Human Chronic Myeloid Leukemia Cells Research Articles

Hydroxyurea Induces Growth Inhibition in BCR-ABL1 T315I+ Clones and Synergizes with Ponatinib in Killing TKI-Resistant CML Cells

In chronic myeloid leukemia (CML), the occurrence of BCR-ABL1 T315I is associated with resistance against first- and second-generation BCR-ABL1 tyrosine kinase inhibitors (TKI). Ponatinib is a third generation TKI that exerts strong anti-neoplastic effects in advanced CML and is capable of suppressing the kinase activity of BCR-ABL1 T315I. However, therapy with ponatinib is associated with potentially severe side effects. In addition, resistance against ponatinib may develop in sub-clones carrying multiple (compound) mutations in BCR-ABL1. In addition, BCR-ABL1-independent oncogenic pathways contribute to drug resistance. For these patients, alternative therapies such as stem cell transplantation (SCT) or various drug combinations are often considered.Hydroxyurea (HU) is used for initial or palliative cytoreduction in CML. However, the effects of HU on TKI-resistant mutant sub-clones have not been examined so far. The aims of this study were to explore the effects of HU on CML clones carrying BCR-ABL1 T315I as individual mutation or in compound-context, and to investigate anti-leukemic effects of the drug combination ponatinib+HU. In in vitro studies, primary patient-derived cells, human CML cell lines (K562, KU812, KCL-22), and Ba/F3 cells expressing wild type (wt) BCR-ABL1, BCR-ABL1 T315I, or BCR-ABL1 compound mutants involving T315 were examined. Cell proliferation was quantified by measuring 3H-thymidine uptake. Drug effects on competitive clonal growth were analyzed by mixing two Ba/F3 clones, one expressing BCR-ABL1 T315I with GFP and one BCR-ABL1 T315I/E255V labeled by tdTomato, at a 1:1 ratio. Then, cells were exposed to HU, pontinib, or HU+ponatinib for 72 hours, and the percentage of viable cells in each clone was analyzed by flow cytometry. The in vivo response of primary CML cells carrying BCR-ABL1 T315I to HU was examined in 4 TKI-resistant CML patients who were treated with HU (1-3 g/day) for up to 18 months. In these patients, we measured white blood counts (WBC), differential counts, and BCR-ABL1 transcript levels in peripheral blood (PB) by qPCR. The percentage of BCR-ABL1 T315I compared to total BCR-ABL1 was determined by ligation-dependent PCR. In all 4 patients treated with HU, WBC and total BCR-ABL1 mRNA levels remained stable for 3-12 months. Surprisingly, in 3 of 4 patients, the leukemic sub-clone expressing BCR-ABL1 T315Iwas no longer detectable after HU-treatment. After 3 months, 2/4 patients received allogenic SCT. In the other 2 patients, the disease remained stable for 6 and 12 months, respectively. In our in vitro studies, HU was found to inhibit the growth of all BCR-ABL1+ cell lines, including K562 (IC50: 1120±89 µM), KU812 (IC50: 216±32 µM), and KCL-22 (IC50: 196±23 µM) as well as Ba/F3 cells harboring BCR-ABL1 T315I as single mutation (IC50: 74±25 µM) or as compound together with E255V (IC50: 86±2 µM), F311L (IC50: 76±20 µM), F359V (IC50: 69±10 µM), or G250E (IC50: 89±4 µM). Interestingly, Ba/F3 cells exhibiting BCR-ABL1 T315I alone or in compound configuration were more sensitive to HU compared to Ba/F3 cells expressing wt BCR-ABL1 (IC50: 236±49 µM). As expected, HU was also found to inhibit growth of primary CML cells. In subsequent experiments, HU and ponatinib were found to synergize with each other in inhibiting growth of K562, KU812, and KCL-22 cells as well as Ba/F3 cells carrying BCR-ABL1 T315I (Figure) or BCR-ABL1-T315I/F359V. In cell mix experiments, ponatinib exerted strong growth-inhibitory effects on Ba/F3-T315I cells but not on Ba/F3-T315I/E255V cells, whereas HU was found to produce stronger effects on Ba/F3-T315I/E255V cells, and only the combination of both drugs resulted in complete suppression of both cell lines. In conclusion, HU exerts strong sub-clone-specific anti-neoplastic effects in TKI-resistant CML cells, both in patients with BCR-ABL1 T315I+CML and in various cell line models, including sub-clones harboring BCR-ABL1 T315I as single mutation or in compound configuration. In addition, we show that HU and ponatinib produce strong synergistic anti-neoplastic effects on TKI resistant CML cells, including sub-clones carrying T315I. These observations may have clinical implications and may pave the way for more effective sub-clone-eradicating but also palliative or bridging-to-SCT concepts in advanced CML. Clinical studies are now warranted to define the exact value of the drug combination ponatinib+HU in TKI-resistant CML. [Display omitted] DisclosuresSperr:Novartis: Honoraria; Amgen: Honoraria, Research Funding. Lion:Ariad: Honoraria; Amgen: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Hoermann:Ariad: Honoraria; Novartis: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Deininger:CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

Cantharidin Overcomes Imatinib Resistance by Depleting BCR-ABL in Chronic Myeloid Leukemia.

Cantharidin (CTD) is an active compound isolated from the traditional Chinese medicine blister beetle and displayed anticancer properties against various types of cancer cells. However, little is known about its effect on human chronic myeloid leukemia (CML) cells, including imatinib-resistant CML cells. The objective of this study was to investigate whether CTD could overcome imatinib resistance in imatinib-resistant CML cells and to explore the possible underlying mechanisms associated with the effect. Our results showed that CTD strongly inhibited the growth of both imatinib-sensitive and imatinib-resistant CML cells. CTD induced cell cycle arrest at mitotic phase and triggered DNA damage in CML cells. The ATM/ATR inhibitor CGK733 abrogated CTD-induced mitotic arrest but promoted the cytotoxic effects of CTD. In addition, we demonstrated that CTD downregulated the expression of the BCR-ABL protein and suppressed its downstream signal transduction. Real-time quantitative PCR revealed that CTD inhibited BCR-ABL at transcriptional level. Knockdown of BCR-ABL increased the cell-killing effects of CTD in K562 cells. These findings indicated that CTD overcomes imatinib resistance through depletion of BCR-ABL. Taken together, CTD is an important new candidate agent for CML therapy.

A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe.

Curcumin is extensively investigated as a good chemo-preventive agent in the development of many cancers and particularly in leukemia, including treatment of chronic myelogenous leukemia and it has been proposed as an adjuvant for leukemia therapies. Human chronic myeloid leukemia cells (K562), were treated with 20 μM of curcumin, and we found that a subpopulation of these cells were arrested and accumulate in the G2/M phase of the cell cycle. Characterization of this cell subpopulation showed that the arrested cells presented nuclear morphology changes resembling those described for mitotic catastrophe. Mitotic cells displayed abnormal chromatin organization, collapse of the mitotic spindle and abnormal chromosome segregation. Then, these cells died in an apoptosis dependent manner and showed diminution in the protein levels of BCL-2 and XIAP. Moreover, our results shown that a transient activation of the nuclear factor κB (NFκB) occurred early in these cells, but decreased after 6 h of the treatment, explaining in part the diminution of the anti-apoptotic proteins. Additionally, P73 was translocated to the cell nuclei, because the expression of the C/EBPα, a cognate repressor of the P73 gene, was decreased, suggesting that apoptosis is trigger by elevation of P73 protein levels acting in concert with the diminution of the two anti-apoptotic molecules. In summary, curcumin treatment might produce a P73-dependent apoptotic cell death in chronic myelogenous leukemia cells (K562), which was triggered by mitotic catastrophe, due to sustained BAX and survivin expression and impairment of the anti-apoptotic proteins BCL-2 and XIAP.

4-methylumbelliferone and imatinib combination enhances senescence induction in chronic myeloid leukemia cell lines.

Chronic myeloid leukemia (CML) is a myeloproliferative syndrome characterized by the presence of the Philadelphia chromosome which encodes a constitutively activated tyrosine kinase (BCR-ABL). The first line treatment for CML consists on BCR-ABL inhibitors such as Imatinib. Nevertheless, such treatment may lead to the selection of resistant cells. Therefore, it is of great value to find molecules that enhance the anti-proliferative effect of first-line drugs. Hyaluronan is the main glycosaminglican of the extracellular matrix which is involved in tumor progression and multidrug resistance. We have previously demonstrated that the inhibition of hyaluronan synthesis by 4-methylumbelliferone (4MU) induces senescence and can revert Vincristine resistance in CML cell lines. However, the effect of 4MU on Imatinib therapy remains unknown. The aim of this work was to determine whether the combination of 4MU with Imatinib is able to modulate the proliferation as well as apoptosis and senescence induction in human CML cell lines. For this purpose the ATCC cell line K562, and its multidrug resistant derivate, Kv562 were used. Cells were exposed to 4MU, Imatinib or a combination of both. We demonstrated that 4MU and Imatinib co-treatment abrogated the proliferation of both cell lines. However, such co-treatment did not increase the levels of apoptosis when compared with the treatment with Imatinib alone. For both cell lines the mechanisms of tumor suppression involved was senescence, since the combination of 4MU and Imatinib arrested the cell cycle and increased senescence associated β-galactosidase activity and senescence associated heterochromatin foci presence when compared to each drug alone. Moreover, 4MU, Imatinib and 4MU+Imatinib decreased pAkt/Akt ratio in both cell lines and reduced the pERK/ERK ratio only in K562 cells. These findings highlight the potential use of 4MU together with Imatinib for CML therapy.

Report on maloine, a new alkaloid discovered from G. maloi: Structural characterization and biological activity

Gymnospermium maloi Kit Tan, & Shuka is a new endemic species of the genus Gymnospermium Spach which has been described recently from the southern part of Albania. The members of this genus are poorly studied for what it concern the secondary metabolites in general and the class of alkaloids in particular. In fact from Gymnospermium genus, there are only few alkaloids characterized, (namely albertramine, albertidine, and albertine) isolated from G. albertii. Until now the chemical composition and the structure elucidation of other possible secondary metabolites, especially alkaloids, remain largely unknown. Here we report, for the first time, the structure of a new alkaloid isolated from G. maloi, designated by us as maloine, and obtained by the use of 2D homonuclear and heteronuclear NMR spectroscopy, FTIR, UV, Fluorescence and HPLC/MS spectra. The biological activity of the crude extract of Gymnospermium maloi and of its alkaloid maloine, was evaluated in vitro on human chronic myeloid leukemia cell line K562 and results herewith reported.

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.

Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments.

Design, synthesis and evaluation of the multidrug resistance-reversing activity of pyridine acid esters of podophyllotoxin in human leukemia cells

Multidrug resistance (MDR) is the main cause for chemotherapeutic failure in cancer treatment. To overcome MDR, a serious of pyridine acid esters of podophyllotoxin was synthesized and their antiproliferation activities were evaluated against two human chronic myeloid leukemia cell lines in vitro. Most of them exhibited potent growth inhibition with IC50 values in the nanomolar range as well as markedly reduced resistance factors. The most potent compound, Y8 exhibited an IC50 of 0.046±0.003μM against resistance K562/ADR cells, showing more significant than that of adriamycin and etoposide, respectively. Furthermore, Y8 efficiently triggered cell cycle arrest at S phase and simultaneously induced apoptosis in K562/ADR cells. Meanwhile, Y8 also regulated the expression levels of cell cycle- and apoptosis-related proteins. Additionally, Y8 stimulated the ERK1/2 signalling and reduced the expression of Pgp protein. Finally, on the basis of results obtained using U0126, an ERK1/2 inhibitor, the ERK1/2 signalling pathway was proposed for the multidrug resistance-reversing effect of Y8 in K562/ADR cells. Together, Y8 could be a novel potential MDR reversal agent for the treatment of drug-resistant leukemia.

Aromatic heterocyclic esters of podophyllotoxin exert anti-MDR activity in human leukemia K562/ADR cells via ROS/MAPK signaling pathways

Currently, multi-drug resistance (MDR) to antineoplastic drugs is a major obstacle to successful treatment of carcinoma. Looking for novel agents with anti-MDR activity is an effectively way to overcome cancer drug resistance. Our previous study showed that podophyllotoxin derivative exhibited potent anti-proliferative effect and down-regulated the expression level of P-gp in K562/ADR cells, which probably was related with the MAPK pathways. However, the relation of P-gp expression and MAPK pathways still remains unclear. In this study, a series of aromatic heterocyclic esters of podophyllotoxin were synthesized and their anticancer effects were evaluated against two human chronic myeloid leukemia cell lines (K562 and K562/ADR), simultaneously, the initial structure-activity relationship was summarized. The most potent compound, Z5, displayed an IC50 value of 0.032 ± 0.006 μM against K562/ADR cells, with a lower resistant factor value of 1.280. Treatment of K562/ADR cells with Z5 caused S cell cycle arrest through reductions in cyclinA, cyclinB1, CDK1 and CDK2 levels. Moreover, Z5 treatment resulted in the induction of apoptosis as characterized by DNA staining, flow cytometry analysis and cleavage of procaspases-3, -8, -9 and PARP. Notably, Z5 significantly inhibited P-gp expression in K562/ADR cells. Additionally, Z5 also caused reactive oxygen species (ROS) generation, which was further demonstrated by preincubation with the antioxidant N-acetylcysteine (NAC). Western blotting revealed that Z5 markedly stimulated the MAPK pathways, including ERK1/2, JNK and P38, however, the mechanisms were prevented by NAC. Finally, the employment of NAC and MAPK inhibitors (U0126, SP600125 and SB203580) remarkably blocked the S phase arrest, apoptosis and down-regulation of P-gp induced by Z5. Taken together, Z5 strongly possessed the potential anti-MDR activity in K562/ADR cells through ROS/MAPK pathways-dependent S phase arrest, apoptosis and down-regulation of P-gp expression.

Reprogramming of Notch1-induced acute lymphoblastic leukemia cells into pluripotent stem cells in mice

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells using the reprogramming factors (Oct4, Sox2, Klf4 and c-Myc, also called OSKM).1 Cellular reprogramming and oncogenesis share many common features. The application of the iPS technology in cancers help us better understand the mechanism underlying the initiation and progression of cancer. Therefore, defining the reprogramming potential of cancer cells would provide unique opportunities to reveal epigenetic mechanisms and develop novel therapeutics for cancer. Because the reprogramming efficiency of cancer cells is paradoxically much lower than that of normal cells in general, only some handful types of cancer cells have been explored using the iPS technology.2 To date, in the hematopoietic system, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, human chronic myeloid leukemia cells, juvenile myelomonocytic leukemia cells and primary murine mixed lineage leukemia-AF9 acute myeloid leukemia cells have been successfully generated into iPS cells.3, 4, 5, 6 However, whether the primary malignant leukemic T cells can be reprogrammed into the iPS cells is still a mystery.

Effect of PKF118-310 on apoptosis of leukemia K562 cells and its mechanism

Objective To investigate the effect of small molecule antagonists PKF118-310 on apoptosis of human chronic myeloid leukemia cell line K562 and its mechanism. Methods After the treatment of PKF118-310 at different concentration, the proliferation inhibition in K562 cells was detected by MTT, the β-catenin/TCF transcription complex in the nucleus was observed by immunofluorescence, the apoptosis was detected by flow cytometry, and the expressions of caspase-3, caspase-8, XIAP, bcl-2, β-catenin, TCF, c-myc and cyclin D1 were detected by Western blot. Results PKF118-310 inhibited the proliferation of K562 cells. The median inhibitory concentration (IC50) of PKF118-310-treated K562 cells for 24 h, 48 h and 72 h were 5.388, 3.290, 1.566 μmol/L, respectively. The β-catenin/TCF transcription complex in the nucleus was observed. After the treatment with 1.6 and 3.2 μmol/L PKF118-310 for 48 h, the apoptosis rates of K562 cells were (48.0±0.9) % and (80.2±1.2) %, respectively, and were higher than that in the control group [(1.2±0.6) %] (both P < 0.05). After the treatment of K562 cells with PKF118-310 at different concentration for 72 h, the expression levels of caspase-3 and caspase-8 were increased (both P < 0.05), and the expression levels of XIAP, bcl-2, β-catenin, TCF, c-myc and cyclin D1 were significantly decreased compared with control group (all P< 0.05). Conclusion PKF118-310 can inhibit K562 cells'proliferation, and induce the apoptosis. Key words: Leukemia, myeloid, chronic; K562 cells; Wnt/β-catenin signaling pathway; PKF118-310; Apoptosis

Part-1: Design, synthesis and biological evaluation of novel bromo-pyrimidine analogs as tyrosine kinase inhibitors

A novel series of 5-bromo-pyrimidine derivatives (5a-l, 6a-h, 9a-m and 10a-d) were synthesized through multi step reactions starting from 5-bromo-2,4-dichloro pyrimidine. The newly synthesized compounds were characterized using elemental analysis and spectral data (IR, 1H NMR, 13C NMR and LC-MS) analysis. The titled compounds were evaluated for their in vitro cytotoxic activity against tumor cell lines panel consisted of HCT116 (human colon cancer cell line), A549 (human lung cancer cell line), K562 (human chronic myeloid leukemia cell line), U937 (human acute monocytic myeloid leukemia cell line), and L02 (human normal cell line) by using MTT assay Mosmann's method. As most of the compounds are highly potent against K562 cells, all the synthesized compounds were evaluated for Bcr/Abl tyrosine kinase inhibitory activity by using well-established ADP-Glo assay method. Dasatinib was utilized as positive control to validate in both biological evaluations.The biological activity revealed that the compounds 5c, 5e, 6g, 9e, 9f and 10c were potent Bcr/Abl kinase inhibitors among the titled compounds. Thus these compounds may be promising lead compounds to be developed as an alternative for current Dasatinib therapy.

Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells.

Although dasatinib is effective in most imatinib mesylate (IMT)-resistant chronic myeloid leukemia (CML) patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT). Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN) was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

Use of deferasirox, an iron chelator, to overcome imatinib resistance of chronic myeloid leukemia cells.

Background/Aims:The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies.Methods:We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib.Results:Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-κB (NF-κB) and β-catenin.Conclusions:We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-κB and β-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML.

Abstract 46: Functional analysis of long noncoding RNAs in Bcr-Abl-mediated tumorigenesis

Abstract Human genome is pervasively transcribed and the majority does not have apparent protein coding potential, which is defined as noncoding RNA (ncRNA). Long noncoding RNAs (lncRNAs) are greater than 200 nucleotides in length, transcribed by RNA polymerase II or III, and poorly conserved. Aberrant expression of numerous functional lncRNAs is associated with various human cancers. Chronic myeloid leukemia (CML) is a hematologic malignancy associated with a translocation between chromosome 9 and 22 that results in the formation of bcr-abl hybrid gene which occurs in over 90% of CML cases. However, the role of lncRNAs in Bcr-Abl-mediated chronic myeloid leukemia (CML) is not fully understood. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using a microarray of cDNAs encoding lncRNAs. The microarray analysis revealed 338 upregulated lncRNAs and 108 downregulated lncRNAs after silencing Bcr-Abl in human CML cells. To identify functional lncRNAs that are important in Bcr-Abl-mediated transformation, we selected the most significantly affected lncRNAs for further studies. These lncRNAs were examined by ORF finder from NCBI and protein-coding potential was determined. We identified several lncRNAs that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Expression of these lncRNAs was further examined in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Experiments including ectopic overexpression or knockdown of these lncRNAs were performed to address the role and mechanism of how these lncRNAs function in the leukemic cells. Together, these results reveal that several lncRNAs are critically involved in Bcr-Abl-mediated cellular transformation and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia. Note: This abstract was not presented at the conference. Citation Format: Guijie Guo, Xuefei Wang, Jilong Chen. Functional analysis of long noncoding RNAs in Bcr-Abl-mediated tumorigenesis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 46.

Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line.

First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro transfection results from BCPV-siRNA, a newly developed biodegradable transfection agent, BCPV, is being probed for transfection performance in an animal model.

Arene ruthenium(ii) complexes with chalcone, aminoantipyrine and aminopyrimidine based ligands: synthesis, structure and preliminary evaluation of anti-leukemia activity

A series of arene ruthenium(ii) complexes withN-monodentate (AAP) andN,O- andN,N-bidentate chelating ligands (AAPS, ADABS, AAPPA and P2P) have been synthesized and evaluated for preliminary antileukemia activity against K562 (Human chronic myeloid leukemia cell line).

Quantitative Proteomics Analysis of Leukemia Cells.

Chronic myeloid leukemia (CML) is driven by the oncogenic fusion kinase Bcr-Abl, which organizes its own signaling network with various proteins. These proteins, their interactions, and their role in relevant signaling pathways can be analyzed by quantitative mass spectrometry (MS) approaches in various models systems, e.g., in cell culture models. In this chapter, we describe in detail immunoprecipitations and quantitative proteomics analysis using stable isotope labeling by amino acids in cell culture (SILAC) of components of the Bcr-Abl signaling pathway in the human CML cell line K562.

Blockade of the interaction between Bcr-Abl and PTB1B by small molecule SBF-1 to overcome imatinib-resistance of chronic myeloid leukemia cells

In this study, a synthetic steroidal glycoside SBF-1 had strong and preferential antitumor effects on the human chronic myeloid leukemia (CML) cell line K562 and its imatinib-resistant form K562/G. SBF-1 induced apoptosis in both cell lines without any effect on cell cycle arrest. It also inhibited the activation of PI3K/Akt pathway members, such as PI3K and Akt, as well as downstream targets mTOR and Bcl-2. Moreover, the degradation of the Bcr-Abl protein was induced by SBF-1 in a concentration- and time-dependent manner. Using a pull-down assay, SBF-1 was found to bind to both Bcr-Abl and PTP1B and disrupted the interaction between them. SBF-1 triggered the degradation of Bcr-Abl through ubiquitination via the lysosome pathway. Taking together these findings, this study, for the first time, suggests that the blockade of the interaction between Bcr-Abl and PTP1B may be a feasible strategy for the treatment of CML, especially CML with resistance to Bcr-Abl kinase inhibitor imatinib. Our study also indicates that SBF-1 may serve as a leading compound for novel anti-CML therapeutic agents.

Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells

Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis.