si-1, 30±6% and Si-2, 8±3% inhibition compared with Ctrl, p<0.05, n=4; in combination with IM, Si-1, 33±5% and Si-2, 9±4% inhibition compared with Ctrl, 8±2% inhibition). SIRT1 inhibition also reduced the number of cells generated in culture from CML progenitors (20-fold reduction in cells generated at day 14 with Si-1 compared to controls). Relatively greater inhibition of myeloid compared with erythroid cell growth was seen (at day 14, CD33+ cells with Si-1 0.42±0.21×106 and Ctrl 1.72 ± 0.61 ×106; CD11b+ cells with Si-1 0.32±0.19 ×106 and Ctrl 9.46 ±1.33 ×106). SIRT-1 inhibition also reduced the number of colonies generated from CML CD34+ cells in methylcellulose progenitor culture by itself (17±6% CFC with Si-1 compared to controls, p=0.005, n=3), and in combination with IM (3±1% CFC with Si-1 compared to 23±8% with Ctrl, p=0.03, n=4). The greater effects of Si-1 compared to Sir-2 shRNA in the above studies suggest that near complete knock-down of SIRT-1 is required to alter CML progenitor proliferation and survival. The above results indicate that the stress response gene SIRT1 contributes to enhanced survival and proliferation of CML progenitors and protects CML progenitors from IM-induced apoptosis. SIRT1 inhibition may provide a novel and effective strategy to eradicate residual CML progenitors in combination with IM. Disclosures: No relevant conflicts of interest to declare.

Role of NKG2D in Cytokine-Induced Killer (CIK) Cells Against Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5119-5119
Author(s):  
Xu-zhang Lu ◽  
Bao-An Chen ◽  
Lin-di Ma ◽  
Xiao-hui Cai ◽  
Min Zhou
Keyword(s):  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Nkg2d Ligands ◽  
Cik Cells ◽  
Ifn Γ ◽  
Nkg2d Receptor

Abstract Abstract 5119 Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells. Disclosures: No relevant conflicts of interest to declare.

Resveratrol Enhances NKG2D-Mediated Cytotoxicity Against Leukemia Cells by Upregulating Both NKG2D Receptor on NK Cells As Well As NKG2D Ligands on Target Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3236-3236
Author(s):  
Luis J. Espinoza ◽  
Akiyoshi Takami ◽  
Katsuya Nakata ◽  
Ly Quoc Trung ◽  
Kayoko Yamada ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Receptor Expression ◽  
Leukemia Cells ◽  
Target Cells ◽  
Lymphoid Leukemia ◽  
Nkg2d Ligands ◽  
Nkg2d Receptor

Abstract Abstract 3236 NKG2D is a powerful activating receptor expressed by natural killer (NK) cells that promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). Pharmacological induction of NKG2D-Ls in malignant cells has been an attractive therapeutic approach but has gained poor clinical utility because currently available NKG2D-Ls inducers have been hampered either by their limited efficacy or by their associated toxicity. Resveratrol (RVT), a compound derived from several natural sources, has proved in vivo and in vitro potent anti-tumor effects against various cancers. Extensive research in the last decade has shown that such effects are mediated by targeting various molecules involved in the regulation of proliferation and cell survival and those include, NFκB, STAT3, ATM/ATR and ERK1/2. To date, it is unknown whether RVT has any effect on NKG2D-Ls expression. We report here NKG2D-Ls induction by RVT in a broad range of leukemia cells. RVT upregulated the NKG2D-Ls MICA/B, ULBP1, ULBP2 and ULBP3 in the myeloid leukemia cells OUN-1, NB4, THP-1 and KG1 and upregulated MICA/B, ULBP-1 and ULBP3 ligands in the lymphoid leukemia cells Jurkat and Molt-4. The upregulation of NKG2D-Ls by RVT was associated with increased transcription of each NKG2D-L gene. Ligand upregulation induced by RVT was prevented by cell pretreatment with caffeine, and inhibitor of ATM/ATR, which is the main signal regulator of NKG2D-Ls expression. Leukemia cells treated with RVT were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the treatment of NK cells with anti-NKG2D monoclonal antibodies. Interestingly, the same concentration of RVT that effectively induced NKG2D-Ls in tumor cells, consistently upregulated NKG2D receptor expression in primary NK cells from healthy individuals and in the NK cell lines NKL and NK-92 and this effect was also associated with enhanced NKG2D-mediated NK cells cytotoxicity. RVT-induced NKG2D receptor enhancement in NK cells associated with the activation of the MAP kinase ERK1/2 and was prevented by the ERK1/2 specific inhibitor PD98059. Thus, RVT represents the first identified agent capable of activating both arms of the NKG2D axis. Since several clinical trials on RVT are ongoing, these previously unrecognized properties of this non toxic compound have an attractive immunotherapeutic potential. Disclosures: No relevant conflicts of interest to declare.

γ-IFN May Act As a Positive Regulator to Hematogenesis Through PI3K/Akt and JAK2/STAT5 Pathways

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4785-4785
Author(s):  
Hong Liu ◽  
Lu Zhou ◽  
Han Wang
Keyword(s):  
Stem Cells ◽  
Aplastic Anemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Regulatory Factor ◽  
Hematopoietic Stem ◽  
Positive Regulator ◽  
Proliferative Effect

Abstract Abstract 4785 It is known to us all that γ-IFN is a growth inhibitory to hematogenesis and plays a critical role in the pathophysiology of aplastic anemia(AA). However, γ-IFN actually is a bilateral regulatory factor. In certain experimental situations, it can facilitate the proliferation and growth of hematopoitic stem cells. Interferon regulatory factor-1 (IRF-1) and IRF-2 are the key regulators of IFN system and IRF-1 has been shown to participate in development of CD8+ T cells, Th1 differentiation, and NK cell development. Thus, IRF-1 may has crucial functions against hematopoietic stem and/or progenitors cells. To investigate the function of IRF-1and the molecular mechanism of proliferative effect of γ-IFN, IRF-1-siRNA was designed to knocked out IRF-1 gene in 32D cells, mouse multipotent progenitor cells, and the apoptosis was assayed by flow cytometry 48 hr after incubation with γ-IFN in three different concentrations. It showed that after IRF-1 gene was knocked out, γ-IFN reversed its action on hematogenesis and apoptosis,it potentiated the proliferation and growth of 32D cells. Interestingly, at this situations, the inhibitory effect of γ-IFN on Akt activation was abolished completely and reversed to enhanced Akt phosphorylation significantly. The expression of pStat5 also changed when γ-IFN only exert proliferative effect.Nevertheless,The negative effect of γ-IFN on Erk1/2 activation was not blocked after IRF-1 gene was knocked out. Taken together, γ-IFN can enhance or antagonize cell proliferation depending on the function of IRF-1 and may act as a positive regulator to hematogenesis through PI3K/Akt and JAK2/STAT5 signaling pathways. Our findings may contribute to understanding the decreased number of stem cells characteristic of aplastic anemia and will provide novel therapeutic strategies for this disease. Disclosures: No relevant conflicts of interest to declare.

Manumycin Induces Apoptosis of Leukemic Stem Cells Through Down-Regulation of Bcl2

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4738-4738
Author(s):  
Miaorong She ◽  
Jinggao Li ◽  
Xilin Chen ◽  
Xin Du ◽  
Wei Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Malignant Disease ◽  
Conflicts Of Interest ◽  
Soft Agar ◽  
Leukemic Stem Cells ◽  
Farnesyltransferase Inhibitor ◽  
Down Regulation ◽  
New Class ◽  
Dose Dependent

Abstract Abstract 4738 Acute myeloid leukemia (AML) is a serious and often lethal hematopoietic malignant disease arising from stem cells. LSCs play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population for ultimately curing leukemia. Unfortunately, very few agents have been shown to directly target the LSC population. The present studies demonstrate that manumycin, a farnesyltransferase inhibitor, resulted in viability decrease in a dose-dependent and soft agar clonogenic formation inhibition in LSCs isolated from KG1a cell line. Furthermore, manumycin induces LSC apoptosis in a dose-dependent. The molecular mechanism of manumycin mediated apoptosis is associated with down regulation of Bcl2 and Bcl-xL. In conclusion, manumycin triggers apoptosis in LSC and represents a potentially important new class of drugs for LSC-targeted therapy. Disclosures: No relevant conflicts of interest to declare.

Effective and Selective Elimination of CML Stem Cells Using Novel Ethacrynic Acid Derivatives

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4508-4508
Author(s):  
Su Chu ◽  
YinWei Ho ◽  
Guisen Zhao ◽  
Tessa L. Holyoake ◽  
Samuel Waxman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Progenitor Cells ◽  
Ethacrynic Acid ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Cd34 Cells ◽  
Induced Apoptosis

Abstract Tyrosine kinase inhibitors (TKI) are highly effective in the treatment of CML but do not eliminate primitive, quiescent leukemia stem cells (LSC), which persist as a potential source of leukemia relapse. Additional strategies to enhance eradication of LSC are required to increase the possibility of treatment free remissions for CML patients. Glutathione S-transferase P1-1 (GSTP1-1) is a Phase II detoxifying enzyme which is overexpressed in several cancers and causes drug resistance. The diuretic Ethacrynic acid (EA) is a GSTP1-1 activity inhibitor and also induces celldeath in malignant cells at high concentrations. We have synthesized two EAoxadiazole analogs, 6U and 6S, which demonstrate enhancedpro-apoptotic effects in CML K562 cells expressing high levels of GSTP1-1. Previously we found that 6U and 6S induced apoptosis through downregulation of anti-apoptotic protein MCL-1 in addition to their GSTP1-1 activity inhibition. We extended these observations to primary CML stem/progenitor cells. We observed increased expression of GSTP1-1 mRNA and protein, associated with increased expression of MCL-1, BCL2 and BCL-xL, in chronic phase (CP) and blast crisis (BC) CML compared to normal CD34+ cells. Treatment of CP CML CD34+ cells with 6U or 6S (1 to 6µM) for 24 to 48 hours resulted in a significant dose-dependent induction of apoptosis, inhibition of cell growth, and reduction in colony forming cell (CFC) numbers, with 6U demonstrating greater efficacy than 6S. Treatment with 6U did not induce significant apoptosis of normal (NL) CD34+ cells at doses below 4µM. 6U induced significantly less apoptosis in NL compared with CML CD34+ cells (2µM, p<0.05). We further tested the activity of 6U against purified CML and normal CD34+CD38- stem/primitive progenitors and CD34+CD38+ committed progenitors with or without the BCR-ABL TKI Dasatinib (DAS). 6U treatment induced apoptosis of CML, but not normal, CD34+CD38- and CD34+CD38+ cells (Table). Combination of 6U with DAS (50nM) selectively enhanced apoptosis of CML compared to normal cells, including quiescent, slowly dividing CML LSC that are resistant to TKI-induced apoptosis (p≤0.01). Treatment with 6U alone or with DAS, significantly increased G1, and decreased S/G2/M phase of CML, but not in normal CD34+ cells, and reduced CFC growth from CML CD34+CD38+ cells (Table). CML, but not normal CD34+ cells, treated with 6U, with or without DAS, prior to transplant, showed significantly reduced engraftment in NSG mice, indicating selective inhibition of in vivo repopulating CML LSC (Table). Treatment with 6U was also effective in inducing apoptosis and inhibiting CFC growth in BC CML progenitor cells (Table). 6U treatment resulted in down-regulation of GSTPI1-1 and MCL-1 protein expression in CP and BC CML, but not in normal CD34+ cells. Interestingly 6U treatment also reduced BCR-ABL protein expression in CP and BC CML CD34+ cells. We conclude that CML CP and BC LSC express high levels of GSTP1-1 and anti-apoptotic proteins, which can be targeted by the novel EA derivative 6U through a new mechanism. Since 6U has significantly lesser effects on normal stem cells, it may offer a promising and innovative approach to selectively target CP and BC CML LSC in combination with TKI inhibitors. Abstract 4508. Table CML CP Normal CML BC Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Apoptosis (normal, CP CML: CD34+CD38-; CML BC CD34+) 3.4± 0.9 15.9±6.7 9.4± 2.6 47.4±13.6 ** 3.3± 0.9 5.1± 1.0 1.6± 0.2 7.0± 1.2 * 3.4± 0.7 30±12.7 10.6±1.8 43.3±14.1 ** CFU-GM (normal, CP CML: CD34+CD38+; CML BC CD34+) 71.3± 7.8 7± 3.2 ** 21± 7.3 ** 5 ± 2.3 ** 121±19.3 102.7±6.2 134.3±15.9 103±5.1 288.5±89.4 26.5±11.3 *** 82.7±33.1 ** 8 ± 3.6 *** NSG engraftment (CD34+) 1.8± 0.3 0.4± 0.1 *** 0.8± 0.3 ** 0.4± 0.04 *** 68.2± 4.9 61± 2.2 68.1± 2.9 64.2± 3.9 Data shown are mean ± SEM of 3-6 samples. Significance, compared to controls. *p≤0.05,**p≤0.01, ***p≤0.001 Disclosures No relevant conflicts of interest to declare.

scholarly journals ANTI-CANCER EFFICIENCY OF NATURAL KILLER CELLS DIFFERENTIATED FROM HUMAN ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS AND TRANSFECTED WITH miRNA150

2017 ◽  
Vol 39 (3) ◽  
pp. 212-218 ◽  
Author(s):  
A Karlitepe ◽  
H Kabadayi ◽  
S Vatansever ◽  
M Gurdal ◽  
C Gunduz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Nk Cell ◽  
Human Adipose Tissue ◽  
Suppressor Gene ◽  
Pancreatic Cancer Cells ◽  
Anti Cancer ◽  
Induced Apoptosis

Aim: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). Methods: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RTPCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were determined by TUNEL and RT-PCR. Results: In miR150-transfected cells, the increased expression of NK cell-specific genes such as GZMB, KIR2DL2, CD16, CD56, NKG2D, NKp46 and increased immunoreactivity of NK cell-specific surface marker CD314 (NKG2D) were evident. TUNEL assays showed that NK-like cells with/without transfection induced apoptosis in PANC1 cells in the same manner. The decrease in oncogene expression and the increase in the tumor suppressor gene expression in PANC1 cells upon co-culture with NK-like cells differentiated from AD-MSCs were more prominent following miRNA150 transfection. Conclusion: It was shown in vitro that NK-like cells could be obtained by differentiation from AD-MSCs and their efficiency could be increased via miR150 transfection. The results are encouraging for further clinical studies in improvement of immunotherapeutic approaches for cancer therapy.

scholarly journals Hydroxychloroquine Sensitizes Chronic Myeloid Cell to γ9δ2T Cell Mediated Lysis Independent of Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4244-4244
Author(s):  
Biqing Han ◽  
Yanmin Zhao ◽  
Yu Lin ◽  
Limengmeng Wang ◽  
Binsheng Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Leukemia Stem Cells ◽  
Immune Recognition ◽  
Γδt Cells ◽  
Nkg2d Ligands ◽  
Γδt Cell ◽  
Autophagy Inhibitor ◽  
Autophagy Inhibition

Abstract Introduction Tyrosine kinase inhibitor (TKI) has greatly improved survival in CML. However, relapse remains a great challenge because of the persistent of leukemia stem cells. Recently autophagy is revealed to be a mechanism of drug resistance of leukemia stem cells. Targeting autophagy in the treatment of CML has been highlighted. Hydroxylchloroquine (HCQ), the only autophagy inhibitor approved for clinical use, has drawn more and more attention. Combining TKI with HCQ, primary CML stem cells can be almost completely eliminated in vitro. What's more, a phaseⅡ clinical research is going on in the University of Glasgow. HCQ as autophagy inhibitor can also augment the anticancer activity of some chemotherapeutics such as histone deacetylase inhibitor, IFNα etc and overcome drug resistance in CML. HCQ has offered the hope that CML may be cured. But seldom do we know whether HCQ exerts anticancer effects through other mechanisms besides autophagy inhibition. Especially the mechanisms underlying the ability of HCQ to modulate the immune visibility of CML are unknown. In our research, we revealed HCQ can sensitize CML to γδT cell mediated lysis through ULBP4 translocation, which proved to be autophagy independent. Results To test the cytotoctity of γδT cells to CML cells. CML cell lines K562 ( imatinib sensitive) and K562/GO1( imatibin resistant) were firstly exposed to nontoxic HCQ, then cocultured with γδT cells from peripheral blood of healthy donors. Compared to untreated cells, pretreated K562 and K562/GO1 were more sensitive to γδT cell mediated lysis. The mortality increased from 35.9% to 47.1% in K562 (P<0.05) and from 31.7% to 46.6% in K562/GO1 (P<0.05) at effecter target ratio 20:1. To evaluate the activation of γδT cells , we tested the degranulation of γδT cells co-cultured with treated or untreated CML. Consistent with the cytotoxity assay,compared to untreated CML HCQ pretreated CML accelerated γδT cells degranulation from 12.45% to 23.05(P<0.05) in K562 and from 18.3% to 28.2% (P<0.05) in K562/GO1 respectively. And we also confirmed this in primary CML stem cells (CD34+) from 3 CML patients. To test whether HCQ sensitized CML to γδT cells by autophagy inhibition, we detected ATG protein LC3 P62 and Bclin1 as well as autophagosome in CML. HCQ treatment can induce the accumulation of LC3Ⅱ P62 and autophagosome in a time dependence manner, which means HCQ can block autophagy in CML. We further conducted an ATG7 knockdown assay by shRNA to block autophagy in CML. ATG7 knockdown can significantly block autophagy in CML, but without effects on CML sensitivity to γδT cells. Moreover, after ATG7 knockdown, HCQ can still sensitize CML to γδT. This implied an autophagy independence manner of HCQ in sensitizing CML to γδT cell mediated lysis. Next we detected the expression of NKG2D ligands on CML cytomembrane after HCQ exposure, which are important for cancer cell immune recognition by γδT cells. Among all NKG2D ligands including MICA/B ULBP1 2 3 4 5, we found only ULBP4 was up regulated after HCQ treatment depend on exposure time. The fact is ULBP4 doesn't express on the cytomembrane of K562 and K562/GO1. After exposure to HCQ for 4 8 and 12 hours, the cells expressed ULBP4 on cytomenbrane raised from 0.84% to 68.14% 87% and 86.4% in K562 (P<0.001) and raised from 1.2% to 54.6% 84.3% and 87.1% in K562/GO1(P<0.001). To test whether HCQ sensitize CML to γδT due to the ULBP4 upregulation on cytomembrane, blocking NKG2D mAb were used to block the binding of NKG2D with its ligands. After that, HCQ exposure can't augment the sensitivity of CML to γδT cells. This means HCQ sensitize CML to γδT cells through up-regulating ULBP4 on cytomembrane. To know how HCQ unregulates ULBP4 on cytomembrane. We checked the mRNA and protein of ULBP4, and found HCQ did not influence the transcription and translation of ULBP4. We further tested the expression of ULBP4 both in CML cytomembrane and cytoplasm, found ULBP4 exists in cytoplasm and doesn't express on cytomembrane untill HCQ exposure. Immunofluorescene showed that HCQ treatment induce ULBP4 transfer from cytoplasm to cytomembrane. Conclusion Our data shows the first time HCQ can sensitize CML to γδT cell mediated lysis in an autophagy independence manner. In CML, ULBP4 intracellular retention may be a way of immune escape. HCQ can induce ULBP4 translocation from cytoplasm to cytomenbrane through which sensitizes CML to γδT cell mediated lysis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Blocking TNF and IL-1 Sensitizes AML Stem Cells to NF-κB Inhibitor Treatment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2302-2302
Author(s):  
Volk Andrew ◽  
Jing Li ◽  
Jun Zhang ◽  
Joseph Cannova ◽  
Caiqin Hao ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Conflicts Of Interest ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Enhanced Sensitivity ◽  
Repressive Effect ◽  
Inhibitor Treatment

Abstract Background: NF-κB inhibition selectively eliminates leukemia stem cells (LSCs) but has less effect on healthy hematopoietic stem cells (HSCs), suggesting an advantageous target for leukemia therapy. However, NF-κB inhibition alone does not have a significant effect on in vivo models of leukemia. We have reported that many types of AML cells produce tumor necrosis factor-α (TNF) which appears to protect LSCs from NF-κB inhibition by stimulating JNK-AP1 survival/proliferative signaling parallel to NF-κB. Complete inactivation of TNF signaling cannot fully reproduce the effects of JNK-AP1 inhibition, suggesting that some other inflammatory cytokine(s) might also compensate for NF-κB inhibition through activation of JNK-AP1. We found that IL-1α and β (IL-1) is such a cytokine. Methods: TNF and IL-1 expression was examined in 430 AML samples by microarray and verified by qRT-PCR. IL-1α and β protein levels in the sera of AML patients and medium from cultured AML cells were examined by ELISA. MLL-AF9-transduced murine AML cells, human AML cell lines and primary patient samples were used in this study. TNF signal was blocked by genetic deletion of receptors Tnfr1 and 2, or treatment with a TNF-specific monoclonal antibody. IL-1 signal was blocked by shRNA knockdown of IL-1R or treatment with an IL-1R antagonist. The effects of IL-1 and TNF signal co-inactivation on growth and response to NF-κB inhibition in AML cells were evaluated by in vitro culture and colony-forming assay, and in vivo transplantation/leukemogenesis assays. Results: We found that most types of human AML cells both express and produce TNF and IL-1. Treatment with these cytokines stimulated the growth and colony-forming ability of AML cells but repressed the growth of normal HSPC in in vitro culture. Inactivation of either the TNF or IL-1 signaling axes suppressed the growth of and significantly sensitized AML stem cells to NF-κB inhibition both in vitro by targeting clonogenic AML progenitor cells, and in vivo through compromising leukemogenesis. Simultaneous inactivation of both TNF and IL-1 resulted in an enhanced repressive effect on AML cells compared to inactivation of either individual cytokine’s receptors alone. AML cells with inactivation of both TNF and IL-1 signals showed significantly lower clonogenic ability in vitro, further reduced leukemogenic capacity in vivo and enhanced sensitivity to NF-kB inhibitor treatment both in vitro and in vivo. Mechanistically, we found that both TNF and IL-1 stimulate JNK-AP1 and NF-κB, two parallel survival signaling pathways in AML cells, but induce JNK-mediated death signaling in normal HSPCs. Conclusion: Both TNF and IL-1 provide protection to LSCs from the effects of NF-κB inhibitor treatment in an autocrine manner and contribute to repression of normal hematopoiesis in a paracrine fashion. This protection is mediated by JNK-AP1. Our studies show inhibiting both TNF and IL-1 inflammatory signals can block this effect, and may be a better strategy to successfully treat AML when combined with NF-κB inhibitors. Disclosures No relevant conflicts of interest to declare.

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