Imatinib-resistant Chronic Myeloid Leukemia Cells Research Articles

"Combination treatments of imatinib with astaxanthin and crocin efficiently ameliorate antioxidant status, inflammation and cell death progression in imatinib-resistant chronic myeloid leukemia cells".

Imatinib resistance remains a major obstacle in the treatment of chronic myelogenous leukemia (CML). Crocin (CRC) and astaxanthin (ATX) are phytochemicals with anti-cancer properties. This study aimed to explore the effects of combination treatment of Imatinib with CRC and ATX on Imatinib-resistant K562 (IR-K562) cells. After the establishment of IR-K562 cells, growth inhibitory activity was determined by the MTT assay. To test the regeneration potential, a colony formation assay was performed. Cell cycle analyses were examined by flow cytometry. Cell injury was evaluated by lactate dehydrogenase (LDH) leakage. Real-time PCR was applied to assess the expression of IL6, TNF-α, STAT3, BAD, CASP3, TP53, and Bcl-2 genes. Caspase-3 activity was determined by a colorimetric assay. Antioxidant activity was measured using a diphenylpicrylhydrazyl (DPPH) assay. After 48h of treatment, ATX (IC50 = 30µM) and CRC (IC50 = 190µM) significantly inhibited cell proliferation and colony formation ability, induced G1 cell cycle arrest and cell injury, upregulated the expression of apoptosis-associated genes, and downregulated the expression of anti-apoptotic and inflammatory genes. The combination of IM with ATX and/or CRC synergistically reduced cell viability (combination index [CI] < 1). Our data suggest that IM shows better therapeutic efficacy at lower doses when combined with ATX and/or CRC.

Isoalantolactone mediates the degradation of BCR-ABL protein in imatinib-resistant CML cells by down-regulating survivin

ABSTRACT Isoalantolactone (Iso) is a bioactive lactone isolated from the root of Inula helenium L, which has been reported to have many pharmacological effects. To investigate the role and mechanism of isoalantolactone in chronic myeloid leukemia (CML), we first investigated isoalantolactone’s anti-proliferative effects on imatinib-sensitive and imatinib-resistant CML cells by CCK8. Flow cytometry was used to detect isoalantolactone-induced cell apoptosis. Survivin was overexpressed in KBM5 and KBM5T315I cells using the lentivirus vector pSIN-3×flag-PURO. In KBM5 and KBM5T315I cells, shRNA was used to knockdown survivin. Cellular Thermal Shift Assay (CETSA) was used to detect the interaction between isoalantolactone and survivin. The ubiquitin of survivin induced by isoalantolactone was detected through immunoprecipitation. Quantitative polymerase-chain reaction (Q-PCR) and western blotting were used to detect the levels of mRNA and protein. Isoalantolactone inhibits the proliferation and promotes apoptosis of imatinib-resistant CML cells. Although isoalantolactone inhibits the proteins of BCR-ABL and survivin, it cannot inhibit survivin and BCR-ABL mRNA levels. Simultaneously, it was shown that isoalantolactone can degrade survivin protein by increasing ubiquitination. It was demonstrated that isoalantolactone-induced survivin mediated downregulation of BCR-ABL protein. It was also revealed that isoalantolactone triggered BCR-ABL protein degradation via caspase-3. Altogether, isoalantolactone inhibits survivin through the ubiquitin proteasome pathway, and mediates BCR-ABL downregulation in a caspase-3 dependent manner. These data suggest that isoalantolactone is a natural compound, which can be used as a potential drug to treat TKI-resistant CML.

Telomere loss is accompanied by decreased pool of shelterin proteins TRF2 and RAP1, elevated levels of TERRA and enhanced glycolysis in imatinib-resistant CML cells

Telomere length may be maintained by telomerase nucleoprotein complex and shelterin complex, namely TRF1, TRF2, TIN2, TPP1, POT1 and RAP1 proteins and modulated by TERRA expression. Telomere loss is observed during progression of chronic myeloid leukemia (CML) from the chronic phase (CML-CP) to the blastic phase (CML-BP). The introduction of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), has changed outcome for majority of patients, however, a number of patients treated with TKIs may develop drug resistance. The molecular mechanisms underlying this phenomenon are not fully understood and require further investigation. In the present study, we demonstrate that IM-resistant BCR::ABL1 gene-positive CML K-562 and MEG-A2 cells are characterized by decreased telomere length, lowered protein levels of TRF2 and RAP1 and increased expression of TERRA in comparison to corresponding IM-sensitive CML cells and BCR::ABL1 gene-negative HL-60 cells. Furthermore, enhanced activity of glycolytic pathway was observed in IM-resistant CML cells. A negative correlation between a telomere length and advanced glycation end products (AGE) was also revealed in CD34+ cells isolated from CML patients. In conclusion, we suggest that affected expression of shelterin complex proteins, namely TRF2 and RAP1, TERRA levels, and glucose consumption rate may promote telomere dysfunction in IM-resistant CML cells.

Alvespimycin Inhibits Heat Shock Protein 90 and Overcomes Imatinib Resistance in Chronic Myeloid Leukemia Cell Lines.

Heat shock protein 90 (HSP90) facilitates folding and stability and prevents the degradation of multiple client proteins. One of these HSP90 clients is BCR-ABL, the oncoprotein characteristic of chronic myeloid leukemia (CML) and the target of tyrosine kinase inhibitors, such as imatinib. Alvespimycin is an HSP90 inhibitor with better pharmacokinetic properties and fewer side effects than other similar drugs, but its role in overcoming imatinib resistance is not yet clarified. This work studied the therapeutic potential of alvespimycin in imatinib-sensitive (K562) and imatinib-resistant (K562-RC and K562-RD) CML cell lines. Metabolic activity was determined by the resazurin assay. Cell death, caspase activity, mitochondrial membrane potential, and cell cycle were evaluated by means of flow cytometry. Cell death was also analyzed by optical microscopy. HSPs expression levels were assessed by western blotting. Alvespimycin reduced metabolic activity in a time-, dose-, and cell line-dependent manner. Resistant cells were more sensitive to alvespimycin with an IC50 of 31 nM for K562-RC and 44 nM for K562-RD, compared to 50 nM for K562. This drug induced apoptosis via the mitochondrial pathway. In K562 cells, alvespimycin induced cell cycle arrest in G0/G1. As a marker of HSP90 inhibition, a significant increase in HSP70 expression was observed. Our results suggest that alvespimycin might be a new therapeutic approach to CML treatment, even in cases of resistance to imatinib.

Differentiation of imatinib -resistant chronic myeloid leukemia cells with BCR-ABL-T315I mutation induced by Jiyuan Oridonin A.

Chronic myeloid leukemia (CML) results from BCR-ABL oncogene, which blocks CML cells differentiation and protects these cells from apoptosis. T315I mutated BCR-ABL is the main cause of the resistance mediated by imatinib and second generation BCR-ABL inhibitor. CML with the T315I mutation has been considered to have poor prognosis. Here, we determined the effect of Jiyuan oridonin A (JOA), an ent-kaurene diterpenoid compound, on the differentiation blockade in imatinib-sensitive, particularly, imatinib-resistant CML cells with BCR-ABL-T315I mutation by cell proliferation assay, apoptosis analysis, cell differentiation analysis, cell cycle analysis and colony formation assay. We also investigated the possible molecular mechanism by mRNA sequencing, qRT-PCR and Western blotting. We found that JOA at lower concentration significantly inhibited the proliferation of CML cells expressing mutant BCR-ABL (T315I mutation included) and wild-type BCR-ABL, which was due to that JOA induced the cell differentiation and the cell cycle arrest at G0/G1 phase. Interestingly, JOA possessed stronger anti-leukemia activity than its analogues such as OGP46 and Oridonin, which has been investigated extensively. Mechanistically, the cell differentiation mediated by JOA may be originated from the inhibition of BCR-ABL/c-MYC signaling in CML cells expressing wild-type BCR-ABL and BCR-ABL-T315I. JOA displayed the activity of inhibiting the BCR-ABL and promoted differentiation of not only imatinib -sensitive but also imatinib -resistant cells with BCR-ABL mutation, which could become a potent lead compound to overcome the imatinib -resistant induced by inhibitors of BCR-ABL tyrosine kinase in CML therapy.

Privileged fragment-based design, synthesis and in vitro antitumor activity of imatinib analogues.

Based on the privileged fragment-based drug design strategy, a series of imatinib analogues bearing the moiety of 3-(2-amino-2-oxoacetyl)-1H-indole were designed and synthesized, and the in vitro antitumor activity of these compounds was detected by MTT method using K562 (human myeloid leukemia) and K562R (imatinib-resistant chronic myeloid leukemia) cell lines. Molecular docking was used to preliminarily explain the possible binding modes. The most potent compound I2 exhibited better antitumor activity than those of imatinib against K562 and K562R cancer cells with IC50 values of 0.8 μM and 0.7 μM.

Curcumin Decreases Viability and Inhibits Proliferation of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia Cell Lines.

Chronic myeloid leukemia (CML) is a myeloproliferative haematological malignancy characterized by constitutive activation of BCR-ABL1 tyrosine kinase in the majority of patients. BCR-ABL1 expression activates signaling pathways involved in cell proliferation and survival. Current treatment options for CML include tyrosine kinase inhibitors (TKI) with resistance as a major issue. Various treatment options for overcoming resistance are being investigated. Among them, phytochemical curcumin could play an important role. Curcumin has been found to exhibit anti-cancerous effects in various models, including CML, through regulation of multiple molecular signaling pathways contributing to tumorigenesis. We have evaluated curcumin's effects on imatinib-sensitive LAMA84S and K562, as well as imatinib-resistant LAMA84R cell lines. Our results indicate a significant dose-dependent decrease in cell viability and proliferation of imatinib-sensitive and imatinib-resistant cell lines after curcumin treatment. Suppression of key signaling molecules regulating metabolic and proliferative events, such as Akt, P70S6K and NF-kB, was observed. Increased expression of caspase-3 suggests the potential pro-apoptotic effect of curcumin in the imatinib-resistant CML model. Additional in silico molecular docking studies revealed binding modes and affinities of curcumin with different targets and the results are in accordance with in vitro findings. Altogether, these results indicate the potential role of curcumin in the treatment of CML.

Antileukemic Activity of hsa-miR-203a-5p by Limiting Glutathione Metabolism in Imatinib-Resistant K562 Cells.

Imatinib has been the first and most successful tyrosine kinase inhibitor (TKI) for chronic myeloid leukemia (CML), but many patients develop resistance to it after a satisfactory response. Glutathione (GSH) metabolism is thought to be one of the factors causing the emergence of imatinib resistance. Since hsa-miR-203a-5p was found to downregulate Bcr-Abl1 oncogene and also a link between this oncogene and GSH metabolism is reported, the present study aimed to investigate whether hsa-miR-203a-5p could overcome imatinib resistance by targeting GSH metabolism in imatinib-resistant CML cells. After the development of imatinib-resistant K562 (IR-K562) cells by gradually exposing K562 (C) cells to increasing doses of imatinib, resistant cells were transfected with hsa-miR-203a-5p (R+203). Thereafter, cell lysates from various K562 cell sets (imatinib-sensitive, imatinib-resistant, and miR-transfected imatinib-resistant K562 cells) were used for GC-MS-based metabolic profiling. L-alanine, 5-oxoproline (also known as pyroglutamic acid), L-glutamic acid, glycine, and phosphoric acid (Pi)-five metabolites from our data, matched with the enumerated 28 metabolites of the MetaboAnalyst 5.0 for the GSH metabolism. All of these metabolites were present in higher concentrations in IR-K562 cells, but intriguingly, they were all reduced in R+203 and equated to imatinib-sensitive K562 cells (C). Concludingly, the identified metabolites associated with GSH metabolism could be used as diagnostic markers.

The Activity of Novel BCR-ABL Small-Molecule Degraders Containing Pyrimidine Rings and Their Role in Overcoming Drug Resistance.

Inducing protein degradation by proteolysis-targeting chimeras (PROTACs) has gained tremendous momentum in the field for its promise in the discovery and development of new therapies. Based on our previously reported PROTAC BCR-ABL degraders, we designed and synthesized additional 4 PROTAC compounds with a novel linker that contains pyrimidine rings. Molecular and cellular studies have shown that different linkers affect the degradation activity of small-molecule degraders on the target protein of BCR-ABL. We screened out a lead compound, DMP11, with stable physicochemical properties and high activity. Preliminary evaluation of its pharmacodynamics in vitro model showed that it has a good inhibitory effect on imatinib-resistant chronic myeloid leukemia cell lines, as has been shown in animal models. Our preliminary research into the mechanism of DMP11 found that DMP11 can overcome drug resistance by simultaneously inhibiting the targets of BCR-ABL and SRC-family kinase (SFK).

KIF23 promotes autophagy-induced imatinib resistance in chronic myeloid leukaemia through activating Wnt/β-catenin pathway.

Imatinib, an inhibitor of tyrosine kinase, shows remarkable efficacy in chronic myeloid leukaemia (CML). Autophagy protects tumour cells against chemotherapeutic stimulation and contributes to imatinib resistance in CML. Kinesin family member 23 (KIF23) is involved in cytokinesis and associated with autophagy. The role of KIF23 in autophagy-induced imatinib resistance in CML was investigated. First, to induce drug resistance, CML cells were exposed to increasing concentrations of imatinib. The concentration of imatinib resistance in CML cells was screened through upregulation of 50% inhibitory concentration (IC50 ) values. KIF23 was elevated in imatinib-resistant tissues and cells of CML. Second, knockdown of KIF23 reduced IC50 values of imatinib-resistant CML cells to imatinib. Moreover, silence of KIF23 also suppressed cell proliferation and promoted apoptosis of imatinib-resistant CML cells. Third, immunofluorescence analysis showed that the number of LC3 bright spots in imatinib-resistant CML cells was reduced by silence of KIF23. Knockdown of KIF23 upregulated p62 expression and downregulated the expression ratio of LC3-II to LC3-I in imatinib-resistant CML cells. Last, silence of KIF23 decreased nuclear β-catenin and increased cytoplasmic β-catenin in imatinib-resistant CML cells. Activator of Wnt/β-catenin attenuated KIF23 silence-induced increase of apoptosis and decrease of autophagy in imatinib-resistant CML cells. In conclusion, loss of KIF23 repressed autophagy-induced imatinib resistance in CML cells through inactivation of Wnt/β-catenin pathway.

Ketoconazole Reverses Imatinib Resistance in Human Chronic Myelogenous Leukemia K562 Cells.

Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the oncogene BCR-ABL1, which encodes an oncoprotein with tyrosine kinase activity. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor, performs exceptionally well with minimal toxicity in CML chemotherapy. According to clinical trials, however, 20–30% of CML patients develop resistance to imatinib. Although the best studied resistance mechanisms are BCR-ABL1-dependent, P-glycoprotein (P-gp, a drug efflux transporter) may also contribute significantly. This study aimed to establish an imatinib-resistant human CML cell line, evaluate the role of P-gp in drug resistance, and assess the capacity of ketoconazole to reverse resistance by inhibiting P-gp. The following parameters were determined in both cell lines: cell viability (as the IC50) after exposure to imatinib and imatinib + ketoconazole, P-gp expression (by Western blot and immunofluorescence), the intracellular accumulation of a P-gp substrate (doxorubicin) by flow cytometry, and the percentage of apoptosis (by the Annexin method). In the highly resistant CML cell line obtained, P-gp was overexpressed, and the level of intracellular doxorubicin was low, representing high P-gp activity. Imatinib plus a non-toxic concentration of ketoconazole (10 μM) overcame drug resistance, inhibited P-gp overexpression and its efflux function, increased the intracellular accumulation of doxorubicin, and favored greater apoptosis of CML cells. P-gp contributes substantially to imatinib resistance in CML cells. Ketoconazole reversed CML cell resistance to imatinib by targeting P-gp-related pathways. The repurposing of ketoconazole for CML treatment will likely help patients resistant to imatinib.

Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation

BackgroundTyrosine kinase inhibitors have achieved quite spectacular advances in the treatment of chronic myeloid leukemia (CML), but disease progression and drug resistance that related to the T315I mutation, remain major obstacles. Dendritic cell-derived exosomes (Dex) induce NK cell immunity, but have yet to achieve satisfactory clinical efficacy. An approach to potentiate antitumor immunity by inducing both NK- and T-cell activation is urgently needed. Retinoic acid early inducible-1γ (RAE-1γ), a major ligand of natural killer group 2 member D (NKG2D), plays an important role in NK-cell and T-lymphocyte responses. We generated RAE-1γ enriched CML-specific Dex (CML-RAE-1γ-Dex) from dendritic cells (DCs) pulsed with lysates of RAE-1γ-expressing CML cells or T315I-mutant CML cells, aiming to simultaneously activate NK cells and T lymphocytes.MethodsWe generated novel CML-RAE-1γ-Dex vaccines, which expressed RAE-1γ, and were loaded with CML tumor cell lysates. NK cells or T lymphocytes were coincubated with CML-RAE-1γ-Dex vaccines. Flow cytometry was performed to evaluate the activation and proliferation of these immune cells. Cytokine production and cytotoxicity toward CML cells with or without the T315I mutation were detected by ELISPOT, ELISA and LDH assays. CML models induced by BCR-ABL or BCR-ABLT315I were used to determine the immunological function of Dex in vivo.ResultsHerein, CML-RAE-1γ-Dex were prepared. CML-RAE-1γ-Dex effectively enhanced the proliferation and effector functions of NK cells, CD4+ T cells and CD8+ T cells, which in turn produced strong anti-CML efficacy in vitro. Moreover, CML-RAE-1γ-Dex-based immunotherapy inhibited leukemogenesis and generated durable immunological memory in CML mouse models. Similar immune responses were also observed with imatinib-resistant CML cells carrying the T315I mutation.ConclusionsThis approach based on CML-RAE-1γ-Dex vaccines may be a promising strategy for CML treatment, especially for cases with the T315I mutation.

The HSP90 inhibitor KW-2478 depletes the malignancy of BCR/ABL and overcomes the imatinib-resistance caused by BCR/ABL amplification

BackgroundWith the widespread clinical application of tyrosine kinase inhibitors (TKIs), an increasing number of chronic myeloid leukaemia (CML) patients have developed resistance or intolerance to TKIs. BCR/ABL is the oncoprotein of CML. HSP90 is an essential chaperone of BCR/ABL and plays an important role in protein folding and the function of BCR/ABL. Therefore, inhibiting the chaperone function of HSP90 may be an effective strategy for CML treatment and to overcome TKI resistance.MethodsThe effect of KW-2478 on CML cell viability, apoptosis and cell cycle progression was detected by CCK-8 assay or flow cytometry. The levels of BCR/ABL, HSP90 and other signalling proteins were detected by western blots. The mitochondrial membrane potential was detected by flow cytometry combined with JC-1 staining. The interaction between BCR/ABL and HSP90α was detected by coimmunoprecipitation. The effect of KW-2478 on BCR/ABL carcinogenesis in vivo was investigated in CML-like mouse models.ResultsKW-2478 inhibited growth and induced apoptosis of CML cells. KW-2478 inhibited the chaperone function of HSP90α and then weakened the BCR/ABL and MAPK signalling pathways. This treatment also caused an increase in p27 and p21 expression and a decrease in cyclin B1 expression, which led to G2/M phase arrest. The mitochondrial pathway was primarily responsible for KW-2478-induced apoptosis. KW-2478 had a synergistic effect with imatinib in growth inhibition. Notably, KW-2478 had a stronger effect on growth inhibition, apoptosis induction and cell cycle arrest of K562/G01 cells than K562 cells. KW-2478 could effectively prolong the mouse lifespan and alleviate disease symptoms in CML-like mouse models.ConclusionsThis finding demonstrated that KW-2478 had anticancer properties in imatinib-sensitive and imatinib-resistant CML cells and illustrated the possible mechanisms. This study provides an alternative choice for CML treatment, especially for TKI-resistant patients with BCR/ABL amplification and TKI-intolerant patients.

Combination of Elacridar with Imatinib Modulates Resistance Associated with Drug Efflux Transporters in Chronic Myeloid Leukemia.

Multidrug resistance (MDR) development has emerged as a complication that compromises the success of several chemotherapeutic agents. In chronic myeloid leukemia (CML), imatinib resistance has been associated with changes in BCR-ABL1 and intracellular drug concentration, controlled by SLC and ABC transporters. We evaluate the therapeutic potential of a P-glycoprotein and BCRP inhibitor, elacridar, in sensitive (K562 and LAMA-84) and imatinib-resistant (K562-RC and K562-RD) CML cell lines as monotherapy and combined with imatinib. Cell viability was analyzed by resazurin assay. Drug transporter activity, cell death, cell proliferation rate, and cell cycle distribution were analyzed by flow cytometry. Both resistant models presented an increased activity of BCRP and P-gP compared to K562 cells. Elacridar as monotherapy did not reach IC50 in any CML models but activated apoptosis without cytostatic effect. Nevertheless, the association of elacridar (250 nM) with imatinib overcomes resistance, re-sensitizing K562-RC and K562-RD cells with five and ten times lower imatinib concentrations, respectively. Drug combination induced apoptosis with increased cleaved-caspases-3, cleaved-PARP and DNA damage, reduced cell proliferation rate, and arrested CML cells in the S phase. These data suggest that elacridar combined with imatinib might represent a new therapeutic option for overcoming TKI resistance involving efflux transporters.

Impact of metformin on tyrosine kinase inhibitor response in chronic myeloid leukemia.

ObjectiveOral tyrosine kinase inhibitors (TKIs) are first line therapy for chronic myeloid leukemia (CML). A complete cytogenetic response (CCyR) correlates with increased overall survival, however only 66%–88% of patients achieve CCyR after one year of TKI treatment. Because TKI therapy alone cannot eliminate CML stem cells, strategies aimed at achieving faster and deeper responses are needed to improve long-term survival. Metformin is a widely prescribed glucose-lowering agent for patients with diabetes and in preclinical studies, has been shown to suppress cell viability, induce apoptosis, and downregulate the mTORC1 signaling pathway in imatinib resistant CML cell lines (K562R). This study aims to investigate the utility of metformin added to TKI therapy in patients with CML.Data SourcesAn observational study at an academic medical center (Salt Lake City, UT) was performed for adults with newly diagnosed, chronic-phase CML to evaluate attainment of CCyR from TKI therapy with or without concomitant metformin use. Descriptive analyses were used to describe baseline characteristics and attainment of response to TKI therapy.Data SummaryFifty-nine patients were evaluated. One hundred percent (5 of 5) in the metformin group and 73.6% (39 of 54) in the non-metformin group achieved CCyR. Approximately 20% of patients in both groups relapsed (defined by a loss of CCyR during study) after a median 34.5 months of follow-up.ConclusionsFuture research is warranted to validate these findings and determine the utility of metformin added to TKI therapy.

Simvastatin potentiates the cell-killing activity of imatinib in imatinib-resistant chronic myeloid leukemia cells mainly through PI3K/AKT pathway attenuation and Myc downregulation

Constitutively activated BCR-ABL kinase is considered the driver event responsible in the initiation and development of chronic myeloid leukemia (CML). The advent of the first BCR-ABL inhibitor imatinib has significantly improved the clinical outcome of CML cases. However, resistance to imatinib occurs in 25–30% of CML patients. Due to the lack of effective therapeutic strategies, novel treatment approaches are urgently required for imatinib-resistant CML. Simvastatin, a well-known HMG-CoA reductase inhibitor that confers tremendous clinical benefits in cardiovascular diseases, has attracted mounting attentions for its potent antitumor effects on multiple tumor types. In this study, we demonstrated that simvastatin monotherapy was effective in diminishing cell viability in both imatinib-sensitive and imatinib-resistant CML cells, including T351I mutated cells, with the latter being less vulnerable to the simvastatin than the former. Notably, we found that simvastatin acted as a robust cytotoxic sensitizer of imatinib to kill imatinib-resistant and T315I mutated CML cells in vitro and in vivo. Mechanistically, the cooperative interaction of simvastatin and imatinib was associated with the inactivation of the PI3K/Akt signaling pathway, which was a classical downstream pro-survival cascade of the BCR-ABL kinase. In addition, this drug combination obviously decreased Myc expression through attenuation of canonical Wnt/β-catenin signaling and increased H3K27 trimethylation. Taken together, we provide attractive preclinical results for the combinatorial regimen of simvastatin and imatinib against imatinib-resistant and T315I mutated CML cells. This combined regimens warrants further clinical investigations in patients with imatinib-resistant CML.

Research on the Mechanism of SIRT1/AMPK Inducing Resistance to Imatinib in K562 Cells By Inhibiting Srebp to Promote Lipid Metabolism

Background Leukemia stem cells (LSC) have the characteristics of independent of BCR-ABL kinase activity and metabolic reprogramming to support self-renewal. They are the root cause of the occurrence of chronic myeloid leukemia (CML), mediating TKI resistance and leading to clonal evolution. LSC self-renewal depends on the activity of SIRT1, the enhanced of SIRT1 induces LSC mitochondrial metabolic reprogramming. Studies have shown that SIRT1 and abnormally activated lipid metabolism are important reasons for inducing CML resistance to imatinib.Objective Study the mechanism of SIRT1 regulating lipid metabolism and intervention strategies under imatinib therapy.Method Liquid chromatography coupled with mass spectrometry (LC-MS) were used to analysis the lipid metabolism in imatinib-resistant CML cell line K562-R under low-glucose conditions. RT-qPCR and Western blot were used to detect the key enzymes related to lipid metabolism, and the SIRT1 inhibitor TV-6 was given to study the regulation mechanism of SIRT1 on lipid metabolism.Result Firstly, we found that the glycolysis/gluconeogenesis pathway and fatty acid metabolism were abnormal in K562-R cells by metabolomics, compared with normal culture conditions, the expression of SIRT1 and AMPK were significantly increased, the expression of fatty acid synthesis-related genes (SREBP, FASN, ACC, SCD1) was significantly reduced, and the fatty acid β oxidation rate-limiting enzyme CPT-1 is significantly increased inK562-R cells under low-glucose conditions. Therefore, in the low-glucose environment, the expression of the above genes was reversed after the application of the SIRT1 inhibitor TV-6.Conclusion K562-R imatinib-resistant cells can down-regulate the expression of SREBP through up-regulated SIRT1/AMPK pathway under low-glucose conditions, thereby inhibiting the expression of transcription factors involved in lipogenesis, reducing fatty acid synthesis, and providing the imatinib-resistant cells with the energy which required for proliferation.Keyword Chronic myeloid leukemia; Metabolomics; Lipid metabolism; SIRT1/AMPK DisclosuresNo relevant conflicts of interest to declare.

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.