Polycomb Genes Are Associated with Response to Imatinib in Chronic Myeloid Leukemia
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Research Article For reprint orders, please contact: [email protected] 7 Research Article 2015/07/28 Polycomb genes are associated with response to imatinib in chronic myeloid leukemia Epigenomics Aim: Imatinib is a tyrosine kinase inhibitor that has revolutionized the treatment Francesco Crea1,2,3, Antonello of chronic myeloid leukemia (CML). Despite its efficacy, about a third of patients Di Paolo4, Hui Hsuan Liu1, discontinue the treatment due to therapy failure or intolerance. The rational Marialuisa Polillo4, 1 identification of patients less likely to respond to imatinib would be of paramount Pier-Luc Clermont , Francesca Guerrini5, Elena Ciabatti5, clinical relevance. We have shown that transmembrane transporter hOCT1 genotyping Federica Ricci5, Claudia predicts imatinib activity. In parallel, Polycomb group genes (PcGs) are epigenetic Baratè5, Giulia Fontanelli5, repressors implicated in CML progression and in therapy resistance. Patients & Sara Barsotti5, Riccardo methods: We measured the expression of eight PcGs in paired pre- and post-imatinib Morganti6, Romano Danesi4, bone marrow samples from 30 CML patients. Results: BMI1, PHC3, CBX6 and CBX7 Yuzhuo Wang1,2, Mario expression was significantly increased during imatinib treatment. Post-treatment levels Petrini5, Sara Galimberti*,‡,5 of CBX6 and CBX7 predicted 3-month response rate. Measurement of post-treatment & Cheryl D Helgason**,‡,1 BMI1 levels improved the predictive power of hOCT1 genotyping. Conclusion: These 1Experimental Therapeutics, BCCA Research Centre, 675 W 10th Avenue, results suggest that the expression levels of PcGs might be useful for a more accurate Vancouver, BC, V5Z 1L3, Canada risk stratification of CML patients. 2Vancouver Prostate Centre, Vancouver General Hospital, 2660 Oak Street, Keywords: BMI1 • CBX6 • CBX7 • imatinib • pharmacoepigenetics • Polycomb Vancouver, BC, V6H 3Z6, Canada 3Department of Life Health & Chemical Sciences, The Open University, UK Imatinib is the first tyrosine kinase inhibitor second-generation TKIs provided clinicians 4Department of Clinical & Experimental (TKI) successfully employed in the clini- with valuable therapeutic alternatives, thus Medicine, Section of Pharmacology, cal setting [1] . This small molecule inhibits having a positive impact on prognosis [5]. University of Pisa, Pisa, Italy 5 the constitutive kinase activity of the BCR– The identification of factors predicting Department of Clinical & Experimental Medicine, Section of Hematology, ABL protein, thereby inducing apoptosis in imatinib response could support rational University of Pisa, Pisa, Italy Philadelphia chromosome-positive chronic therapeutic decisions and foster therapeu- 6Department of Clinical & Experimental myeloid leukemia (CML) cells. Clinical tri- tic tailoring. For this reason, several groups Medicine, Section of Statistics, University 5 als demonstrated that imatinib treatment have investigated the role of genetic vari- of Pisa, Pisa, Italy was superior to conventional cytotoxic drugs ants in predicting CML patients’ response *Author for correspondence: Tel.: +39 347 0038656 as first-line therapy for CML [2]. A recent to imatinib [6]. We have recently associated Fax: +39 050 993378 update reported an 89% overall survival rate transmembrane transporter hOCT1 germ- 2015 [email protected] in CML patients treated with imatinib for line variants with imatinib pharmacokinet- **Author for correspondence: 5 years [3]. Despite these superlative results, ics and therapeutic activity [7]. Despite these Tel.: +39 604 675 8011 objective response to imatinib is quite vari- promising results, no genetic factor has been Fax: +39 604 675 8019 [email protected] able, and about 30% of patients must dis- conclusively linked to the clinical efficacy of ‡Authors contributed equally continue the treatment due to adverse events imatinib. or unsatisfactory clinical outcome [1] . It has Emerging evidence indicates that epigen- been shown that CML patients achieving etic factors could be at least as important a complete hematological response (CHR) as genetic ones in determining therapeutic within 3 months from the initiation of responses [8]. In particular, Polycomb group therapy are more likely to achieve complete genes (PcGs) are epigenetic effectors involved cytogenetic response (CCyR) and better in the progression of both solid and hemato- part of long-term clinical outcome [4]. The advent of logical malignancies [9]. These proteins are 10.2217/epi.15.35 © 2015 Future Medicine Ltd Epigenomics (2015) 7(5), 757–765 ISSN 1750-1911 757 Research Article Crea, Di Paolo, Liu et al. organized in two multimeric polycomb repressive com- ment. Twenty milliliter of EDTA-anticoagulated plexes (PRC1 and PRC2), which mediate gene silenc- bone marrow was used for total RNA extraction after ing and control cell specification in physiological condi- buffy coat preparation. Bone marrow was employed tions. According to the most widely accepted molecular for cytogenetic analysis, as stated by the international model, PRC2 catalyzes the trimethylation of histone guidelines [16], and for PcG expression analysis. Con- H3-Lys27, and subsequently PRC1 binds to this epigen- comitantly, 20 ml of peripheral blood was employed etic mark and catalyzes histone H2a monoubiquitina- for RNA extraction, banking and for analysis of the tion. These developmental regulatory mechanisms are BCR–ABL1/ABL1 International Scale (IS) ratio by the deregulated in cancer cells, leading to the coordinated GeneXpert Technology (Cepheid, Maurent-Scopont, silencing of several tumor-suppressor genes. Notably, France) after 3 and 6 months of therapy, as indicated by PRCs silence target loci in a context-specific manner European guidelines [17] . For 23 patients, we obtained that arises from their extensive combinatorial complex- paired pre- and post-imatinib bone marrow samples ity. For example, a BMI1+/PCGF2- PRC1 drives breast harvested 3 months after start of imatinib treatment. cancer tumorigenesis and metastatic progression, while The study was approved by the Ethics Committee of a BMI1-/PCGF2+ complex exerts tumor-suppressive Pisa University Hospital and by the University of Brit- functions [10] . Selected PRC members have been impli- ish Columbia/British Columbia Cancer Agency REB. cated in chemotherapy resistance [11] and have been pro- All patients gave informed consent to the study. Clini- posed as innovative therapeutic targets in hematological cal outcome data were recorded throughout the whole and solid malignancies [9]. duration of the follow-up. The clinical characteristics of In CML, the PRC1 member BMI1 has been identi- the patients are reported in Supplementary Table 1 (see fied as a prognostic marker [12] and as a downstream tar- online at www.futuremedicine.com/doi/full/10.2217/ get of the BCR–ABL tyrosine kinase [13] . BMI1 expres- epi.15.35). sion is increasingly higher in peripheral blood samples from unaffected, chronic phase CML and blastic phase Quantitative PCR CML patients [13] . In addition, BMI1 expression is suf- Gene-expression levels of PRC1 components were ficient to reprogram B-lymphoid progenitors, thereby assayed using reverse transcribed cDNA and SYBR generating B-cell acute lymphoid leukemia-initiating green primers following a previously described pro- cells [14] . Despite this evidence, no study has investi- cedure for amplification and relative gene-expression gated the role of BMI1 or any other PRC1 member in quantification [16] . GAPDH was used as a reference CML patients’ response to imatinib treatment. gene. Expression values were calculated using the Here, we describe pre- and post-treatment expres- 2-ΔΔCt method and normalizing the measurement sion levels of eight PRC1 members, in 30 CML to the highest Ct in the cohort. Primers sequences are patients treated with imatinib in the context of the reported in Supplementary Table 2. TIKlet study [15] . The primary aims of the present study were to Statistical analyses demonstrate that PcG expression can be measured in All statistical analyses were performed using SPSS 21.0 bone marrow samples obtained during routine clinical (SPSS Inc, Chicago, IL, USA) or GraphPad Prism 6.0 procedures and PcG expression levels in bone marrow software. samples are associated with imatinib clinical activity. At the third month of treatment, all cases were scored Our analysis identified the coordinated regulation of for hematological, cytogenetic and molecular response four PcGs upon TKI treatment, and suggested for the (MR). CHR was defined as white blood cells (WBC) first time that post-therapeutic levels of PcGs might be <10 × 109/l, platelets (PLT) <450 × 109/l and absence of used as predictors of therapeutic efficacy. We believe immature cells and palpable splenomegaly. Major cyto- that our preliminary analysis paves the way for more genetic response (MCyR) corresponded to <35% Phil- extensive studies examining the utilization of PcGs as adelphia-positive metaphases, while CCyR identified predictors of imatinib response in CML. cases without any Philadelphia-positive metaphases. The MR was scored according to the logarithmic Patients & methods reduction of the BCR–ABL1/ABL1 ratio in respect of Patients the 100% value of the diagnosis (MR1 = reduction of Thirty consecutive CML patients observed at the 1 log; MR3 = reduction of 3 logs). Hematology Division of the Pisa University, Italy, In order to ensure our measurements took into between January 2011 and June 2013 were enrolled in account differences