Treatment of patients with Chronic Myeloid Leukemia in chronic phase (CML-CP) with tyrosine kinase inhibitors (TKIs) showed a substantially improving of patient life expectancy. However,it is becoming evident that persistent leukemic stem cells, which are in-sensitive to TKIs in their quiescent state, can lead to CML recurring.Nanog is a pluripotency gene associated to a vital role in neoplasia, correlating with cell proliferation, clonogenic growth, tumorigenicity, and therapeutic resistance. Our group carried out microarray experiments on Ph+ KCL22 cell line with a sensible (Kcl22-S) or resistant (Kcl22-R) phenotype to Imatinib (Ima). The gene expression of Nanog was significantly increased in the Kcl22-R. Thus, we sought to investigate the role of Nanog in the TKI resistance observed in patients with CML-CP. Methods: Real Time RT-PCR (RT-qPCR) for the expression of Nanog was con- ducted on Ph+ K562 cell line treated with increasing doses of Ima. Western blotting (WB) analysis was conducted for the protein expression of Nanog on K562 cells treated with 5uM Ima. RNA was purified from mononuclear cells of 27 CML patients at diagnosis and after 3 months of TKI treatment. Patients were monitored by RT-qPCR for the expres- sion of the fusion BCR-ABL mRNA. RT-qPCR for the expression of Nanog, was conducted. RT-qPCR results were normalized by the ex- pression of Gus mRNA (Normalized mRNA copy Number: NCN). Re- sults: We observed a significant increase of Nanog mRNA expression in K562 cells treated with 0.5 uM of Imatinib. Moreover, we were also able to observe a significant increase of Nanog protein expression in K562 cells treated with 1-5uM Imatinib by WB. In peripheral blood samples of CML patients at diagnosis, we observed a significant higher mRNA expression of Nanog in No-Optimal Responder compared to Optimal Responder patients (NANOg mRNA: 0.3±0.25 NCN by GUS mRNA vs 0.6±0.7 NCN by GUS mRNA) Conclusions and Summary: These data sug- gest that the expression analysis of Nanog at CML patient baseline, may assist in the early prediction of molecular response in patients treated with TKI. Further studies are ongoing to functionally evaluate whether Nanog is regulated by endogenous or exogenous signals in leukemic cells and evaluate the role of Nanog stemness power in induction of trans- formation of hematopoietic stem cell.

NANOG: ITS ROLE IN THE TKI RESISTANCE OBSERVED IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

CARUSO, SIMONA;ERRICHIELLO, SANTA;PISANO, IDA;QUINTARELLI, CONCETTA;IZZO, BARBARA;PANE, FABRIZIO
2015

Abstract

Treatment of patients with Chronic Myeloid Leukemia in chronic phase (CML-CP) with tyrosine kinase inhibitors (TKIs) showed a substantially improving of patient life expectancy. However,it is becoming evident that persistent leukemic stem cells, which are in-sensitive to TKIs in their quiescent state, can lead to CML recurring.Nanog is a pluripotency gene associated to a vital role in neoplasia, correlating with cell proliferation, clonogenic growth, tumorigenicity, and therapeutic resistance. Our group carried out microarray experiments on Ph+ KCL22 cell line with a sensible (Kcl22-S) or resistant (Kcl22-R) phenotype to Imatinib (Ima). The gene expression of Nanog was significantly increased in the Kcl22-R. Thus, we sought to investigate the role of Nanog in the TKI resistance observed in patients with CML-CP. Methods: Real Time RT-PCR (RT-qPCR) for the expression of Nanog was con- ducted on Ph+ K562 cell line treated with increasing doses of Ima. Western blotting (WB) analysis was conducted for the protein expression of Nanog on K562 cells treated with 5uM Ima. RNA was purified from mononuclear cells of 27 CML patients at diagnosis and after 3 months of TKI treatment. Patients were monitored by RT-qPCR for the expres- sion of the fusion BCR-ABL mRNA. RT-qPCR for the expression of Nanog, was conducted. RT-qPCR results were normalized by the ex- pression of Gus mRNA (Normalized mRNA copy Number: NCN). Re- sults: We observed a significant increase of Nanog mRNA expression in K562 cells treated with 0.5 uM of Imatinib. Moreover, we were also able to observe a significant increase of Nanog protein expression in K562 cells treated with 1-5uM Imatinib by WB. In peripheral blood samples of CML patients at diagnosis, we observed a significant higher mRNA expression of Nanog in No-Optimal Responder compared to Optimal Responder patients (NANOg mRNA: 0.3±0.25 NCN by GUS mRNA vs 0.6±0.7 NCN by GUS mRNA) Conclusions and Summary: These data sug- gest that the expression analysis of Nanog at CML patient baseline, may assist in the early prediction of molecular response in patients treated with TKI. Further studies are ongoing to functionally evaluate whether Nanog is regulated by endogenous or exogenous signals in leukemic cells and evaluate the role of Nanog stemness power in induction of trans- formation of hematopoietic stem cell.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/629947
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