Imatinib-resistant Chronic Myeloid Leukemia Cells Research Articles

CircRNA Microarray Profiling Reveals hsa_circ_0058493 as a Novel Biomarker for Imatinib-Resistant CML

Background: CircRNA has appeared as a critical molecular in the development of various cancers. However, the cellular function of circRNAs and exosomal circRNAs has not been well explored in Chronic myeloid leukemia (CML). Methods: Differentially expressed circRNAs were identified by a human circRNA microarray analysis. The expression of hsa_circ_0058493 in peripheral blood mononuclear cells (PBMCs) and exosomes was verified using quantitative real-time PCR. Short hairpin RNAs against hsa_circ_0058493 were constructed to silence the expression of circ_0058493. CCK8, flow cytometry and EdU assay were performed to investigate the biological functions of circ_0058493. Results: Hsa_circ_0058493 was significantly overexpressed in the PBMCs of CML patients and high level of circ_0058493 was associated with the poor clinical efficacy of imatinib. Silencing the expression of circ_0058493 significantly inhibited the development of imatinib-resistant CML cells. miR-548b-3p was overexpressed in circ_0058493-downregulated CML cells. Bioinformatic analysis revealed that circ_0058493 might exert its regulatory function acting as a “sponge” of miR-548b-3p. Moreover, hsa_circ_0058493 was significantly enriched in the exosomes derived from imatinib-resistant CML cells. Conclusion: Hsa_circ_0058493 in PBMCs could be a promising prognostic biomarker and might provide a therapeutic target for CML treatment.

Vitamin K3 (menadione) Induces Cytotoxicity in Chronic Myeloid Leukemia Stem Cells By Upregulating DYRK2 through the Inhibition of SIAH2 Ubiquitin Ligase

Leukemic stem cells (LSCs) generated by the transformation of normal hematopoietic stem cells (HSCs) by BCR-ABL are elusive targets that can initiate and sustain leukemia owing to their unique capacity to regenerate themselves during cell division (self-renewal) and differentiate into a bulk of leukemia cells. Although chronic myeloid leukemia (CML) can be successfully managed using tyrosine kinase inhibitors (TKIs), drug discontinuation trials have shown that 30-60% of patients, depending on the drug used for first- and second-line treatment, can remain in treatment-free remission. Thus, discontinuation is still considered experimental and there are not safety guidelines to stop treatment. A better understanding of self-renewal mechanisms in LSCs will support the development of LSC-specific drugs to induce treatment-free remission in chronic-phase patients by eradicating BCR-ABL-positive LSCs. We recently discovered in mouse models that deletion of the Klf4 gene encoding for the transcription factor KLF4 severely abrogated progression of BCR-ABL(p210)-induced CML by impairing survival and self-renewal of LSCs but not normal hematopoietic stem cells (HSCs). Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated DYRK2 protein levels, which was associated with p53-mediated apoptosis and inhibition of self-renewal via depletion of c-Myc protein in LSCs and not in normal HSCs. Here, we report that this mechanism is active in bone marrow cells from chronic-phase CML patients and that stabilization of DYRK2 protein by inhibition of the ubiquitin E3 ligase with vitamin K3 (VK3) has the potential therapeutic approach. Treatment of CML cell lines and bone marrow cells from CML patients with VK3 induced dose-dependent cytotoxicity and apoptosis. This was correlated with increased DYRK2 expression, depletion of c-Myc, and activation of p53-mediated apoptosis. Most importantly, VK3 induced DYRK2 expression and abrogated capacity of purified CD34+ CML stem cells, but not normal CD34+ cells, to generate colonies in methylcellulose assays. Cytotoxicity of VK3 largely depended on the expression of DYRK2, the reduced form menadiol inhibited cell viability similarly to menadione (VK3), and the presence of p53 enhanced cytotoxicity by inducing apoptosis in addition to inhibition of self-renewal. VK3 augmented cytotoxicity of Imatinib in CML cell lines and inhibited cell survival in Imatinib-resistant CML cells. The ability to target CML LSCs was confirmed by in vivo administration of VK3 in BCR-ABL induced mouse model followed by transplantation of leukemic bone marrow in secondary recipient mice. Altogether, our results suggest that stabilization of DYRK2 by inhibition of SIAH2 can induce apoptosis in bulk leukemia cells and LSCs by activating p53 and depleting c-Myc. DisclosuresNo relevant conflicts of interest to declare.

Targeting HSPA8 inhibits proliferation via downregulating BCR-ABL and enhances chemosensitivity in imatinib-resistant chronic myeloid leukemia cells

The resistance to tyrosine kinase inhibitors is currently a major problem for chronic myeloid leukemia (CML) treatment and HSPA8 is highly expressed and a hallmark of poor prognosis in several human cancers. However, its role in imatinib-resistant CML (IR-CML) cells remains undetermined. Here, we determined HSPA8 was overexpressed in IR-CML cells and associated with imatinib resistance. HSPA8 ablation could downregulate BCR-ABL/STAT5 and BCR-ABL/AKT signaling pathways, dramatically induce proliferation inhibition, autophagy, G0/G1 phase cell cycle arrest but not apoptosis in IR-CML cells. Significantly, HSPA8 ablation enhanced the antitumor activity of imatinib via promoting apoptosis in vitro and vivo. These findings unraveled that HSPA8 ablation inhibits proliferation via downregulating BCR-ABL and enhances chemosensitivity of imatinib in IR-CML cells, which investigate the role and molecular mechanism of HSPA8 in IR-CML cells and suggest that HSPA8 may be a potential target for IR-CML treatment.

Altered apoptotic protein expressions characterize the survival of Bcr-Abl-independent drug-resistant chronic myeloid leukemia cell line

Apoptosis is a programmed cellular process that occurs in pathological and physiological pathways and it is one of the most studied topics in cell biology. To understand the underlying mechanism of apoptosis plays an important role in the molecular pathogenesis of many diseases including cancers. Chronic myeloid leukemia (CML) is a clonal myeloproliferative malignancy arising from the neoplastic transformation of the hematopoietic stem cell. Here, we used a Bcr-Abl-independent, imatinib-resistant K562 subpopulation (K562-IR) generated and characterized earlier in our laboratory. We showed that the proteins Bcl-2, Bim, RIP, p-MAPK(Erk1/Erk2) and NF-кB which play critical roles in cell death pathways are downregulated, Lamin A/C protein expression is upregulated in K562-IR derivative cells compared to K562 ancestral cells. Our data provide new information on the expression of apoptotic molecules in a Bcr-Abl-independent imatinib-resistant CML cell line.

Metabolic Plasticity Is an Essential Requirement of Acquired Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.

Simple SummaryTyrosine kinase inhibitors (TKIs), such as imatinib, have become the standard initial treatment of choice for chronic myeloid leukemia (CML) patients. However, one obstacle to face is that a significant proportion of patients presents poor response to TKIs, or acquires resistance resulting in disease relapses. Mutations in BCR-ABL1 protein are a well described mechanism of resistance but other not well established mechanisms outside BCR-ABL1 mutations are emerging as important in the acquisition of resistance. Abnormal metabolism of CML cells that acquire resistance to imatinib has been pointed out as a putative downstream key event, but deep studies aimed to unveil metabolic adaptations associated with acquired resistance are still lacking. Here, we perform an exhaustive study on metabolic reprogramming associated with acquired imatinib resistance and we identify metabolic vulnerabilities of CML imatinib resistant cells that could pave the way for new therapies targeting TKI failure. Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.

Effects of Twist1 on drug resistance of chronic myeloid leukemia cells through the PI3K/AKT signaling pathway

This study aimed to investigate the effects of Twist1 on the drug resistance of chronic myeloid leukemia (CML) cells through the PI3K/AKT signaling pathway. K562 and KCL-22 cells were modeled for imatinib resistance, so as to analyze the effects of inhibiting Twist1 and the pathway on the therapeutic effect of imatinib on imatinib-resistant CML cells, and to find the mechanism of action of Twist1 on affecting the resistance. After the CML cells were successfully resistant to imatinib, Twist1 expression increased again in the cells and the PI3K/AKT signaling pathway was further activated. After the silence of the Twist1 expression, the imatinib-resistant CML cells were more sensitive to imatinib, and the PI3K/AKT signaling pathway was inhibited, and the expression level of p-AKT protein significantly reduced. According to further experiments, imatinib enhanced its inhibitory effect on the growth of the imatinib-resistant CML cells after the activation of the pathway was inhibited by an LY3023414 inhibitor. In conclusion, Twist1 and the PI3K/AKT signaling pathway are over-activated during the formation of the CML cells resistant to imatinib. The silence of Twist1 can reverse the resistance through the pathway.

Cross-talk between Bcr-abl and the Thioredoxin System in Chronic Myeloid Leukaemia: Implications for CML Treatment.

Chronic myeloid leukaemia (CML) is currently treated with inhibitors of the CML specific oncoprotein, bcr-abl. While this strategy is initially successful, drug resistance can become a problem. Therefore, new targets need to be identified to ensure the disease can be appropriately managed. The thioredoxin (Trx) system, comprised of Trx, thioredoxin reductase (TrxR), and NADPH, is an antioxidant system previously identified as a target for therapies aimed at overcoming drug resistance in other cancers. We assessed the effectiveness of TrxR inhibitors on drug resistant CML cells and examined links between TrxR and the bcr-abl cell-signalling pathway. Two TrxR inhibitors, auranofin and [Au(d2pype)2]Cl, increased intracellular ROS levels and elicited apoptosis in both sensitive and imatinib resistant CML cells. Inhibition of TrxR activity by these pharmacological inhibitors, or by specific siRNA, also resulted in decreased bcr-abl mRNA and protein levels, and lower bcr-abl downstream signalling activity, potentially enhancing the effectiveness of TrxR inhibitors as CML therapies. In addition, imatinib resistant CML cell lines showed upregulated expression of the Trx system. Furthermore, analysis of datasets showed that CML patients who did not respond to imatinib had higher Trx mRNA levels than patients who responded to treatment. Our study demonstrates a link between the Trx system and the bcr-abl protein and highlights the therapeutic potential of targeting the Trx system to improve CML patients’ outcomes.

Lyn regulates creatine uptake in an imatinib-resistant CML cell line

BackgroundImatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells. MethodsStable isotope metabolic labeling, media creatine depletion, and Na+/K+-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na+/K+-ATPase and creatine uptake. ResultsInhibition of the Na+/K+-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na+/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na+/K+-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na+/K+-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na+/K+-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells. ConclusionsThese data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity. General significanceThese studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.

Downregulation of the histone methyltransferase SETD2 promotes imatinib resistance in chronic myeloid leukaemia cells.

ObjectivesEpigenetic modifiers were important players in the development of haematological malignancies and sensitivity to therapy. Mutations of SET domain‐containing 2 (SETD2), a methyltransferase that catalyses the trimethylation of histone 3 on lysine 36 (H3K36me3), were found in various myeloid malignancies. However, the detailed mechanisms through which SETD2 confers chronic myeloid leukaemia progression and resistance to therapy targeting on BCR‐ABL remain unclear.Materials and methodsThe level of SETD2 in imatinib‐sensitive and imatinib‐resistant chronic myeloid leukaemia (CML) cells was examined by immunoblotting and quantitative real‐time PCR. We analysed CD34+CD38− leukaemic stem cells by flow cytometry and colony formation assays upon SETD2 knockdown or overexpression. The impact of SETD2 expression alterations or small‐molecule inhibitor JIB‐04 targeting H3K36me3 loss on imatinib sensitivity was assessed by IC50, cell apoptosis and proliferation assays. Finally, RNA sequencing and ChIP‐quantitative PCR were performed to verify putative downstream targets.ResultsSETD2 was found to act as a tumour suppressor in CML. The novel oncogenic targets MYCN and ERG were shown to be the direct downstream targets of SETD2, where their overexpression induced by SETD2 knockdown caused imatinib insensitivity and leukaemic stem cell enrichment in CML cell lines. Treatment with JIB‐04, an inhibitor that restores H3K36me3 levels through blockade of its demethylation, successfully improved the cell imatinib sensitivity and enhanced the chemotherapeutic effect.ConclusionsOur study not only emphasizes the regulatory mechanism of SETD2 in CML, but also provides promising therapeutic strategies for overcoming the imatinib resistance in patients with CML.

USP10 modulates the SKP2/Bcr-Abl axis via stabilizing SKP2 in chronic myeloid leukemia

Constitutive activation of tyrosine kinase Bcr-Abl is the leading cause of the development and progression of chronic myeloid leukemia (CML). Currently, the application of tyrosine kinase inhibitors (TKIs) targeting the Bcr-Abl is the primary therapy for CML patients. However, acquired resistance to TKIs that develops overtime in the long-term administration renders TKIs ineffective to patients with advanced CML. Therefore, increasing studies focus on the amplified expression or activation of Bcr-Abl which is proposed to contribute to the advanced phase. Here, we show that S-phase kinase-associated protein 2 (SKP2) acts as a co-regulator of Bcr-Abl by mediating its K63-linked ubiquitination and activation. Further investigations show that USP10 as a novel deubiquitinase of SKP2 amplifies the activation of Bcr-Abl via mediating deubiquitination and stabilization of SKP2 in CML cells. Moreover, inhibition of USP10 significantly suppresses the proliferation of both imatinib-sensitive and imatinib-resistant CML cells, which likely depends on SKP2 status. This findings are confirmed in primary CML cells because these cells are over-expressed with USP10 and SKP2 and are sensitive to a USP10 inhibitor. Taken together, the present study not only provides a novel insight into the amplified activation of Bcr-Abl in CML, but also demonstrates that targeting the USP10/SKP2/Bcr-Abl axis is a potential strategy to overcome imatinib resistance in CML patients.

Andrographolide and its potent derivative exhibit anticancer effects against imatinib-resistant chronic myeloid leukemia cells by downregulating the Bcr-Abl oncoprotein

Chronic myelogenous leukemia (CML) is clinically treated with imatinib, which inhibits the kinase activity of the Bcr-Abl oncoprotein. However, imatinib resistance remains a common clinical issue. Andrographolide, the major compound of the medicinal plant Andrographis paniculata, was reported to exhibit anticancer activity. In this study, we explored the therapeutic potential of andrographolide and its derivative, NCTU-322, against both imatinib-sensitive and imatinib-resistant human CML cell lines. Both andrographolide and NCTU-322 downregulated the Bcr-Abl oncoprotein in imatinib-resistant CML cells through an Hsp90-dependent mechanism similar to that observed in imatinib-sensitive CML cells. In addition, NCTU-322 had stronger effects than andrographolide on downregulation of Bcr-Abl oncoprotein, induction of Hsp90 cleavage and cytotoxicity of CML cells. Notably, andrographolide and NCTU-322 could induce differentiation, mitotic arrest and apoptosis of both imatinib-sensitive and imatinib-resistant CML cells. Finally, the anticancer activity of NCTU-322 against imatinib-resistant CML cells was demonstrated in vivo. In summary, our data demonstrated that andrographolide and NCTU-322 inhibit Bcr-abl function via a mechanism different from that of imatinib, and they induced multiple anticancer effects in both imatinib-sensitive and resistant CML cells. Our findings demonstrate that andrographolide and NCTU-322 are potential therapeutic agents again CML.

(-)-Epigallocatechin-3-gallate induces cell apoptosis in chronic myeloid leukaemia by regulating Bcr/Abl-mediated p38-MAPK/JNK and JAK2/STAT3/AKT signalling pathways.

Epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent of green tea, possesses remarkable chemopreventive and therapeutic potential against various types of cancer, including leukaemia. However, the molecular mechanism involved in chronic myeloid leukaemia (CML), especially imatinib-resistant CML cells, is not completely understood. In the present study, we investigated the effect of EGCG on the growth of Bcr/Abl+ CML cell lines, including imatinib-resistant cell lines and primary CML cells. The results revealed that EGCG could inhibit cell growth and induce apoptosis in CML cells. The mechanisms involved inhibition of the Bcr/Abl oncoprotein and regulation of its downstream p38-MAPK/JNK and JAK2/STAT3/AKT pathways. In conclusion, we documented the anti-CML effects of EGCG in imatinib-sensitive and imatinib-resistant Bcr/Abl+ cells, especially T315I-mutated cells.

Overexpression of miR-202 resensitizes imatinib resistant chronic myeloid leukemia cells through targetting Hexokinase 2.

Chronic myeloid leukemia (CML) is a myeloproliferative disease which uniquely expresses a constitutively active tyrosine kinase, BCR/ABL. As a specific inhibitor of the BCR-ABL tyrosine kinase, imatinib becomes the first choice for the treatment of CML due to its high efficacy and low toxicity. However, the development of imatinib resistance limits the long-term treatment benefits of it in CML patients. In the present study, we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell lines and the possible mechanisms involved in this process. We found miR-202 was down-regulated in seven CML cell lines by quantitative reverse-transcription PCR (qRT-PCR) analysis. Overexpression of miR-202 significantly suppressed proliferation rates of CML cells. By establishing imatinib resistant cell lines originating from K562 and KU812 cells, we observed expressions of miR-202 were down-regulated by imatinib treatments and imatinib resistant CML cell lines exhibited lower level of miR-202. On the contrary, imatinib resistant CML cell lines displayed up-regulated glycolysis rate than sensitive cells with the evidence that glucose uptake, lactate production, and key glycolysis enzymes were elevated in imatinib resistant cells. Importantly, the imatinib resistant CML cell lines were more sensitive to glucose starvation and glycolysis inhibitors. In addition, we identified Hexokinase 2 (HK2) as a direct target of miR-202 in CML cell lines. Overexpression of miR-202 sensitized imatinib resistant CML through the miR-202-mediated glycolysis inhibition by targetting HK2. Finally, we provided the clinical relevance that miR-202 was down-regulated in CML patients and patients with lower miR-202 expression displayed higher HK2 expression. The present study will provide new aspects on the miRNA-modulated tyrosine kinase inhibitor (TKI) sensitivity in CML, contributing to the development of new therapeutic anticancer drugs.

Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases

BackgroundThe bcr-abl fusion gene is the pathological origin of chronic myeloid leukemia (CML) and plays a critical role in the resistance of imatinib. Thus, bcr-abl disruption-based novel therapeutic strategy may warrant exploration. In our study, we were surprised to find that the characteristics of bcr-abl sequences met the design requirements of zinc finger nucleases (ZFNs).MethodsWe constructed the ZFNs targeting bcr-abl with high specificity through simple modular assembly approach. Western blotting was conducted to detect the expression of BCR-ABL and phosphorylation of its downstream STAT5, ERK and CRKL in CML cells. CCK8 assay, colony-forming assay and flow cytometry (FCM) were used to evaluate the effect of the ZFNs on the viablity and apoptosis of CML cells and CML CD34+ cells. Moreover, mice model was used to determine the ability of ZFNs in disrupting the leukemogenesis of bcr-abl in vivo.ResultsThe ZFNs skillfully mediated 8-base NotI enzyme cutting site addition in bcr-abl gene of imatinib sensitive and resistant CML cells by homology-directed repair (HDR), which led to a stop codon and terminated the translation of BCR-ABL protein. As expected, the disruption of bcr-abl gene induced cell apoptosis and inhibited cell proliferation. Notably, we obtained similar result in CD34+ cells from CML patients. Moreover, the ZFNs significantly reduced the oncogenicity of CML cells in mice.ConclusionThese results reveal that the bcr-abl gene disruption based on ZFNs may provide a treatment choice for imatinib resistant or intolerant CML patients.

Antitumor Effects of Blocking Protein Neddylation in T315I-BCR-ABL Leukemia Cells and Leukemia Stem Cells.

Imatinib revolutionized the treatment of chronic myeloid leukemia (CML), but drug resistance and disease recurrence remain a challenge. In this study, we suggest a novel strategy based on blocking protein neddylation to address BCR-ABL point mutations and leukemia stem cells (LSC) that lie at the root of imatinib-resistant recurrences. On the basis of the finding that the NEDD8-activating enzyme subunit NAE1 is overexpressed in CML cells, we hypothesized that the function of certain neddylation-dependent protein substrates might be targeted to therapeutic ends in imatinib-resistant CML cells and LSCs. In support of this hypothesis, we demonstrated that the NAE1 inhibitor MLN4924 induced G2-M-phase arrest and apoptosis in bulk CML cells with wild-type p53, regardless of their T315I mutation status in BCR-ABL. Moreover, MLN4924 inhibited the survival and self-renewal of primary human CML CD34+ cells and LSCs in CML-bearing mice via accumulation of p27kip1 in the nucleus. Notably, p27kip1 silencing attenuated the suppressive effect of MLN4924 on the maintenance of LSCs in CML-bearing mice. Taken together, our findings offer a preclinical proof of concept for targeting protein neddylation as a novel therapeutic strategy to override mutational and LSC-derived imatinib resistance in CML.Significance: These findings highlight a mediator of protein neddylation, a type of protein turnover mechanism, as a viable therapeutic target against imatinib-resistant forms of chronic myelogenous leukemia. Cancer Res; 78(6); 1522-36. ©2018 AACR.

Exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediate a horizontal transfer of drug-resistant trait by delivering miR-365

Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem/progenitor cells. Majority of patients can be effectively treated with tyrosine kinase inhibitors (TKIs) such as imatinib, but a portion of patients will develop drug resistance. Accumulated evidences have identified exosomes in cancer as promoters of tumor progression. Herein, we found that exosomes derived from imatinib resistant CML cells can be internalized into sensitive CML cells and confer drug-resistance traits. We also demonstrated a significant higher level of miR-365 in exosomes derived from drug-resistant CML cells compared with those from sensitive ones using microarray and qRT-PCR. The imatinib sensitive CML cells transfected with pre-miR-365 displayed lower chemosensitivity and apoptosis rate compared with controls. We further confirmed that exosomal transfer of miR-365 induced drug resistance by inhibiting expression of pro-apoptosis protein in sensitive CML cells. In conclusion, our study reveals that exosomes mediate a horizontal transfer of drug-resistant trait in chronic myeloid leukemia cell by delivering miR-365.

Correction: MPT0B169, a New Antitubulin Agent, Inhibits Bcr-Abl Expression and Induces Mitochondrion-Mediated Apoptosis in Nonresistant and Imatinib-Resistant Chronic Myeloid Leukemia Cells.

[This corrects the article DOI: 10.1371/journal.pone.0148093.].

Comparative effect of imatinib and ponatinib on autophagy and miRNome in chronic myeloid leukemia

BCR-ABL tyrosine kinase inhibitors (TKIs) are selective therapies for the patients with Chronic Myeloid Leukemia (CML). Imatinib and ponatinib have remarkable long-term efficacy on a major molecular response. Although TKI related induction of cytotoxicity and apoptosis have been clearly investigated in molecular levels, their comparative effect on autophagy and miRNome are largely unknown. This study aimed to investigate the involvement of alterations of miRNA expressions in CML progression, and how imatinib and ponatinib affect this process, by comparing CML, imatinib-resistant CML and leukemia stem cells (LSC). Cytotoxicity analysis was conducted by WST-1, apoptosis was evaluated by AnnexinV, autophagy was analyzed by Tb/GFP TR-FRET LC3B assay and changes in miRNomes were evaluated with microarray method. Ponatinib showed higher cytotoxicity and apoptosis at far fewer concentrations than imatinib. Both imatinib and ponatinib was able to trigger autophagy in imatinib-resistant K562ima3 cell line but not in LSC. We pointed that imatinib and ponatinib caused significant miRNA profile alterations, especially in the expressions of miR-214-pre, miR-218, miR-19a-5p, miR-19b-1-5p, miR-27b-pre, miR-23b-pre, miR-320e, miR-200a-pre, miR-508-3p, miR-33-pre and miR-766. This study is the first comparative miRNome analysis of CML, resistant CML and LSCs following the imatinib or ponatinib treatment and may guide to identify new markers for diagnosis, follow-up of the disease and to develop novel therapeutic strategies if supported by preclinical studies.

Erratum to: A novel tubulin polymerization inhibitor, MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells.

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.

Time-series analysis in imatinib-resistant chronic myeloid leukemia K562-cells under different drug treatments.

SummaryChronic myeloid leukemia (CML) is characterized by the accumulation of active BCR-ABL protein. Imatinib is the first-line treatment of CML; however, many patients are resistant to this drug. In this study, we aimed to compare the differences in expression patterns and functions of time-series genes in imatinib-resistant CML cells under different drug treatments. GSE24946 was downloaded from the GEO database, which included 17 samples of K562-r cells with (n=12) or without drug administration (n=5). Three drug treatment groups were considered for this study: arsenic trioxide (ATO), AMN107, and ATO+AMN107. Each group had one sample at each time point (3, 12, 24, and 48 h). Time-series genes with a ratio of standard deviation/average (coefficient of variation) >0.15 were screened, and their expression patterns were revealed based on Short Time-series Expression Miner (STEM). Then, the functional enrichment analysis of time-series genes in each group was performed using DAVID, and the genes enriched in the top ten functional categories were extracted to detect their expression patterns. Different time-series genes were identified in the three groups, and most of them were enriched in the ribosome and oxidative phosphorylation pathways. Time-series genes in the three treatment groups had different expression patterns and functions. Time-series genes in the ATO group (e.g. CCNA2 and DAB2) were significantly associated with cell adhesion, those in the AMN107 group were related to cellular carbohydrate metabolic process, while those in the ATO+AMN107 group (e.g. AP2M1) were significantly related to cell proliferation and antigen processing. In imatinib-resistant CML cells, ATO could influence genes related to cell adhesion, AMN107 might affect genes involved in cellular carbohydrate metabolism, and the combination therapy might regulate genes involved in cell proliferation.