Nilotinib interferes with cell cycle, ABC transporters and JAK-STAT signaling pathway in CD34+/lin- cells of patients with chronic phase chronic myeloid leukemia after 12 months of treatment

RESEARCH ARTICLE Nilotinib interferes with cell cycle, ABC transporters and JAK-STAT signaling pathway in CD34+/lin- cells of patients with chronic phase chronic myeloid leukemia after 12 months of treatment a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Trojani A, Pungolino E, Dal Molin A, Lodola M, Rossi G, D’Adda M, et al. (2019) Nilotinib interferes with cell cycle, ABC transporters and JAK-STAT signaling pathway in CD34+/lincells of patients with chronic phase chronic myeloid leukemia after 12 months of treatment. PLoS ONE 14(7): e0218444. https://doi.org/ 10.1371/journal.pone.0218444 Editor: Francesco Bertolini, European Institute of Oncology, ITALY Alessandra Trojani ID1*, Ester Pungolino1, Alessandra Dal Molin2☯, Milena Lodola1, Giuseppe Rossi3, Mariella D’Adda3, Alessandra Perego4, Chiara Elena5, Mauro Turrini6, Lorenza Borin7, Cristina Bucelli8, Simona Malato9, Maria Cristina Carraro10, Francesco Spina11, Maria Luisa Latargia12, Salvatore Artale13, Pierangelo Spedini14, Michela Anghilieri15, Barbara Di Camillo2☯, Giacomo Baruzzo2☯, Gabriella De Canal16, Alessandra Iurlo ID8, Enrica Morra17, Roberto Cairoli1 1 Division of Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milano, Italy, 2 Department of Information Engineering, University of Padova, Padova, Italy, 3 Department of Hematology, ASST Spedali Civili, Brescia, Italy, 4 Internal Medicine-Haematology, Desio Hospital, Desio, Italy, 5 Hematology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 6 Division of Hematology, Department of Internal Medicine, Valduce Hospital, Como, Italy, 7 Hematology Division, San Gerardo Hospital, Monza, Italy, 8 Hematology Division, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy, 9 Hematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milano, Italy, 10 Hematology and Transfusion Medicine, Sacco Hospital, Milano, Italy, 11 Division of Hematology– Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy, 12 ASST Valle Olona Ospedale di Circolo, Busto Arsizio, Italy, 13 ASST Valle Olona Sant’Antonio Abate, Gallarate, Italy, 14 Division of Hematology, Hospital of Cremona, Cremona, Italy, 15 ASST Lecco, Lecco, Italy, 16 Pathology Department, Cytogenetics, ASST Grande Ospedale Metropolitano Niguarda, Milano, Italy, 17 Executive Committee, Rete Ematologia Lombarda, Italy ☯ These authors contributed equally to this work. * Received: November 27, 2018 Accepted: June 3, 2019 Published: July 18, 2019 Copyright: © 2019 Trojani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The microarray data is available upon request due to ethical restrictions imposed by The Ethics Committee ASST Grande Ospedale Metropolitano Niguarda (Milan, Italy). Patients agreed to sign the informed consent document that did not allow the publication of patient-related data. In order to protect patient confidentiality, microarray data cannot be made publicly available. Interested researchers may contact Abstract Chronic myeloid leukemia (CML) is characterized by the constitutive tyrosine kinase activity of the oncoprotein BCR-ABL1 in myeloid progenitor cells that activates multiple signal transduction pathways leading to the leukemic phenotype. The tyrosine-kinase inhibitor (TKI) nilotinib inhibits the tyrosine kinase activity of BCR-ABL1 in CML patients. Despite the success of nilotinib treatment in patients with chronic-phase (CP) CML, a population of Philadelphia-positive (Ph+) quiescent stem cells escapes the drug activity and can lead to drug resistance. The molecular mechanism by which these quiescent cells remain insensitive is poorly understood. The aim of this study was to compare the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells from CP-CML patients at diagnosis and after 12 months of nilotinib treatment by microarray, in order to identify gene expression changes and the dysregulation of pathways due to nilotinib action. We selected BM CD34+/lin- cells from 78 CP-CML patients at diagnosis and after 12 months of first-line nilotinib therapy and microarray analysis was performed. GEP bioinformatic analyses identified 2,959 differently expressed probes and functional clustering determined some significantly enriched PLOS ONE | https://doi.org/10.1371/journal.pone.0218444 July 18, 2019 1 / 15 Gene profiling of patients with chronic myeloid leukemia at diagnosis vs. 12 months of nilotinib for microarray data access requests. All other data is available in the manuscript and as supplemental materials. Laboratory protocols are deposited here: http://dx.doi.org/10.17504/protocols.io.yncfvaw. Funding: Novartis funded reagents and materials for the experiments, and provided nilotinib for this study. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. There was no additional external funding received for this study. Competing interests: The authors have declared that non-financial competing interests exist. Novartis funded reagents and materials for the experiments in our laboratory, and provided nilotinib for this study. Novartis had no role in study design, data collection, analysis and interpretation, decision to publish or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There was no additional external funding received for this study. pathways between diagnosis and 12 months of nilotinib treatment. Among these pathways, we observed the under expression of 26 genes encoding proteins belonging to the cell cycle after 12 months of nilotinib treatment which led to the up-regulation of chromosome replication, cell proliferation, DNA replication, and DNA damage checkpoint at diagnosis. We demonstrated the under expression of the ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCD3 encoding proteins which pumped drugs out of the cells after 12 months of nilotinib. Moreover, GEP data demonstrated the deregulation of genes involved in the JAK-STAT signaling pathway. The down-regulation of JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, and SOS1 key genes after 12 months of nilotinib could demonstrate the up-regulation of cell cycle, proliferation and differentiation via MAPK and PI3K-AKT signaling pathways at diagnosis. Introduction CML results from unfaithful repaired DNA damage in a hematopoietic stem cell, but specific features of leukemic stem cells (LSCs) have not yet been fully understood. Several studies demonstrated that LSCs show a strong resistance to therapies in TKI-treated CML patients due to their ability to activate specific signaling biological pathways [1]. Although nilotinib is highly effect (...truncated)