Research On Mechanism Of Resistance To Tyrosine Kinase Inhibitors In Chronic Myeloid Leukemia Based On Deep Sequeincng And Multi-omics Approach

Background The widespread use of tyrosine kinase inhibitors(TKIs)have significantly improved the remission rate and long-term survival of patients with chronic myeloid leukemia(CML).However,there are still a minority of patients with disease progression and refractory recurrence due to drug intolerance or drug resistance,which is also the difficulty and research focus in the clinical treatment of CML.At present,the mechanisms of drug resistance to TKI are mainly divided into two types:BCR-ABLI-dependent and non-BCR-ABL1-dependent.BCR-ABL1 dependence in drug-resistance is mainly about kinase region mutation and BCR-ABL1 gene amplification,while the mechanism of non-BCR-ABL1-dependent drug resistance is more complex.Recent studies have found that there is a disorder of energy metabolism and hyper glycolysis in leukemia cells of TKI-resistant CML patients,and imatinib(IM)treatment can normalize energy metabolism in sensitive cells to some extent,while high glycolysis metabolism has been maintained in IM-resistant CML cells.We explored the characteristics of energy metabolism of BCR-ABL 1-independent TKI resistance in CML leukemia cells and the role of energy metabolism in the process in this research.Besides,we detected and analyzed the mutations of cancer-related genes in TKI-resistant or intolerant CML patients.MethodDeep sequencing and liquid chromatography mass spectrometry(LC-MS)were used to compare the multi-omics analysis between IM-resistant CML cell line K562-R and sensitive CML cell line K562-S,both of which are without BCR-ABL1 kinase region mutation.We analyzed energy metabolism in cells and found out the target gene,PKM2,verifying it in cytological experiments.Besides,based on the second-generation sequencing technique,the cancer-related gene mutations in TKI-resistant or intolerant CML patients were studied.ResultFirstly,it was found that the glycolysis/gluconeogenesis pathway was abnormal in K562-R.And the expression of PKM,one of the isozymes of pyruvate kinase(PK),and 6-phosphate fructose kinase-2(PFK-2)were up-regulated.A stable cell line with low expression of PKM2 was established by CRISPR/Cas9 technique.It was found that the sensitivity of IM-resistant cell line K562-R to IM was restored with a low expression of PKM2 protein,which confirmed that the high expression of PKM2 was closely related to IM drug-resistanceIn addition,glutathione catabolism is exuberant in K562-R,and the proportion of sources of intracellular reduced equivalent NADPH have changed.The metabolism of intracellular pentose phosphate pathway was weakened,and more NADPH comes from the conversion of isocitrate to ?-ketoglutaric acid.In the genetic study of TKI-resistant or intolerant CML patients,it was found that ABL1 KD mutations played a dominant role in TKIs resistance,while CUXI,KIT and GATA2 mutations are more important in TKIs intolerance.The number of transcription factor gene mutations in TKIs-resistant patients was significantly more than that in TKIs-resistant patients,suggesting that transcriptional genes are essential in TKIs intolerance.ASXL1 and TET2 mutations are closely related to the disease progression of CML patients.Although they are all ARCH mutations,the effect of poor prognosis cannot be ignoredConclusionIn the IM-resistant cell K562-R,the disorder of energy metabolism is more serious and cannot be corrected and improved by IM,in which PKM2 plays an essential role.Genomic risk assessment can improve the risk stratification of clinical diagnosis and more accurately identify patients who are resistant or intolerant to TKIs in the era of TKIs treatment.