Discovery Of A Novel Spliceosome Of ABL Gene(ABL^{Δexon7+35INS}) And The Association With TKIs Resistance In Chronic Myeloid Leukemia

Objective:Tyrosine kinase inhibitors（TKIs） resistance in patients with chronic myeloid leukemia（CML） of targeted therapy are more and more attention. Our study screened ABLΔexon7 and ABL35 INS in normal people, CML patients in remission and patients with TKIs resistance. According as the break characteristic based on the BCR/ABL gene, using new primer, to find the differences between ABL spliceosomeof fusion type（BCR/ABL） and wild type（ABL） in CML patients..Predict the protein construct of ABL transcript, to explore whether the ABL spliceosome are contribute to TKIs resistance. It will contribute to clarify the new drug resistance mechanism in addition to the point mutation in BCR/ABL kinase domain and develop the new targeted drugs. Methods:1. We screened ABLΔexon7 and ABL35 INS in 74 normal people and 76 CML patients（53 patients in remission and 23 patients with TKIs resistance） by using polyacrylamide gel electrophoresis combined with cloning sequencing.2. According as the break characteristic based on the BCR/ABL gene, using new primer design way and confirm the linear amplification cycle, to find the differences between ABL spliceosome of fusion type（BCR/ABL） and wild type（ABL） in CML patients by the ratio of optical density.3. Predict the biological functions of ABL spliceosome in protein level, to explore whether the spliceosome are contribute to TKIs resistance. Results:1.A novel spliceosome ABL^{Δexon7+35INS}（ABLΔexon7 and ABL35 INS exist at the same time） was identified and the mutation was detected on 8（10.8%） among 74 normal people, 4（7.5%） among 53 remission patients and 2（8.7%） among 23 resistant patients.Statistical analysis has no difference. And found that the new spliceosome is the main existence formof ABL35 INS, the incidence rate is 87.5%（14/16）, suggesting that ABL35 INSare often accompany with exon7 deletion.2.While 47（63.5%） cases express ABLΔexon7 and 8（10.8%） cases express ABL35 INS in 74 healthy people, 30（56.6%） cases express ABLΔexon7 and 5（9.4%） cases express ABL35 INS in 53 remission patients, 12（52.2%） cases express ABLΔexon7 and 3（13.0%） cases express ABL35 INS in 23 resistant patients. Three kinds of spliceosome in all group have no statistical difference.3. Detecting the relative expression of ABL^{Δexon7+35INS}, ABLΔexon7 and ABL35 INS spliceosome in BCR/ABL and ABL gene in 25 new cases, analysis showed no significant differences（P > 0.05）.4. ABL^{Δexon7+35INS}, ABLΔexon7 and ABL35 INS have similar functional domains, may eventually encoding the same proteins. Conclusion:1. This research found a novel spliceosome of ABL gene(ABL^{Δexon7+35INS}), which was not reported ever. We also found ABL35 INS are often accompany with exon7 deletion.2. The expression of ABL^{Δexon7+35INS}, ABLΔexon7 and ABL35 INS in BCR/ABL and ABL genehave no significant differences.3. The spliceosome of ABL^{Δexon7+35INS}, ABLΔexon7 and ABL35 INSmay not contribute to TKIs resistance.