Nilotinib Deregulates Cell Cycle Checkpoints, ABC Transporters Genes and JAK-STAT Signaling Pathway of CD34+/Lin- Cells in Chronic-Phase Chronic Myeloid Leukemia (CP-CML) Patients after 12 Months of Treatment
Authors: Trojani Alessandra , Pungolino Ester , Rossi Giuseppe , D'adda Mariella , Lodola Milena , Dal Molin Alessandra , Baruzzo Giacomo , Perego Alessandra , Turrini Mauro , Elena Chiara , Borin Lorenza Maria , Iurlo Alessandra , Malato Simona , Spina Francesco , Latargia Maria Luisa , Spedini Pierangelo , Artale Salvatore , Anghilieri Michela , Maria Cristina Carraro , Bucelli Cristina , De Canal Gabriella , Morra Enrica , Cairoli Roberto 
Published: October 2019

Abstract
Introduction
Chronic myeloid leukemia (CML) is a stem cell disease characterized by the constitutive activity of the oncoprotein BCR-ABL that activates multiple signal transduction pathways. Tyrosine-kinase inhibitor (TKI) nilotinib successfully inhibits the activation and the proliferative function of BCR-ABL in patients with CP-CML. Despite the success of nilotinib, some patients become refractory suggesting the presence of a population of Philadelphia positive (Ph+) quiescent stem cells escaping the drug activity. Thus, the molecular mechanisms underlying CML remain poorly understood.
In this study, we enrolled 87 CP-CML patients (Pungolino et. al. Am J Hematol. 2018). Samples were collected on the behalf of the Rete Ematologica Lombarda (REL) the PhilosoPhi34 study (EudraCT: 2012-005062-34), which included 15 centers from Italy. We undertook gene expression profiling (GEP) of selected bone marrow (BM) CD34+/lin- cells of 80 patients at diagnosis vs. the same patients after 12 months of nilotinib to investigate gene expression changes induced by the treatment.
Methods
We isolated CD34+/lin- cells from BM samples in 87 patients at diagnosis whereas the same cells were also selected from 80/87 patients after 3, 6 and 12 months of nilotinib (Trojani et. al. Cancer Biomark. 2017). BM mononuclear cells (MNCs) as well as BM CD34+/lin- cells of all 80 CML patients were counted at diagnosis and during the treatment with nilotinib (at 3, 6, and 12 months, respectively). Standard FISH tested isolated BM CD34/lin- cells for the 87 patients at diagnosis, and for 80/87 patients after 3, 6 and 12 months of nilotinib treatment, respectively.
Therefore, we performed GEP analyses of selected BM CD34+/lin- cells of 80/87 patients at diagnosis vs. the same patients after 12 months of nilotinib treatment. Then, we executed bioinformatic preprocessing and correction for batch effects on raw microarray data. Finally, we conducted differential expression analysis and significantly perturbed genes were subjected to functional clustering.
Results
We observed a wide variability of the number of BM MNCs as well as the number of the BM CD34+/lin- cells among the 80 CP-CML patients at diagnosis and after 3, 6 and 12 months of nilotinib for each patient (Table 1). Figure 1 showed that the number of the BM CD34+/lin- cells dramatically decreased between the diagnosis and after 3 as well as 6 months of nilotinib. We noticed that the BM CD34+/lin- cells slightly increased between 6 and 12 months of nilotinib which might be caused by the gradual repopulation of the normal CD34+/lin- cells in the bone marrow as FISH results suggested.
FISH analysis detected CD34+/lin- Ph+ cells in 87 CP-CML patients at diagnosis. No positive Ph+ nuclei were detected on CD34+/lin- cells of 79/80 patients after 12 months of treatment (to categorize a sample as negative, at least 200 nuclei were examined). All of these 79 patients achieved at least complete cytogenetic response. 1/80 patient relapsed at 12 months. We conducted GEP analyses on 78 subjects because, due to experimental issues, two patients were not considered for differential expression analyses, as the microarray CEL files of the 12 months' samples were corrupted and missed probe intensities for most of the probes.
GEP analyses determined 2,959 significantly differently expressed probes between diagnosis and after 12 months of nilotinib treatment. Functional clustering identified some pathways significantly enriched between diagnosis and 12 months of nilotinib. Among these pathways, we found that ABCC4, ABCC5, and ABCD3 genes associated with ATP-binding cassette (ABC) transporters were up regulated at diagnosis. GEP results highlighted that 26 genes belonging to cell cycle, mitosis, DNA damage and repair were over expressed at diagnosis. Moreover, GEP data demonstrated that JAK-STAT signaling pathway was deregulated: JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, SOS1 were over expressed whereas IL22RA1 was under expressed at diagnosis vs. 12 months of nilotinib, respectively.
Conclusions
In summary, we reported that BM CD34+/lin- cells from CP-CML patients after 12 months of nilotinib were characterized by changes of expression of genes involved in cell cycle checkpoints and mitosis, ABC transporters genes that pump drugs outside form the cells, and JAK-STAT signaling pathway genes responsible for the proliferation, differentiation and cell survival in CML.

Disclosures
Rossi: Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.