Date Published: March 16, 2017
Publisher: Public Library of Science
Author(s): Muhammad Rauzan, Charles T. H. Chuah, Tun Kiat Ko, S. Tiong Ong, Giovanni Maga.
Chronic myeloid leukemia (CML) treatment has been improved by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate (IM) but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM) gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat) is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance.
Chronic myeloid leukemia (CML) is a disease defined by the presence of the BCR-ABL fusion protein, a constitutively active kinase produced by the 9,22 translocation which is sufficient to transform hematopoietic cells . ABL-specific tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), have significantly improved CML treatment, and prevent transformation to the deadly blast phase of the disease [2, 3]. However, patients with suboptimal TKI responses are at risk of developing TKI-resistance and progressing to blast phase . We previously described a germline intronic deletion polymorphism in the BCL2-like 11 (BIM) gene that was sufficient to mediate TKI resistance in CML . In cells which harbor the BIM deletion polymorphism, splicing of BIM pre-mRNA is biased toward the inclusion of exon 3 (E3) and exclusion of exon 4 (E4). Since the pro-apoptotic BH3 domain is encoded by E4, the deletion promotes the expression of non-apoptotic BIM isoforms (which retain E3 and encode the non-functional BIMγ protein) over pro-apoptotic isoforms (which exclude 3 and include E4, and encode the pro-apoptotic BIMEL, BIML, and BIMS proteins), thereby impairing the pro-apoptotic TKI response and confering partial TKI-resistance .
The presence of the BIM deletion polymorphism can confer TKI resistance in CML by maintaining a higher ratio of anti-apoptotic exon 3- to pro-apoptotic exon 4-containing BIM transcripts . SB939, however, can reverse the effect by reducing the ratio of exon 3- to exon 4-containing BIM transcripts (Fig 1A) which, translated to a significant increase in the induction of apoptosis in CML cells with the BIM deletion polymorphism (Fig 1B and 1C). Thus, SB939 corrects BIM pre-mRNA splicing in CML cell lines with the BIM deletion polymorphism in favour of apoptosis. We have previously shown that CML cells with the BIM deletion polymorphism were more resistant to imatinib when compared to their wildtype counterpart . Unlike SB939, imatinib does not appear to change ratio of exon 3- to exon 4-containing BIM transcripts (Fig 2A) but it can increase the basal level of total BIM proteins which is further enhanced when combined with SB939 (Fig 2B). Furthermore, the observed inhibition of BCR-ABL activity by SB939 is consistent with previous reports on the ability of HDAC inhibitors to regulate the stability of BCR-ABL that is mediated by HDAC6 & HSP90 [16, 17]. Thus, SB939 in combination with imatinib can result in further enhancement of apoptosis in CML cell lines with the BIM deletion polymorphism (Figs 2B, 2C, 3C and 3E). Our findings are consistent with a previous report involving the HDAC inhibitor vorinostat and an EGFR TKI gefitinib on non-small cell lung cancer (NSCLC) with the BIM deletion polymorphism where vorinostat was also able to reduce the ratio of exon 3- to exon 4-containing BIM transcripts and thus, resensitized NSCLC cells with BIM deletion polymorphism to gefitinib . CML patients with the BIM deletion polymorphism were found to respond poorly to standard dose of imatinib when compared to those without the deletion polymorphism . SB939 alone can induce apoptosis in a dose-dependent manner in primary CML cells with the BIM deletion polymorphism (Fig 4C). More importantly, our colony-formation assays showed that SB939 decreases the viability of primary CML progenitors and in combination with IM, can further reduce the viability of primary CML progenitors especially in those with the BIM deletion polymorphism that are more resistant to both SB939 and IM (compare Fig 4B to 4D). In summary, our results indicate that SB939 overcomes the BIM deletion polymorphism-induced TKI resistance and SB939 should be considered as a therapeutic strategy for CML patients with TKI resistance associated with this deletion polymorphism.