KDM6A promotes imatinib resistance through YY1-mediated transcriptional upregulation of TRKA independently of its demethylase activity in chronic myelogenous leukemia.

Rationale: Despite landmark therapy of chronic myelogenous leukemia (CML) with tyrosine kinase inhibitors (TKIs), drug resistance remains problematic. Cancer pathogenesis involves epigenetic dysregulation and in particular, histone lysine demethylases (KDMs) have been implicated in TKI resistance. We sought to identify KDMs with altered expression in CML and define their contribution to imatinib resistance. Methods: Bioinformatics screening compared KDM expression in CML versus normal bone marrow with shRNA knockdown and flow cytometry used to measure effects on imatinib-induced apoptosis in K562 cells. Transcriptomic analyses were performed against KDM6A CRISPR knockout/shRNA knockdown K562 cells along with gene rescue experiments using wildtype and mutant demethylase-dead KDM6A constructs. Co-immunoprecipitation, luciferase reporter and ChIP were employed to elucidate mechanisms of KDM6A-dependent resistance. Results: Amongst five KDMs upregulated in CML, only KDM6A depletion sensitized CML cells to imatinib-induced apoptosis. Re-introduction of demethylase-dead KDM6A as well as wild-type KDM6A restored imatinib resistance. RNA-seq identified NTRK1 gene downregulation after depletion of KDM6A. Moreover, NTRK1 expression positively correlated with KDM6A in a subset of clinical CML samples and KDM6A knockdown in fresh CML isolates decreased NTRK1 encoded protein (TRKA) expression. Mechanistically, KDM6A was recruited to the NTRK1 promoter by the transcription factor YY1 with subsequent TRKA upregulation activating down-stream survival pathways to invoke imatinib resistance. Conclusion: Contrary to its reported role as a tumor suppressor and independent of its demethylase function, KDM6A promotes imatinib-resistance in CML cells. The identification of the KDM6A/YY1/TRKA axis as a novel imatinib-resistance mechanism represents an unexplored avenue to overcome TKI resistance in CML.

[Allicin enhances cytotoxicity of CPT-11 to colon cancer LoVo cell in vitro].

OBJECTIVE: To study the effect of allicin on human colon cancer cell line LoVo and the combined effect of allicin and CPT-11 on this cancer cell line. METHOD: The LoVo cells were cultured in vitro and treated with allicin in different concentrations. MTT assay was used to test dynamically the cell growth inhibiting effect. Apoptosis induction (Annexin-V-FITC/PI) and modulation of DNA cell cycle were measured by flow cytometry. The change of cytotoxicity of CPT-11 after combination of allicin at the concentration of 4.0, 8.0 mg x L(-1) were investigated. RESULT: Allicin had inhibitive effect on growth of LoVo cells in a dose and time dependent manner, with IC50 value of 32.23, 10.74, 6.58 mg x L(-1) at 24 h, 48 h and 72 h, respectively. The apoptosis rate of LoVo cells increased progressively as the cells were treated with increasing concentration of allicin in 24 h, while the apoptosis rate achieved peak value when the cells were treated with allicin at the concentration of 8 mg x L(-1) in 48 h. The result indicated the low concentrations of allicin (< 4 mg x L(-1)) lead to G2/M cell cycle arrest, and higer concentrations ( > 4 mg x L(-1)) exert G1 + G2/M cell cycle arrest in 24 h. Compared with single use of CPT-11, the combined use of CPT-11 and allicin (4.0, 8.0 mg x L(-1), respectively) showed increasing cytotoxicity on the LoVo cells, with IC50 of 24 h decreasing from 47.5 to 7.4 and 7.2 mg x L(-1), respectively. CONCLUSION: Allicin has significant anti-proliferation effect on human colon cancer cell line LoVo by induction of apoptosis and arrestment of cell cycle and can enhance the cytotoxicity of CPT-11 on the colon cancer LoVo cell.