Quantitative Proteomic Analysis of Plasma Exosomes to Identify the Candidate Biomarker of Imatinib Resistance in Chronic Myeloid Leukemia Patients

BackgroundImatinib (IM), a tyrosine kinase inhibitor (TKI), has markedly improved the survival and life quality of chronic myeloid leukemia (CML) patients. However, the lack of specific biomarkers for IM resistance remains a serious clinical challenge. Recently, growing evidence has suggested that exosome-harbored proteins were involved in tumor drug resistance and could be novel biomarkers for the diagnosis and drug sensitivity prediction of cancer. Therefore, we aimed to investigate the proteomic profile of plasma exosomes derived from CML patients to identify ideal biomarkers for IM resistance.MethodsWe extracted exosomes from pooled plasma samples of 9 imatinib-resistant CML patients and 9 imatinib-sensitive CML patients by ultracentrifugation. Then, we identified the expression levels of exosomal proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based label free quantification. Bioinformatics analyses were used to analyze the proteomic data. Finally, the western blot (WB) and parallel reaction monitoring (PRM) analyses were applied to validate the candidate proteins.ResultsA total of 2812 proteins were identified in plasma exosomes from imatinib-resistant and imatinib-sensitive CML patients, including 279 differentially expressed proteins (DEPs) with restricted criteria (fold change≥1.5 or ≤0.667, p<0.05). Compared with imatinib-sensitive CML patients, 151 proteins were up-regulated and 128 proteins were down-regulated. Bioinformatics analyses revealed that the main function of the upregulated proteins was regulation of protein synthesis, while the downregulated proteins were mainly involved in lipid metabolism. The top 20 hub genes were obtained using STRING and Cytoscape, most of which were components of ribosomes. Moreover, we found that RPL13 and RPL14 exhibited exceptional upregulation in imatinib-resistant CML patients, which were further confirmed by PRM and WB.ConclusionProteomic analysis of plasma exosomes provides new ideas and important information for the study of IM resistance in CML. Especially the exosomal proteins (RPL13 and RPL14), which may have great potential as biomarkers of IM resistance.

Correction to: Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl

An amendment to this paper has been published and can be accessed via the original article.

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 Disclosures No 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.

Imatinib-Resistant Gastrointestinal Stromal Tumor Presenting as a Large Abdominal Mass

Gastrointestinal stromal tumors (GISTs) are the stromal or mesenchymal neoplasms affecting the gastrointestinal tract. Although they constitute 1% of primary gastrointestinal tumors, they are the most common nonepithelial tumors involving the gastrointestinal tract. They mostly present as overt or occult gastrointestinal bleeding. We present a case in which a 77-year-old female presented with a large abdominal mass. The origin of the mass was unclear on CT and MRI scan of the abdomen. Upper gastrointestinal endoscopic ultrasonography showed a cystic lesion in the perigastric region. A fine-needle biopsy of the lesion was performed, which was consistent with spindle type GIST. After the initial failure of imatinib therapy, the tumor was managed surgically.

Spectrum of BCR-ABL Mutations and Treatment Outcomes in Ethiopian Imatinib-Resistant Patients With Chronic Myeloid Leukemia

PURPOSE Despite the successes achieved in chronic myeloid leukemia (CML) with tyrosine kinase inhibitor (TKI) therapy, resistance remains an obstacle. The most common mechanism of resistance is the acquisition of a point mutation in the BCR-ABL kinase domain. Few studies have reported African patients with CML in regard to such mutations. We here report the types of BCR-ABL mutations in Ethiopian imatinib-resistant patients with CML and their outcome. PATIENTS AND METHODS Patients with CML with a diagnosis of imatinib resistance who were tested for BCR-ABL mutation between 2014 and September 2019 were included. RESULTS A total of 962 cases of CML on imatinib therapy were reviewed and 164 cases of failure were found. Of these, only 31 cases (19%) had mutation analysis performed. Most cases (94%) were secondary failures. At the time of CML diagnosis, the median age was 33 years and the majority presented with features of advanced-phase disease. Of the 31 patients, 22 mutations were found (65%). The types of mutations detected were as follows: non–P-loop mutations 36% (11), P-loop mutations 13% (four), and alternatively spliced BCR-ABL variants 23% (seven). The splice variant frequently detected was BCR-ABL35INS (20%). Twenty-six of the 31 patients (84%) were switched to second-line TKIs, whereas in four patients (13%), imatinib dose escalation was done. Overall, the outcome revealed that 16 patients (52%) were alive with complete hematologic response, whereas 12 patients (39%) had died. All patients who expressed BCR-ABL135INS were treated with second-line TKIs, and two of them (33%) had died because of disease progression. CONCLUSION In Ethiopia, CML affects the young and point mutations were frequently detected in imatinib-resistant patients. BCR-ABL1 35INS was also prevalent and associated with disease progression.