TYROSINE KINASE INHIBITORS DOWNREGULATE MIRNA EXPRESSION IN K562 CELL LINE

FREUE, JM; RUIZ, MS; SANCHEZ, MB; BIANCHINI, M

Congreso; 26th Congress of the European Hematology Association (virtual edition).; 2021

European Hematology Association

Background:  Chronic myeloidleukemia is a clonal disease of the bone marrow characterized by a geneticmarker: the t (9; 22) translocation. This genetic event produces an oncoprotein(BCR-ABL1) with constitutive tyrosine kinase activity which, through thephosphorylation of adapter proteins, leads to an increase in proliferation anddecrease in apoptosis of hematopoietic precursors. Although this genetic translocation was believed to be the onlyresponsible for the pathology, nowadays, other additional molecular events arebeing investigated. Such events could be an alteration in the expression ofmicroRNAs since t (9; 22) generates genomic instability and consequentlyadditional genetic alterations to BCR-ABL1. It is well known that miRNAs aredysregulated y many types of cancer and they are being studied as potentialbiomarkers. Moreover, dysregulation in miRNAs expression could be an eventindependent from the activity of the BCR-ABL1.  Aims: To evaluate the dependence ofmiRNAs expression from BCR-ABL1 activity in vitro. Methods: mirVana miRNA Isolation Kit (Ambion®) was used for RNAextraction from K562 CML cell line previously incubated with either imatinib1000nM, 450nM and 200nM or nilotinib 120nM, 60nM and 30nM according to calculatedIC50 using MTT assay. To confirminhibition of the BCR-ABL1 pathway a western blot for phospho-CRKL was done.For qPCR a protocol that is designed to specifically amplify and quantifymiRNAs using stem-loop primers was used. The following miRNAs were evaluated:hsa-miR-196a-5p, hsa-miR-92b-3p, hsa-miR-126-5p,hsa-miR-125a-5p, hsa-miR-2355-5p, hsa-miR-10a-5p, hsa-miR-132-3p, hsa-let-7a-5pand hsa-miR-182-5p. This panel was base on previous experiments in the lab whichevaluated miRNAs expression in CML patients (leukemic stem cells vshematopoietic stem cells) by next generation sequencing. Statistical analysiswas done with GraphPad v6.0. T-Student test was used to calculate statisticalsignificance between samples. Results: Imatinib at a concentration of 200nM did not inhibit p-CRKL by westernblot. the expression of all miRNAs aforementioned but for miR2355was downregulated after incubation for 48hs with both ITKs. This effect wasmore powerful with nilotinib than imatinib. When incubated with imatinib hsa-miR-126-5p, hsa-miR-182-5p hsa-miR-92b-5pand hsa-miR-196a-5p showed a dose dependent inhibition (Figure 1). Summary/Conclusion: In K562 cell line, a down regulationof miRNA expression was observed when exposed to both drugs, with adose-dependent effect confirmed for some miRNAs incubated with imatinib. Morestudies should be done to evaluate if this effect is also observed in vivo.Eventually, miRNAs could be used as a biomarker of response to ITKs in CMLpatients. The fact that imatinib 200nM did notinhibited BCR-ABL1 but miR92 and miR 126 where still dowregulated could beexplained by off target effect of ITKs.