TKIs induce alternative spliced BCR-ABL Ins35bp variant via inhibition of RNA polymerase Ⅱ on genomic BCR-ABL.

To elucidate dynamic changes in nativeBCR-ABLand alternatively spliced tyrosine kinase inhibitor (TKI)-resistant but function-deadBCR-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. Because both transcripts were amplified together using conventional PCR system for measuring International Scale (IS), deep sequencing method was used for quantifying suchBCR-ABLvariants. At the initial diagnosis, 7 of 9 patients presented a small fraction of cells possessingBCR-ABL(Ins35bp), accounting for 0.8% of the total ISBCR-ABL, corresponding to actualBCR-ABL(Ins35bp)value of 1.1539% IS. TKI rapidly decreased nativeBCR-ABLbut notBCR-ABL(Ins35bp), leading to the initial increase in the proportion ofBCR-ABL(Ins35bp). Thereafter, both nativeBCR-ABLandBCR-ABL(Ins35bp)gradually decreased in the course of TKI treatment, whereas small populations positive for TKI-resistantBCR-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 nativeBCR-ABL(+)clones. However, IS fluctuating at low levels around MR4.0 marked a predominant persistence of cells expressing function-deadBCR-ABL(Ins35bp), suggesting that TKI resumption was unnecessary. We clarified the possible mechanism underlying mis-splicingBCR-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 functionalBCR-ABLextractingBCR-ABL(Ins35bp)would lead us to a correct evaluation of MR status, thus determining the adequate therapeutic intervention.