Abstract
Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers.
Highlights
Small-molecule targeting of mutant oncoproteins in human cancers has resulted in significant improvements in progression free survival (PFS) and overall survival (OS) compared to conventional chemotherapy [1,2,3]
We identified several regions with consecutive and/or overlapping deletions which consistently increased or decreased exon 3 (E3)/exon 4 (E4) ratio as measured by real-time reversetranscription polymerase chain reaction (RT-PCR) using junction reverse primers with proven specificity (Supplementary Figure 1A) which captured the splicing change conferred by the deletion polymorphism (Supplementary Figure 1B)
This study shows that it is feasible to switch BCL-2 interacting mediator of cell death (BIM) splicing so as to enhance production of proapoptotic BIM isoforms, and thereby resensitize resistant BIM deletion-containing chronic myeloid leukemia (CML) cell lines to imatinib
Summary
Small-molecule targeting of mutant oncoproteins in human cancers has resulted in significant improvements in progression free survival (PFS) and overall survival (OS) compared to conventional chemotherapy [1,2,3]. Other TKIs like erlotinib exhibit >70% response rates in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) activating mutations [5,6,7]. Other human cancers like c-KIT-driven gastrointestinal stromal tumours and BRAF-driven melanomas have available TKIs. The BIM (BCL2-Interacting Mediator of cell death, known as BCL2L11) protein is a BH3-only proapoptotic member of the BCL-2 family that is absolutely required for the cancer killing of such drugs via the intracellular or mitochondrial pathway, elicited via upregulation of its expression at different levels [8,9,10,11,12,13,14]. Despite the high overall TKI response rates, significant heterogeneity in both the depth and duration of responses exists [15,16,17]
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