Characterization of T315I BCR-ABL1-mediated drug resistance in chronic myeloid leukemia : new regulatory mechanism and clinical detection assay

Abstract

Although the success of tyrosine kinase inhibitors (TKIs) is evident, 30% of patients treated with imatinib reach complete molecular response (CMR) after 5 years. T315I prevents susceptibility to all TKIs, with the exception of ponatinib. LncRNAs and miRNAs are members of the competitive endogenous RNA (ceRNA) mechanism. This provides a backdrop for extensive exploration of the fundamental functions of non-coding RNAs specifically in TKI-resistance in CML. Additionally, studies have demonstrated the connection of glycolytic metabolism and mitochondrial oxidative phosphorylation to imatinib cytotoxicity in leukemic cells. Furthermore, to improve clinical decisions and overall survival of CML patients, implementing routine monitoring of assays capable of early mutation detection and highly sensitive quantitative techniques are vital. Putting all these together, this study aims to investigate the regulatory pathways involved in resistance mechanisms in BCR-ABL1-T315I and sensitive clinical assay for monitoring drug-resistant mutations in CML patients. K562 cells harbouring T315I mutation (T315I-K562) were validated and verified by several cell viability assays, and the cells were found to be >90% viable. Further validation includes Sanger sequencing, ddPCR, and Western blot, to quantify mutation abundance, and the expression of key downstream enzyme pCRKL, respectively. Imatinib treated T315I-K562 were analyzed using small RNA-sequencing and transcriptome sequencing. DESeq2 extracted 1420 differentially expressed mature miRNAs, and 1641 mRNAs. Several studies have reported lncRNA H19 and MALAT1 to be intricately linked to multiple pathophysiological processes. LncRNA H19-MALAT1-mediated ceRNA network was constructed by performing in-silico analysis by selecting common miRNAs with MREs binding sites on both lncRNA H19 and MALAT1. Subsequently, 65 shared mRNA targets were predicted for the miRNAs, generating a ceRNA network comprising of 2 lncRNAs, 3 miRNAs, and 5 mRNAs. In addition, 15 DEmRNAs dysregulated genes involved in the CML survival and imatinib-resistance were also found to be implicated in several cellular metabolisms and glycolysis. SiRNA lncRNA H19 and MALAT1 were introduced into T315I-K562 cells exposed to imatinib through transfection. Subsequently, changes in cellular metabolism were monitored using Seahorse XFe24 extracellular flux analyzer. A more significant reduction in glycolytic function was observed in T315I K562 cells treated simultaneously with siRNA MALAT1 and imatinib. To develop a sensitive assay for monitoring, Nanopore and ddPCR both were compared using serially diluted T315I-K562 (10% to 0.0001%). Libraries prepared with the amplicons were subjected to MinION sequencer and bioinformatics analysis. The same serially diluted K562 gDNAs were used for conducting ddPCR. Our findings indicate ddPCR can detect and quantify the abundance of T315I mutation until 0.01%, on the other hand, Nanopore sequencing could detect but not accurately quantify the mutations. Putting all these together, DemiRNAs and DemRNAs obtained from sRNA- and transcriptome sequencing reveal potential metabolisms and genes that can be explored. One of those cellular metabolisms explored is glycolysis. Nevertheless, another important issue in TKI resistance is the implementation of a routine assay capable of early detection and quantification of rare and common mutations on the BCR-ABL1 TKD in CML patients.