Human Chronic Myeloid Leukemia Cells Research Articles

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10(6) copies per 50ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG(0) phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1μM and 5μM) showed an increase in events within the subG(0) phase of cell cycle over 96h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10nM) in combination with Imatinib (5μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.

Roles of ceramide synthase and ceramide clearence genes in nilotinib-induced cell death in chronic myeloidleukemia cells

In this study, we aimed to increase the sensitivity of human K562 and Meg-01 chronic myeloid leukemia (CML) cells to nilotinib by targeting bioactive sphingolipids, in addition to investigating the roles of ceramide metabolizing genes in nilotinib induced apoptosis. Cytotoxic effects of nilotinib, C8:ceramide, glucosyle ceramide synthase (GCS) and sphingosine kinase-1 (SK-1) inhibitors were determined by XTT cell proliferation assay and synergism between the agents was determined by isobologram analysis. Also, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results demonstrated that expression levels of longevity assurance (LASS) genes in response to nilotinib were correlated with sensitivity to nilotinib. For the first time, The results of this study showed for the first time that nilotinib induces apoptosis through upregulating ceramide synthase genes and downregulating SK-1 in CML cells in addition to inhibition of BCR/ABL. On the other hand, manipulating bioactive sphingolipids toward generation/accumulation of ceramides increased the apoptotic effects of nilotinib in CML cells.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr‐Abl inhibitor and cooperatively induces glycogen synthase kinase‐3‐regulated apoptosis

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance.

Proliferation-Attenuating and Apoptosis-Inducing Effects of Tryptanthrin on Human Chronic Myeloid Leukemia K562 Cell Line in Vitro

Tryptanthrin, a kind of indole quinazoline alkaloid, has been shown to exhibit anti-microbial, anti-inflammation and anti-tumor effects both in vivo and in vitro. However, its biological activity on human chronic myeloid leukemia cell line K562 is not fully understood. In the present study, we investigated the proliferation-attenuating and apoptosis-inducing effects of tryptanthrin on leukemia K562 cells in vitro and explored the underlying mechanisms. The results showed that tryptanthrin could significantly inhibit K562 cells proliferation in a time- and dose-dependent manner as evidenced by MTT assay and flow cytometry analysis. We also observed pyknosis, chromatin margination and the formation of apoptotic bodies in the presence of tryptanthrin under the electron microscope. Nuclei fragmentation and condensation by Hoechst 33258 staining were detected as well. The amount of apoptotic cells significantly increased whereas the mitochondrial membrane potential decreased dramatically after tryptanthrin exposure. K562 cells in the tryptanthrin treated group exhibited an increase in cytosol cyt-c, Bax and activated caspase-3 expression while a decrease in Bcl-2, mito cyt-c and pro-caspase-3 contents. However, the changes of pro-caspase-3 and activated caspase-3 could be abolished by a pan-caspase inhibitor ZVAD-FMK. These results suggest that tryptanthrin has proliferation-attenuating and apoptosis-inducing effects on K562 cells. The underlying mechanism is probably attributed to the reduction in mitochondria membrane potential, the release of mito cyt-c and pro-caspase-3 activation.

Sphingosine kinase-1 and sphingosine 1-phosphate receptor 2 mediate Bcr-Abl1 stability and drug resistance by modulation of protein phosphatase 2A

AbstractThe mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1−/− MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34+ mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1–derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr–Abl1 thereby overcoming drug resistance.

Inhibition of thioredoxin reductase by auranofin induces apoptosis in adriamycin-resistant human K562 chronic myeloid leukemia cells.

Mammalian thioredoxin reductase (TrxR) catalyzes the NADPH-dependent reduction of oxidized thioredoxin (Trx) and plays a central role in regulating cellular redox homeostasis, cell growth and apoptosis. Increasing evidence shows that TrxR is over-expressed or constitutively active in many tumor cells. Moreover, TrxR appears to contribute to increased tumor cell growth and a resistance to chemotherapy. In this study, we evaluated the activity of TrxR in adriamycin-resistant leukemic cells (K562/ADM) and adriamycin-sensitive parental lines (K562), and found that TrxR activity was higher in the drug resistant cell sublines K562/ADM than in K562 drug sensitive parental cells. Auranofin, a gold(I) compound clinically used as an antirheumatic agent, reduced TrxR activity and was more effective than adriamycin in decreasing cell viability in K562/ADM cells. In addition, auranofin induced apoptosis in dose-dependent manners, accompanied by caspase-3 activation in K562/ADM cells. Our results demonstrate that inhibition of TrxR and induction of apoptosis by auranofin provides its ability in overcoming adriamycin resistance in K562/ADM cells.

Overexpression of IL-32α Increases Natural Killer Cell-mediated Killing through Up-regulation of Fas and UL16-binding protein 2 (ULBP2) Expression in Human Chronic Myeloid Leukemia Cells

IL-32 was recently identified as a proinflammatory cytokine that is induced by IL-18 in natural killer (NK) cells and is highly correlated with inflammatory disorders. However, the relationship between IL-32 and tumor progression is still unknown. In this study, we investigated whether overexpression of IL-32 affects susceptibility of chronic myeloid leukemia (CML) cells to NK cells. Interestingly, IL-32α-overexpressing CML cell lines, K562, Kcl22, and BV173, showed higher NK cell-mediated killing. Flow cytometry analysis revealed that overexpression of IL-32α induced increased expression of Fas and UL16-binding protein 2 (ULBP2) in CML cells. The direct relationship between overexpression of surface molecules by IL-32α and increased NK cell-mediated killing was confirmed by Fas or ULBP2 siRNA transfection. IL-32α-induced Fas and ULBP2 expression are regulated p38 MAPK. In addition, the transcription factor Ets1 plays a key role in ULBP2 specific expression by IL-32α overexpression in ULBP family members. Taken together, these data show that IL-32α stimulates Fas and ULBP2 expression via activation of p38 MAPK, which increases NK susceptibility of CML cells. Enhanced NK cell susceptibility of CML cells by IL-32α overexpression may improve the efficiency of NK cell-based immunotherapy.

Up-regulation of P-glycoprotein confers acquired resistance to 6-mercaptopurine in human chronic myeloid leukemia cells

To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloid leukemia (CML), a 6-MP resistant cell line (K562-MP5) was established by stepwise selection of the CML cell line (K562). The results of the drug sensitivity analysis of the K562-MP5 cell line revealed the cells to be 339-fold more resistant to 6-MP compared with the parental K562 cells. K562-MP5 cells exhibited decreased accumulation and increased efflux of [(14)C]6-MP and its metabolites. In addition, K562-MP5 cells showed increased [(3)H]MTX transport. K562-MP5 cells over-expressed P-glycoprotein (P-gp) and up-regulated MDR1 mRNA levels. Taken together, these results suggest that the up-regulation of P-gp, which contributes to the decreased accumulation by increasing the efflux of 6-MP and its metabolites, underlies the mechanism of 6-MP resistance in K562 cells.

DNA Binding, DNA Cleavage, and Cytotoxicity Studies of Two New Copper (II) Complexes

The DNA binding behavior of [Cu(phen)(phen-dione)Cl]Cl (1) and [Cu(bpy)(phen-dione)Cl]Cl (2) was studied with a series of techniques including UV-vis absorption, circular dichroism spectroscopy, and viscometric methods. Cytotoxicity effect and DNA unwinding properties were also investigated. The results indicate that the Cu(II) complexes interact with calf-thymus DNA by both partially intercalative and hydrogen binding. These findings have been further substantiated by the determination of intrinsic binding constants spectrophotometrically, 12.5 × 10(5) and 5 × 10(5) for 1 and 2, respectively. Our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Circular dichroism results show that complex 1 causes considerable increase in base stacking of DNA, whereas 2 decreases the base stacking, which is related to more extended aromatic area of 1,10-phenanthroline in 1 rather than bipyridine in 2. Slow decrease in DNA viscosity indicates partially intercalative binding in addition to hydrogen binding on the surface of DNA. The second binding mode was also confirmed by additional tests: interaction in denaturation condition and acidic pH. Also, these new complexes induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cells.

Imatinib-induced apoptosis: a possible link to topoisomerase enzyme inhibition

Imatinib is a specific BCR/ABL inhibitor, commonly used for the treatment of chronic myeloid leukaemia (CML), a hematological malignancy resulting from a chromosomal translocation that generates the BCR/ABL fusion protein. Recent studies showed that the imatinib has cytotoxic and apoptotic effects on many BCR/ABL-negative cancers. Numerous compounds with cytotoxic potential exert their functions by interfering with the DNA topoisomerase. In this study, we examined the effects of imatinib on tumour cell-killing in relation to DNA topoisomerase enzyme inhibition. We determined the cytotoxicity by cell proliferation assay (XTT; tetrazolium hydroxide), using the human K562 CML cells, and loss of mitochondrial membrane potential by monitoring the changes in caspase-3 enzyme activity. Type I and II topoisomerase activities were measured by supercoiled plasmid relaxation and minicircle DNA decatenation assays respectively. Imatinib-induced apoptosis and inhibited cell proliferation in a dose-dependent manner. We also found that the imatinib was effective in both type I and type II topoisomerase reactions to a varying degree between 94% and 7% for the concentration range of 1 mm-0.02 mm in a dose-dependent manner. Our results suggest that the inhibition of topoisomerases may be a significant factor in imatinib-induced apoptosis in CML.

BCL6 Is Required for the Maintenance of Leukemia-Initiating Cells In Chronic Myeloid Leukemia

AbstractAbstract 202 Background:Chronic myeloid leukemia (CML) can be effectively treated for many years with tyrosine kinase inhibitors (TKI). However, unless CML patients take TKI-treatment life-long, leukemia will eventually recur, which is attributed to the failure of TKI-treatment to eradicate leukemia stem cells in CML. Relapse from leukemia stem cells in CML often results in TKI-resistant blast crisis, which is fatal within months. Approach:Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in chronic myeloid leukemia (CML). The mechanism of FoxO3A-dependent maintenance of leukemia stem cells remained unclear. Here we identified the BCL6 protooncogene downstream of FoxO3A as a critical effector molecule of self-renewal signaling in CML-initiating cells. BCL6 is known as a proto-oncogene in Diffuse Large B cell Lymphoma (DLBCL), where it functions as transcriptional repressor of p53. BCL6-Null CML cells fail to initiate leukemia:Studying gene expression changes of CML cells in 6 patients before and after treatment with Imatinib, we found that BCL6 mRNA levels were increased by >15-fold in response to Imatinib-treatment. Studying CD34+ CD38- CML cells from leukapheresis samples of two patients and CML cell lines, we found that overnight incubation with Imatinib resulted in a >12-fold increase of BCL6 expression at the mRNA and protein level. Previous studies showed that FoxO factors are required for transcriptional activation of BCL6 and FoxO3A was recently identified as a critical factor of leukemia stem cell maintenance in CML. Here we showed that inducible activation of FoxO3A indeed leads to de novo expression of BCL6 in human CML cells. We next tested the functional significance of BCL6 expression in CML cells in a genetic experiment. To this end, we used a classical mouse model for CML-like leukemia and transformed Lin− Sca-1+ c-kit+ (LSK) cells from BCL6+/+ and BCL6-Null mice with BCR-ABL1. While CML transformation efficiency was similar for BCL6+/+ and BCL6-Null LSK cells, the LSK phenotype was rapidly lost in BCL6-Null CML cells. To elucidate the mechanism of progressive loss of LSK cells in BCL6-Null CML, we performed a systematic analysis of gene expression changes in BCL6+/+ and BCL6-Null CML cells: The ABCG2 transporter, which is required for the side population (SP+) phenotype in LSK cells was reduced by >7-fold in the absence of BCL6. SP+ LSK cells represent a highly drug resistant CML subpopulation with leukemia-initiation capacity. In addition, BCL6-Null CML cells express excessively high levels of p53 at the protein level. A genome-wide mapping approach of BCL6-DNA interactions using ChIP-seq showed that BCL6 strongly binds to and represses the p53 promoter. Importantly, BCL6 expression represents a critical requirement for CML cells to form colonies in semisolid agar. Compared to BCL6+/+ CML cells (>400 colonies/10,000 cells), colony formation by BCL6-Null CML cells was reduced by ≂f100-fold (<5 colonies). After an initially successful engraftment of BCL6-Null CML cells in NOD/SCID mice (as visualized by luciferase bioimaging), BCL6-Null CML cells failed to initiate leukemia, whereas NOD/SCID mice injected with BCL6+/+ CML cells succumbed to the disease. Targeted inhibition of BCL6 for leukemia stem cell eradication in CML:A retro-inverso BCL6 peptide inhibitor (RI-BPI) was recently developed for targeted therapy of DLBCL. Therefore, we tested the potential therapeutic usefulness of RI-BPI for eradication of BCL6-dependent leukemia-initiating cells. Human CML cells were incubated overnight in the presence of 5 μ mol/l RI-BPI or vehicle control. While Hoechst 33342 staining revealed a distinct side population of ≂f2.8% CML cells, overnight incubation with RI-BPI reduced the frequency of ABCG2+ side population cells by more than 30-fold. Likewise, RI-BPI incubation decreased colony formation by >5-fold in human CML cells. Importantly, RI-BPI treatment of CML cells resulted in significantly prolonged overall survival of xenografted NOD/SCID mice and decreased penetrance of leukemia (10 mice/group; p=0.032), Conclusion:Pharmacological inhibition of BCL6 represents a powerful strategy to eradicate leukemia-initiating cells in CML. Clinical validation of this concept could limit the duration of TKI-treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation. Disclosures:Hochhaus:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Human Leukemia Xenotransplantation Into Zebrafish Embryos as An In Vivo Assay for Chemotherapy Drug Response

AbstractAbstract 1704A major challenge in the treatment of human cancer is the variable response to therapy between patients. Any host or tumor-derived factors that produce variations in drug concentration, duration of exposure, or sensitivity of tumour cell populations to drug, may contribute to heterogeneity in the tumour response. The identification and characterization of specific drug-tumor interactions would enable the design of more personalized and targeted treatment with improved outcomes and reduced toxicity. To date, human cancer cell lines and murine models have provided some advances, however these approaches have been hindered by technical limitations, considerable expense and a lack of timely information to directly impact on a given therapeutic window. The zebrafish has emerged as a robust animal model of human malignancy due to conserved genetics and cell biology. In addition, their transparency provides exceptional opportunities for in vivo imaging, such as the unique ability to directly visualize the response of cancer cells to drugs in real time. Using zebrafish as a host organism, we have been developing a quantitative cell proliferation assay to monitor the response of human leukemia cells to anti-cancer agents in an environment that more closely recapitulates the human tumor microenvironment. As proof of principle, we have injected boluses of 50 fluorescently-labeled cells from the human K562 chronic myeloid leukemia cell (CML) line into the yolk sac of 48 hour old casper embryos, a combinatorial zebrafish mutant lacking both melanocytes and iridophores, thus enabling facile cell tracking and imaging. Injected embryos tolerate the presence of human leukemia cells for up to 7 days, during which time the engrafted leukemia cells proliferate and circulate within the embryonic bloodstream. Proliferation of the leukemia cells can be monitored by live-cell microscopy of engrafted embryos and quantified by their dissociation to a single cell suspension at 24 and 72 hours post-injection. Specifically, 20 embryos are dissociated at each time point and the number of leukemia cells is determined from fluorescent micrographs using a semi-automated cell quantification macro executed in ImageJ (NIH). Using this proliferation assay, we observed a reproducible increase in leukemia cell numbers within the embryo of approximately 5-fold after 48 hours. Furthermore, the proliferation of K562 cells in xenotransplanted zebrafish embryos could be significantly inhibited (by 45 ± 3% relative to untreated; p<0.001) following a 48 hour treatment with 20 μM of imatinib mesylate (Gleevec), a drug that targets the characteristic CML BCR-ABL translocation gene product harboured by these cells. In contrast, treatment with a second agent, all trans-retinoic acid (ATRA) that targets the PML-RARA fusion protein found in acute promyelocytic leukemia (APL), had no impact on proliferation. These results validate the use of zebrafish xenotransplantation in studying the response of human leukemia cells to anti-cancer agents and position this model as a unique in vivo tool to determine the sensitivity of primary patient tumor samples to current and novel chemotherapeutics. Disclosures:No relevant conflicts of interest to declare.

Treatment with TKIs Overcomes Imatinib Resistance through the PLCgamma-1 Signaling Pathway In Imatinib Resistant Human CML Cell Lines

AbstractAbstract 4468Suppression of BCR-ABL1 catalytic activity by the tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) has dramatically improved the natural history of Chronic Myeloid Leukemia (CML) and - to date - represents the first and most successful example of effective anti-cancer targeted therapy {Druker, 2009}. Despite the success, patients can become resistant. Since IM-resistance in CML patients occurs despite adequate suppression of BCR-ABL activity, it is likely due to the activation of other pathways, and for this reason we need to discover novel Bcr-Abl independent pathways than can become the targets of resistant cells.Therefore we used immortalized CML human cell lines K562 and LAM84, both sensitive and resistant to Imatinib, to study the signaling in sensitive/resistant cell lines following treatment with 4 different compounds. Reverse-Phase protein microarrays were used to quantitatively map 35 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. Cells were incubated with IM 1uM, Dasatinib 1uM, Nilotinib 1uM or LY-29400210 uM and after 2 or 12 hours were placed in a preservative that suppresses fluctuations in kinase pathway proteins (Espina, 2008).5/35 protein endpoints were linked together and suppressed by Dasatinib, even in the resistant cell line: PLC-y-1-Tyr783, and its upstream (ShCTyr317 SrcTyr416) and downstream targets (mTORSer2448, STAT5Tyr694, ERKThr202/Tyr204) without interfering with AKT activation status on Ser473 compared to Imatinib (p=0.0031 for K562, p= 0.042 for LAM84), Nilotinib (p=0.0034 for K562, p= 0.043 for LAM84), LY-294002 (p=0.0009 for K562, p= 0.015 for LAM84).In Imatinib-sensitive cell line LAM84 there were no differences between Dasatinib and Imatinib in the modulation of the pathway, compared to IM-sensitive K562 cell line (p=0.005), thus confirming the different profile among these two CML models.Dasatinib showed a greater suppression of the PLC-y-1 pathway compared to LY-294002 in both resistant cell lines (K562, p=0.0009 and LAM84, p=0.015).These data confirm and extend the conclusions of Markova et al. (Oncogene 2010) showing PLC-y-1 as a mechanism of death in sensitive cells. Our data showed the mechanism by which second generation TKIs (dasatinib) can overcome Imatinib resistance by suppressing the PLC-y-1 pathway. Disclosures:No relevant conflicts of interest to declare.

Stromal Cell-Derived Secreted Factors Contribute to the Innate Imatinib Resistance of Leukemia Stem Cells In a Genetically Defined Murine Model of Chronic Myeloid Leukemia Blast Crisis

AbstractAbstract 204Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL stimulates mutagenic homologous DNA double-strand break repair via the DNA-end-processing factor CtIP

Expression of BCR-ABL oncoprotein in chronic myeloid leukemia (CML) promotes neoplastic transformation of hematopoietic stem cells through modulation of diverse pathways. CML is a multistep disease, which evolves as a chronic phase and progresses to blast crisis. This progression has been associated with the appearance and accumulation of new cytogenetic anomalies and mutations. The mechanisms underlying the genomic instability promoted by BCR-ABL remain obscure. Through comparative analysis of different DNA double-strand break (DSB) repair mechanisms as a function of the BCR-ABL status in human megakaryocytic and CML cell lines, we found that BCR-ABL upregulates error-prone DSB repair pathways [single-strand annealing (SSA) and non-homologous end joining] rather than the high-fidelity mechanism of homologous recombination. Intriguingly, expression analysis of DSB repair pathway choice determining factors revealed increased levels of the protein CtIP in BCR-ABL-positive cells, particularly in response to irradiation. Moreover, treatment with the BCR-ABL kinase inhibitor, Imatinib Mesylate, abolished CtIP accumulation. When we silenced CtIP expression in cells with functional BCR-ABL, SSA enhancement by BCR-ABL was completely abrogated. Importantly, we also provide evidence that BCR-ABL stimulates DSB end resection, which is mediated by CtIP. Briefly, BCR-ABL promotes mutagenic DSB repair with the DSB end-processing protein CtIP acting as the key mediator downstream of BCR-ABL.

Global Proteome Quantification for Discovering Imatinib-Induced Perturbation of Multiple Biological Pathways in K562 Human Chronic Myeloid Leukemia Cells

Imatinib mesylate, currently marketed by Novartis as Gleevec in the U.S., has emerged as the leading compound to treat the chronic phase of chronic myeloid leukemia (CML), through its inhibition of Bcr-Abl tyrosine kinases, and other cancers. However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular pathways affected by imatinib treatment, we applied mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in K562 cells that were untreated or treated with a clinically relevant concentration of imatinib. Our results revealed that, among the 1344 quantified proteins, 73 had significantly altered levels of expression induced by imatinib and could be quantified in both forward and reverse SILAC labeling experiments. These included the down-regulation of thymidylate synthase, S-adenosylmethionine synthetase, and glycerol-3-phosphate dehydrogenase as well as the up-regulation of poly(ADP-ribose) polymerase 1, hemoglobins, and enzymes involved in heme biosynthesis. We also found, by assessing alteration in the acetylation level in histone H4 upon imatinib treatment, that the imatinib-induced hemoglobinization and erythroid differentiation in K562 cells are associated with global histone H4 hyperacetylation. Overall, these results provided potential biomarkers for monitoring the therapeutic intervention of CML using imatinib and offered important new knowledge for gaining insight into the molecular mechanisms of action of imatinib.

A new abietane diterpene from Glyptostrobus pensilis

A new abietane diterpene, glypensin A ( 1) and four known compounds, 12-acetoxy- ent-labda-8(17), 13 E-dien-15-oic acid ( 2), quercetin 3- O- α- L-arabinofuranoside ( 3), quercetin 3- O- β- D-galactopyranoside ( 4), β-sitosterol ( 5) were isolated from the branches and leaves of Glyptostrobus pensilis (Staut.) Koch. Their structures were determined by MS, 1D- and 2D-NMR means. Compound 1 showed cytotoxicity on human chronic myeloid leukemia cell line K562 (IC 50 = 21.2 μM).

S-trityl- L-cysteine derivative induces caspase-independent cell death in K562 human chronic myeloid leukemia cell line

Effect of CF 3-STLC, a potent kinesin spindle protein (KSP) inhibitor, on K562 human CML cell line was investigated. Treatment with CF 3-STLC induced mitotic arrest of the cell cycle with the appearance of characteristic monoastral spindles, subsequent apoptotic cell death and cleavage of PARP-1, caspase-3, and 4E-BP1. The wide ranging caspase inhibitor z-VAD fmk prevented the cleavage of caspase-3 and 4E-BP1, but failed to attenuate PARP-1 cleavage or cell death triggered by CF 3-STLC. These results suggest that CF 3-STLC can induce apoptotic cell death in a caspase-independent manner, and may work effectively as an anti-cancer agent for hematological malignancies.

Biomechanical alterations of dendritic cells by co-culturing with K562 CML cells and their potential role in immune escape

Dendritic cells (DCs), which are potent antigen presenting cells (APCs), are utilized to deliver the signals essential for the initiation of immune responses. In this study, we used an interdisciplinary approach to characterize the effect of K562 cells, a human chronic myeloid leukemia (CML) cell line, on the biomechanical characteristics and immune functions of DCs. When co-cultured with K562 cells, the biomechanical and immunological characteristics of immature DCs (imDCs) and mature DCs (mDCs) were severely impaired compared with controls. The changes include increased membrane viscoelasticity, reorganized cytoskeleton (F-actin), suppressed capability of antigen uptake, transendothelium migration, and activation of naïve T cells. In exploring the mechanisms of these changes, we identified several genes and proteins by microarray analysis and 2D gel electrophoresis. Changes were found in the cytoskeleton-related genes and proteins (such as cofilin1 and profilin1) and matrix-related genes and proteins (such as TIMP1 and MMP9). These findings provide a molecular basis for the biomechanical and immunological changes of DCs in response to K562 and may help to elucidate the mechanism for tumor immune escape.

Cochliones A–D, four new tetrahydrochromanone derivatives from endophytic Cochliobolus sp.

Four new tetrahydrochromanone derivatives, cochliones A–D (1–4), along with three known metabolites, 4-hydroxybenzaldehyde (5), 4-hydroxy-3-(3-methylbut-2-enyl) benzoic acid (6), and 2,2-dimethyl-2H-chromene-6-carboxylic acid (7), were characterized from the culture of Cochliobolus sp. IFB-E039, a fungus that resides inside the healthy root of Cynodon dactylon (Gramineae). The structures of 1–4 were accommodated by their spectral data (UV, IR, CD, HR-ESI-MS, 1H and 13C NMR, 1H–1H COSY, HMQC, HMBC, and NOESY). The bioassay for the cytotoxic metabolite showed that cochlione C (3) inhibited human breast adenocarcinoma cell line (MCF-7) and human chronic myeloid leukemia cell line (K562) with IC50 values of 21.99 and 4.59 μg/ml, respectively.