Abstract
Baculoviral IAP repeat containing 6 (BIRC6) is a member of the inhibitors of apoptosis proteins (IAPs), a family of functionally and structurally related proteins that inhibit apoptosis. BIRC6 has been implicated in drug resistance in several different human cancers, however mechanisms regulating BIRC6 have not been extensively explored. Our phosphoproteomic analysis of an imatinib-resistant chronic myelogenous leukemia (CML) cell line (MYL-R) identified increased amounts of a BIRC6 peptide phosphorylated at S480, S482, and S486 compared to imatinib-sensitive CML cells (MYL). Thus we investigated the role of BIRC6 in mediating imatinib resistance and compared it to the well-characterized anti-apoptotic protein, Mcl-1. Both BIRC6 and Mcl-1 were elevated in MYL-R compared to MYL cells. Lentiviral shRNA knockdown of BIRC6 in MYL-R cells increased imatinib-stimulated caspase activation and resulted in a ~20-25-fold increase in imatinib sensitivity, without affecting Mcl-1. Treating MYL-R cells with CDK9 inhibitors decreased BIRC6 mRNA, but not BIRC6 protein levels. By contrast, while CDK9 inhibitors reduced Mcl-1 mRNA and protein, they did not affect imatinib sensitivity. Since the Src family kinase Lyn is highly expressed and active in MYL-R cells, we tested the effects of Lyn inhibition on BIRC6 and Mcl-1. RNAi-mediated knockdown or inhibition of Lyn (dasatinib/ponatinib) reduced BIRC6 protein stability and increased caspase activation. Inhibition of Lyn also increased formation of an N-terminal BIRC6 fragment in parallel with reduced amount of the BIRC6 phosphopeptide, suggesting that Lyn may regulate BIRC6 phosphorylation and stability. In summary, our data show that BIRC6 stability is dependent on Lyn, and that BIRC6 mediates imatinib sensitivity independently of Mcl-1 or CDK9. Hence, BIRC6 may be a novel target for the treatment of drug-resistant CML where Mcl-1 or CDK9 inhibitors have failed.
Highlights
Chronic myelogenous leukemia (CML) is a malignancy of myeloid cells characterized by accumulation of mostly myeloid cells in the bone marrow and bloodstream [1,2]
In a previous study we compared the functional kinomes of MYL and MYL-R cells [14] using Multiplexed inhibitor bead (MIB)/MS technology [57] and showed that Lyn was substantially up-regulated in MYL-R compared to MYL cells [14]
MYL-R cells were treated for 1 hour with 10 nM ponatinib, a dual BcrAbl and Lyn inhibitor [12,16], or DMSO and MIB/MS kinome profiling and phosphopeptide enrichment were performed as described in Materials and Methods
Summary
Chronic myelogenous leukemia (CML) is a malignancy of myeloid cells characterized by accumulation of mostly myeloid cells in the bone marrow and bloodstream [1,2]. While successfully treated with the Bcr-Abl kinase inhibitor imatinib, high incidences of disease relapse and drug resistance have been recorded in CML patients [4,5,8,10,11]. Additional evidence suggest that imatinib resistance is due to Bcr-Abl independent mechanisms like enhanced expression of drug exporters (like P-glycoprotein) or activation of alternative kinase signaling cascades [7,14,15]. These challenges have led to the development of second generation (dasatinib and bosutinib) and third generation (ponatinib) inhibitors that target both Bcr-Abl and Src family kinases [8,12,16]. Most patients cannot benefit from this approach due to advanced age at diagnosis or lack of a suitable stem cell donor [3,17]
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