Tyrosine kinase inhibitors induce alternative spliced BCR‐ABLIns35bp variant via inhibition of RNA polymerase II on genomic BCR‐ABL

Abstract

To elucidate dynamic changes in native BCR‐ABL and alternatively spliced tyrosine kinase inhibitor (TKI)‐resistant but function‐dead BCR‐ABL^Ins35bp variant, following commencement or discontinuation of TKI therapy, each transcript was serially quantified in patients with chronic myeloid leukemia (CML) by deep sequencing. Since both transcripts are measured together by conventional polymerase chain reaction as a common International Scale (IS), deep sequencing method was used for quantifying such BCR‐ABL variants. At the initial diagnosis, seven of nine patients presented a small fraction possessing BCR‐ABL^Ins35bp, accounting for 0.8% of the total IS BCR‐ABL, corresponding to an actual BCR‐ABL^Ins35bp value of 1.1539% IS. TKI rapidly decreased native BCR‐ABL but not BCR‐ABL^Ins35bp, leading to an initial increase in the proportion of BCR‐ABL^Ins35bp. Thereafter, both native BCR‐ABL and BCR‐ABL^Ins35bp gradually decreased on the course of TKI treatment, whereas small populations positive for TKI‐resistant BCR‐ABL^Ins35bp continued fluctuating at low levels, possibly underestimating the molecular response (MR). Following TKI discontinuation, sequencing analysis of 54 patients revealed a rapid relapse, apparently derived from native BCR‐ABL⁺ clones. However, IS fluctuating at low levels around MR4.0 marked a predominant persistence of cells expressing function‐dead BCR‐ABL^Ins35bp, suggesting that TKI resumption was unnecessary. We clarified the possible mechanism underlying mis‐splicing BCR‐ABL^Ins35bp, occurring at the particular pseudo‐splice site within intron8, which can be augmented by TKI treatment through inhibition of RNA polymerase II phosphorylation. No mutations were found in spliceosomal genes. Therefore, monitoring IS functional BCR‐ABL extracting BCR‐ABL^Ins35bp would lead us toward a correct evaluation of MR status thus determining an adequate therapeutic intervention.