Differential expression of BIRC family genes in chronic myeloid leukaemia – BIRC3 and BIRC8 as potential new candidates to identify disease progression

Although the therapy of chronic myeloid leukaemia (CML) with tyrosine kinase inhibitors (TKI) is considered a major advance in oncology, a significant group of patients still develops drug resistance and does not benefit from targeted therapy (Marin et al, 2013). These patients are at high risk of progression from chronic phase (CML-CP) to almost inevitably fatal blastic phase (CML-BP) (or blast crisis). Therefore, there is a need to identify suboptimal responders to imatinib who should be switched to secondor third-generation TKI as early as possible (Marin et al, 2013). Although the Sokal and Hasford risk scores remain the most used prognostic indicators at diagnosis, reliable markers of suboptimal response and/or progression of the disease are needed. BCR-ABL1 transcript level monitoring is used to evaluate response to the therapy, and its predictive value at 3 and/or 6 months after starting TKI therapy has recently been shown (Neelakantan et al, 2013). Among other possible candidates, high levels of cancerous inhibitor of PP2A (CIP2A) may be an independent determinant of progression to blast crisis (Lucas et al, 2011). Searching for potential candidates of disease progression we have focused on the BIRC (baculoviral IAP repeat-containing; BIRC) gene family expression in various stages of CML. To date, there is no comprehensive analysis of the whole family of BIRC genes in CML. The BIRC family comprises eight functionallyand structurally-related members. Their common structural feature, a motif termed the baculovirus IAP repeat, is required for their cytoprotective function, hence the majority of BIRC serve as endogenous inhibitors of apoptosis (Deveraux & Reed, 1999). While the first human BIRC gene (originally termed BIRC1, now NAIP, encoding the NLR family, apoptosis inhibitory protein) was described in the neurodegenerative disorder, spinal muscular atrophy (Roy et al, 1995), most recent studies focus on the role of BIRC genes in various types of neoplasms. Overexpression of BIRC genes has been associated with cancer progression, multidrug resistance, poor prognosis and short survival in several cancers including haematological malignancies. One of the most extensively studied members of BIRC family – BIRC5 (BIRC5, also known as survivin) is upregulated in haematological malignancies and solid tumours, and was also shown to be overexpressed in CML-BP in comparison to CML-CP (Hernandez-Boluda et al, 2005). Higher expression of BIRC5 was also linked to higher Sokal score and was positively correlated with P-glycoprotein expression in late CML-CP (Reis et al, 2011). Recently, the disruption of BIRC3, leading to truncated protein expression has been implicated in resistance to fludarabine in chronic lymphocytic leukaemia (CLL) (Rossi et al, 2012). To elucidate the potential role of the BIRC family of genes in CML we investigated the relative expression of all eight known BIRC family members. We analysed sequential samples of cDNA from peripheral blood obtained from CML patients at various stages of the disease. Blood samples were taken after informed consent was obtained and reverse transcription (RT)-quantitative real time polymerase chain reaction (qPCR) experiments were performed according to MIQE (Minimum Information for Publication of qPCR Experiments) guidelines (Bustin et al, 2009). Initially we looked at samples from CML-CP patients at diagnosis and after development of TKI resistance (confirmed as a loss of cytogenetic response, n = 5). Four patients developed resistance to imatinib (one had V299L mutation); the fifth was resistant to imatinib, dasatinib and nilotinib (no mutation detected) (Fig. 1A). We then analysed sequential samples from patients with CML who progressed to either accelerated phase (CMLAccPh) (n = 2) or to CML-BP (n = 4). Contrary to the general view of the role of BIRC genes in tumour progression, we found a significant decrease in BIRC3 and BIRC8 expression in CML-CP after development of TKI resistance (Fig. 1A) and also in progression to CML-AccPh/BP (Fig. 1B). We also observed a marked increase in BIRC5 expression after progression to CML-AccPh/BP (as previously shown by Hernandez-Boluda et al, 2005) (Fig. 1B) but not in CML-CP samples obtained after development of TKI resistance (Fig. 1A). Expression of other BIRC genes, namely: NAIP (BIRC1), BIRC2, XIAP (BIRC4), BIRC6 and BIRC7 was comparable in all the studied stages of the disease (data not shown). To confirm these results analysis of a larger group of patients was performed. Samples were obtained from patients in either CML-CP at diagnosis prior to any treatment (n = 15) or in CML-BP (n = 11) (Fig. 2). To compare BIRC family genes expression in CML with that of normal haematopoietic cells, cDNA from healthy blood donors was also used (n = 10). In accordance with paired samples analysis, we observed downregulation of BIRC3 and BIRC8 expression in CML-BP, while BIRC5 was upregulated in CML-BP patients (as compared to CML-CP and healthy blood donors) (Fig. 2). There was no significant difference in the relative expression of other BIRC family members (data not shown). correspondence