Original Research published: 25 November 2019 doi: 10.3389/fphar.2019.01340 Immediate Effects of Dasatinib on the Migration and Redistribution of Naïve and Memory Lymphocytes Associated With Lymphocytosis in Chronic Myeloid Leukemia Patients Edited by: Carine Michiels, Beatriz Colom-Fernández 1, Anna Kreutzman 1†, Ana Marcos-Jiménez 1†, University of Namur, Valentín García-Gutiérrez 2, Carlos Cuesta-Mateos 1, Itxaso Portero-Sainz 1, Belgium Yaiza Pérez-García 1, Luis Felipe Casado 3, Fermín Sánchez-Guijo 4, Joaquín Martínez-López 5, Reviewed by: Rosa M. Ayala 5, Concha Boqué 6, Blanca Xicoy 7, Isabel Montero 8, César Soto 9, Raquel Paz 10, Jacques Barbet, Gabriela Silva 11, Lorena Vega-Piris 12, Juan Luis Steegmann 11 and Cecilia Muñoz-Calleja 1* Arronax, France Frederick E. Williams, 1 Servicio de Inmunología, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa (IP), Universidad University of Toledo, Autónoma de Madrid (UAM), Madrid, Spain, 2 Servicio Hematología y Hemoterapia, IRYCIS, Hospital Universitario Ramón United States y Cajal, Madrid, Spain, 3 Servicio de Hematología y Hemoterapia, Hospital Virgen de la Salud, Toledo, Spain, 4 Servicio de Hematología y Hemoterapia, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain, 5 Servicio de Hematología y *Correspondence: Hemoterapia, Hospital Universitario 12 de Octubre, Universidad Complutense de Madrid (UCM), CIBERONC, Madrid, Spain, Cecilia Muñoz-Calleja 6 Servicio de Hematología Clínica, Hospital Duran i Reynals, Institut Català d'Oncologia, Barcelona, Spain, 7 Servicio de cmunozc@salud.madrid.org Hematología, Servicio de Hematología Clínica, Institut Català d'Oncologia, Hospital Germans Trias i Pujol, Barcelona, José Carreras Leukemia Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain, 8 Servicio de Hematología, † These authors have contributed Hospital Universitario Virgen del Rocío, Sevilla, Spain, 9 Servicio de Hematología, Hospital Povisa, Vigo, Spain, 10 Servicio equally to this work de Hematología, Hospital Universitario de la Paz, Madrid, Spain, 11 Servicio de Hematología, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain, 12 Unidad de Metodología, Instituto de Specialty section: Investigación Sanitaria Princesa (IP), Madrid, Spain This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Introduction: Dasatinib is a dual SRC/ABL tyrosine kinase inhibitor used to treat chronic Frontiers in Pharmacology myeloid leukemia (CML) that is known to have unique immunomodulatory effects. In Received: 15 April 2019 particular, dasatinib intake typically causes lymphocytosis, which has been linked to Accepted: 22 October 2019 Published: 25 November 2019 better clinical response. Since the underlying mechanisms are unknown and SRC family Citation: kinases are involved in many cell motility processes, we hypothesized that the movement Colom-Fernández B, Kreutzman A, and migration of lymphocytes is modulated by dasatinib. Marcos-Jiménez A, García-Gutiérrez V, Cuesta-Mateos C, Portero-Sainz I, Patients, Materials and Methods: Peripheral blood samples from CML patients treated Pérez-García Y, Casado LF, with second-line dasatinib were collected before and 2 h after the first dasatinib intake, Sánchez-Guijo F, Martínez-López J, Ayala RM, Boqué C, Xicoy B, and follow-up samples from the same patients 3 and 6 months after the start of therapy. Montero I, Soto C, Paz R, Silva G, The migratory capacity and phenotype of lymphocytes and differential blood counts Vega-Piris L, Steegmann JL and Muñoz-Calleja C (2019) Immediate before and after drug intake were compared for all study time-points. Effects of Dasatinib on the Migration Results: We report here for the first time that dasatinib intake is associated with inhibition and Redistribution of Naïve and Memory Lymphocytes Associated of peripheral blood T-cell migration toward the homeostatic chemokines CCL19 and With Lymphocytosis in Chronic CCL21, which control the trafficking toward secondary lymphoid organs, mainly the Myeloid Leukemia Patients. Front. Pharmacol. 10:1340. lymph nodes. Accordingly, the proportion of lymphocytes in blood expressing CCR7, the doi: 10.3389/fphar.2019.01340 chemokine receptor for both CCL19 and CCL21, decreased after the intake including Frontiers in Pharmacology | www.frontiersin.org 1 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes both naïve CD45RA+ and central memory CD45RO+ T-cells. Similarly, naïve B-cells diminished with dasatinib. Finally, such changes in the migratory patterns did not occur in those patients whose lymphocyte counts remained unchanged after taking the drug. Discussion: We, therefore, conclude that lymphocytosis induced by dasatinib reflects a pronounced redistribution of naïve and memory populations of all lymphocyte subsets including CD4+ and CD8+ T-cells and B-cells. Keywords: Dasatinib, lymphocytosis, CCR7, migration, chronic myeloid leukemia INTRODUCTION Later, the same group showed that dasatinib intake induces a rapid, dose-dependent, and substantial mobilization of lymphocytes in Dasatinib is a second generation multitargeted tyrosine kinase blood peaking 1–2 h after oral intake (Mustjoki et al., 2013). More inhibitor (TKI) directed against BCR-ABL, which is approved for the recently, Paydas and Schiffer et al. investigated, in a large group treatment of Philadelphia chromosome positive (Ph+) leukemias, of patients, the actual incidence of lymphocytosis (Paydas, 2014; including newly diagnosed chronic myeloid leukemia (CML) in Schiffer et al., 2016). They confirmed that lymphocytosis is not the chronic phase (Talpaz et al., 2006; Jabbour and Lipton, 2013). In observed with other TKI and verified its association with higher addition to the BCR-ABL fusion protein, dasatinib inhibits with high response rates and significantly longer response duration. In potency several receptor tyrosine kinases (RTK) such as c-KIT or particular, patients with imatinib-intolerant or imatinib-resistant platelet-derived growth factor receptors (PDGFR) as well as a broad chronic phase who developed lymphocytosis after treatment range of cytoplasmic kinases including TEC, SYK, and SRC family with dasatinib had better progression-free and overall survival, kinases (SFK) (Bantscheff et al., 2007; Rix et al., 2007; Steegmann et suggesting a quite specific immunomodulatory effect of dasatinib. al., 2012). SFK are involved in mediating signal transduction from However, the reasons for the benefit of dasatinib-induced cell surface receptors and regulate fundamental cellular processes lymphocytosis and its underlying mechanisms are not clear and including migration, adhesion, invasion, angiogenesis, proliferation, deserve further research. In this study, we hypothesized that and differentiation (Parsons and Parsons, 2004). Therefore, SFK play dasatinib may induce lymphocyte mobilization from lymphoid a major role in the development, growth, progression, and metastasis organs or peripheral tissues, it may inhibit their physiological of a wide variety of human cancers, and their inhibition by dasatinib recirculation from the bloodstream to these organs and tissues, can suppress tumor growth and metastatic dissemination of several or both mechanisms. To our knowledge, there are no reports human cancer cell lines (Montero et al., 2011). Finally, several kinase studying the effects of dasatinib intake on lymphocyte migration targets of dasatinib are known to be important in the immune ex vivo. Therefore, our aim was to study whether the intake of system function. For example, ZAP-70, LCK, FYN, and ITK are dasatinib changes the migratory pattern of the lymphocytes of essential in T-cell signaling, while LYN, SYK, and BTK play key roles the patients as a possible cause of lymphocytosis, to gain insight in B-cell receptor signalosome function (Donato et al., 2003; Pene- into the immunomodulatory effect of this TKI. Dumitrescu and Smithgall, 2010). The physiological recirculation of lymphocytes between peripheral blood (PB), bone marrow, secondary lymphoid organs PATIENTS AND METHODS (SLO) and other tissues is regulated by a variety of chemokines and their corresponding receptors, which participate in the rolling Study Patients and Samples and firm adhesion phases of the lymphocyte extravasation cascade A total of 17 CML patients treated with dasatinib were included through the endothelium (Viola et al., 2008; Alon and Shulman, in this study. The patients were enrolled in the DASAPOST phase 2011). One of the most important chemokine/receptor pairs is II study (NCT01802450), designed to evaluate the efficacy and the CCR7 receptor and its ligands, the homeostatic chemokines safety of treatment change to dasatinib in patients previously CCL19 and CCL21, which mediate the main trafficking pathway treated with first line imatinib who had shown late suboptimal of T-cells from blood to SLO (Moschovakis and Förster, 2012). response (patients with complete cytogenetic response (CCyR) Another important pathway in lymphocyte homing to SLO and without major molecular response (MMR) after at least 18 bone marrow is the CXCR4/CXCL12 axis (Griffith et al., 2014). months of treatment) according to the ELN 09 recommendations. Because of its wide inhibition profile, dasatinib inhibits kinases DASAPOST study was approved by the Clinical Research Ethics also in healthy normal cells, such as lymphocytes, a mechanism Committee of Hospital Universitari Germans Trias i Pujol that probably underlies many of the immunomodulatory effects (Badalona, Barcelona, Spain). Patients were treated with dasatinib attributed to this drug (Steegmann et al., 2016). Among them, (Sprycel) 100 mg QD administered orally as continuous daily in 2009, Mustjoki et al. reported persistent clonal expansion of dosage. A group of healthy donors and a different cohort of CML cytotoxic T-cells or NK-cells in a distinct group of Ph+ patients patients were also included in some experiments. This last cohort receiving dasatinib therapy (Mustjoki et al., 2009; Kreutzman et al., included CML patients treated for more than three months with 2010; Nagata et al., 2010; Kreutzman et al., 2011; Powers et al., 2011). first line or second line dasatinib (100 mg QD) administered Frontiers in Pharmacology | www.frontiersin.org 2 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes orally as continuous daily dosage. The analysis of this second were added into the upper chamber of the transwell and were cohort was approved by the Clinical Research Ethics Committee allowed to migrate for 3h at 37°C in 5% CO2 atmosphere. of Hospital Universitario La Princesa (Madrid, Spain) (Reference The assay included two control wells: input cells (maximum number PI-561). The study was conducted in accordance with of cells = 500,000) and basal migration without chemoattractant the Declaration of the Helsinki principles. Written informed in the lower chamber. After 3 h, the migrated cells in the lower consent was obtained from each patient and healthy controls chamber were collected into tubes and antibodies against CD45, prior to sample collection. All measurements of BCR-ABL/ABL CD3, CD4, CD8, and CD56 were added to each tube. Events levels (international scale, IS) were centralized in a sole EUTOS were acquired for 210 s by flow cytometry (FACSCanto™ II) and (European Treatment and Outcome Study for CML) laboratory. data were analyzed using FACSDiva software. Gates were set as PB samples were obtained the day the patients switched to described in the Immunophenotyping section. dasatinib for DASAPOST patients, and at any time point after Two parameters were used to evaluate lymphocyte migratory three months of dasatinib treatment for patients in the other cohort capacity: the percentage of migrated cell and the migratory described above. For each patient, two samples were collected: one index. The percentage of migrated cells was calculated as the before the dasatinib dose (preintake sample) and the other one 2 h ratio between the number of cells migrated in response to CCL19 after drug intake (postintake sample). Only patients having paired and CCL21, or CXCL12 and the number of cells present in the preintake and postintake samples were included in the analysis. input well. The migratory index was defined as the ratio between Dasatinib used for in vitro assays was purchased from the number of cells migrated in response to each chemokine and Selleckchem, Houston, TX, USA (HPLC purity 99.8%). the number of cells migrated into the basal well. Similarly, in vitro effects of dasatinib were analyzed with PBMC from healthy donors, which were pretreated with dasatinib (100 Immunophenotyping nM) for 1 h and allowed to migrate for 3 h in the presence of the Immunophenotyping of preintake and postintake samples was inhibitor. performed on fresh whole blood within 24 h after extraction using the following monoclonal antibodies (mAbs): CD45-V500 (BD; clone HI30), CD3-PerCP (BD; clone SK7), CD8-APC-H7 (BD; Statistical Analyses clone SK1), CD16-Pacific Blue (BD; clone 3G8), CXCR4 (CD184)- Descriptive analysis of the sample: variables were described by their PECy7 (BD; clone 12G5), CD56-Brilliant Violet 421 (Biolegend; measures of central tendency (mean) and dispersion (standard clone HCD56), CCR7 (CD197)-PE (RyD; clone 150503), CD27-PE deviation (SD)) or standard error of the mean (SEM). Normality (BD; clone M-T271), CD45RA-FITC (BD; clone HI100), CD45RO- was tested with Shapiro-Wilk test and homoscedasticity was tested PECy7 (BD; clone UCHL1). Monoclonal isotype controls (IC) with Levene test. Preintake and postintake samples were compared were used to define basal levels of immunofluorescence. Whole by using both parametric (paired samples t-test) and nonparametric blood was incubated with the antibodies for 15 min followed by (Wilcoxon matched-pairs test) methods as appropriate. Adjusted standard lysis and washing steps. A minimum of 100,000 events p-value (p-adj) was calculated when an adjustment was necessary were acquired with FACSCanto™ II flow cytometer and analyzed due to multiple comparisons (fdr test). P values ≤ 0.05 were using FACSDiva software (both from BD Biosciences, USA). First, considered statistically significant. All analyses were performed lymphocytes were identified as CD45+ and then T, B and NK using GraphPad Prism version 6 software or R version 3.5.2. cells were identified as CD3+, CD19+ and CD3-CD16+CD56+ lymphocytes, respectively. CXCR4 and CCR7 expression was evaluated on CD8+ T-cells (CD3+CD8+) and CD4+ T-cells RESULTS (CD3+CD8-). Finally, naïve (TN), central memory (TCM), effector memory (TEM), and CD45RA+ effector memory (TEMRA) T-cells Dasatinib Intake Inhibits the Migratory were defined as CCR7+CD45RA+, CCR7+CD45RO+, CCR7- Capacity of T-Lymphocytes CD45RO+, and CCR7-CD45RA+ T cells, respectively, following To study the acute effects of dasatinib intake on lymphocyte the strategy showed in Figure 1. CD27 was used to identify naïve migration, we obtained samples from 17 CML patients the day they CD27- B-cells and memory CD27+ B-cells. began dasatinib treatment. PB samples were taken before the first drug intake (preintake) and 2 h later (postintake) from all patients. We first studied the acute effects of the first dasatinib intake on Migration Assay PBMC migration by performing a standard transwell assay using PB mononuclear cells (PBMCs) from preintake and postintake CCL19+CCL21 or CXCL12 as chemoattractants. Migrated cells samples were separated by Ficoll gradient centrifugation and were quantified and phenotyped for the main T lymphocyte serum starved in RPMI+0.1% BSA for 30 min. The chemokines subpopulations (CD4+ and CD8+ T-cells) and NK-cells. CCL19+CCL21 (1 μg/ml) and CXCL12 (0.5 μg/ml) (all Interestingly, the migratory index toward CCL19+CCL21 PeproTech, Rocky Hills, NJ, USA; SDS-PAGE and HPLC purity was significantly reduced in both CD4+ and CD8+ T-cells ≥ 98%) were added to 24-well plates in a final volume of 600 μl. (Figure 2A, upper panels). A similar tendency was found when Polycarbonate filter (5-μm pore size, 6.5-mm membrane, 10-mm the percentage of migration was analyzed, although differences thickness, Costar, Cambridge, MA, USA) transwell-inserts were did not reach significance (p = 0.097 for both CD4+ and CD8+ put on top of the wells and starved cells (5x105 cells in 100 μl) T-cells) (Figure 2A, lower panels). Frontiers in Pharmacology | www.frontiersin.org 3 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes FIGURE 1 | Gating strategy to identify differentiation stages of T-cells. Lymphocytes were selected with a SSC/CD45 gate. Then, CD4+ and CD8+ T-cells were identified as CD3+CD8- or CD3+CD8+ lymphocytes, respectively (upper left). CCR7 expression on CD4+ and CD8+ T-cells is shown in a histogram (upper right). Finally, T-cell maturation stages were defined based on the differential expression of CCR7, CD45RA, and CD45RO on CD8+ (lower left) or CD4+ T-cells (lower right). TN = naïve T-cells (CD45RA+CCR7+); TCM = central memory T-cells (CD45RO+CCR7+); TEM = effector memory T-cells; TEMRA = CD45RA+ effector memory (CD45RA+CCR7-). Conversely, the migration of NK-cells toward CCL19+CCL21 revealed that dasatinib inhibited spontaneous T-cell migration, was not affected by dasatinib intake. Furthermore, there was no whereas chemotaxis toward CCL19, CCL21, and CXCL12 was significant effect on the migration of CD4+ T-cells, CD8+ T-cells, not significantly affected (Figure 2B). and NK-cells toward CXCL12 in postintake samples (Figure 2A). Next, we aimed to analyze whether the inhibition observed in Figure 2A was due to a direct effect of dasatinib on the The First Dasatinib Intake Influences migratory capacity of lymphocytes. To this end, we performed CCR7 Expression on T-Lymphocytes migration experiments with PBMC from healthy donors (n = 11) In view of the previous results, an alternative explanation for the pretreated in vitro with dasatinib for 1 h. This experiment findings shown in Figure 2 could be the presence of a higher Frontiers in Pharmacology | www.frontiersin.org 4 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes FIGURE 2 | Dasatinib intake reduces CD4+ and CD8+ T-cell migration toward CCL19 and CCL21. (A) Blood samples were collected before (pre) and 2 h after (post) the first dasatinib intake in DASAPOST patients. Peripheral blood mononuclear cells (PBMCs) migration was studied by transwell assays, and compared between paired preintake (gray bars) and postintake (black bars) samples from DASAPOST patients (n = 12). The proportion of migrated cells in different cell populations is shown as migratory index (upper panels) and percentage of input (lower panels). (B) Migration transwell experiments with healthy donor PBMCs (n = 11) preincubated for 1 h (black bars) or not (gray bars) with dasatinib in vitro. Data are represented as means + SEM. Statistically significant p-adj values are shown on the graphs. ns, not significant. proportion of CCR7 negative T-cells in the PB of the patients panels). Furthermore, no significant differences were observed in immediately after dasatinib intake. To confirm this hypothesis, NK-cells (data not shown). we determined the percentage of CCR7+ and CXCR4+ cells in both CD4+ and CD8+ T-cell subsets and in NK-cells, both in preintake and postintake samples after the first dasatinib dose. This Dasatinib Favors the Accumulation of analysis first revealed that the percentage of expression of these Effector T-Cells and Memory B-Cells in chemokine receptors was very heterogeneous among patients Peripheral Blood before the first dasatinib intake (Figure 3A). This variability was CCR7 expression in combination with other molecules defines already observed in CML patients at diagnosis (data not shown). T-cell maturation stages. After the observation that dasatinib After dasatinib intake, the percentage of CCR7+ cells in both intake significantly decreased the proportion of CCR7+ T-cells, CD4+ and CD8+ T lymphocytes was significantly lower (Figure we performed a more detailed phenotypic analysis of lymphocyte 3A, upper panels), whereas no significant changes were observed subsets according to the expression of CCR7, CD45RA, and for CXCR4 in both CD4+ and CD8+ T-cells (Figure 3A, lower CD45RO. We observed an important redistribution of the Frontiers in Pharmacology | www.frontiersin.org 5 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes FIGURE 3 | (A) The first dasatinib intake changes the proportion of CCR7 positive T-cells. Percentages of CCR7+ and CXCR4+ lymphocytes in CD4+ and CD8+ T lymphocytes are compared between paired samples collected before (PRE) and 2 h after (POST) the first dasatinib intake from DASAPOST patients (CCR7, n = 17;CXCR4, n = 13). Data are represented as dot plots with lines connecting samples corresponding to the same patient. (B) T- and B-cell subsets redistribute differently in response to dasatinib therapy. Percentages of naïve (CCR7+CD45RA+)(TN), CD45RA+ effector memory (CCR7-CD45RA+)(TEMRA) and central memory (CCR7+CD45RO+)(TCM) CD4+ and CD8+ T-cells in total CD4+ and CD8+ T-cells (upper panels) and total lymphocytes (middle panels) are compared between paired preintake and postintake samples collected before (gray bars) and 2 h after (black bars) the first dasatinib intake from DASAPOST patients (n = 12). Percentages of memory B-cells in total B-cells and total lymphocytes in the same cohort of patients are shown (lower panels). Data in (B) are represented as mean + SEM. Statistically significant p-adj values are shown on the graphs. ns, not significant. Frontiers in Pharmacology | www.frontiersin.org 6 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes different subsets within both CD4+ and CD8+ T-cells. In We next analyzed CCR7 expression in these two groups of particular, dasatinib clearly increased the proportion of terminal patients and found that mobilizers had less CCR7+CD4+ T-cells effector CCR7-CD45RA+ T cells (TEMRA) in both the CD4+ and than non mobilizers (Figure 4D). A similar tendency was found CD8+ T-cell subsets whereas in total lymphocytes only CD8+ for CCR7+CD8+ T-cells, although the difference did not reach TEMRA changed (Figure 3B, upper and middle panels). significance (p = 0.098). Conversely, the proportion of T-cell subsets expressing CCR7, These two groups of patients were compared in terms of the which include central memory CCR7+CD45RO+ (TCM) and naïve migratory capacity of T lymphocytes, since CCR7 expression on CCR7+CD45RA+ (TN) T-cells, significantly diminished: CD8+ those cells tended to be different between the two groups. Both TCM in both total CD8+ T-cells and total lymphocytes, CD4+ TCM CD4+ and CD8+ T-cells showed lower migratory index toward in total lymphocytes, and TN in both CD4+ and CD8+ T-cells and CCL19+CCL21 in mobilizers (Figure 4E), whereas the migratory total lymphocytes (Figure 3B, upper and middle panels). index toward CXCL12 was not significantly different between Similarly, B-cell differentiation stages were identified by preintake and postintake samples from mobilizers (Figure 4E). CD27 expression. We observed higher proportions of memory Interestingly, no differences in CD4+ or CD8+ T-cell migration B-cells in total B-cells and total lymphocytes from the postintake to any of the chemokines tested were observed in non mobilizers samples (Figure 3B, lower panels). patients after dasatinib intake (Figure 4E). Finally, we investigated whether these findings occurred after every drug intake. To this end, preintake and postintake samples from the same patients after 3 and 6 months of treatment and from DISCUSSION AND CONCLUSION patients from another cohort treated for more than 3 months with dasatinib, were also analyzed. Similar as to the first drug intake, In addition to having potent effects on malignant cells, TKI also chemotaxis to CCL19 and CCL21 significantly diminished target kinases in normal cells, a process that is now known to in the postintake samples of patients previously treated with have consequences in the immune system (Ilander et al., 2014). dasatinib: percentage of input 17% in the preintake sample vs. In this context, dasatinib, a second-generation broad-spectrum 10% in the postintake sample (p = 0.0215). Furthermore, we TKI, has many unique effects on the immune system such as a could also confirm a redistribution of maturation stages and well-described rapid, dose-dependent, and substantial increment an accumulation of CD4+ and CD8+ TEMRA lymphocytes in PB of lymphocyte numbers in the PB 1–2 h after an oral intake (data not shown). (Mustjoki et al., 2013). Nevertheless, neither the molecular mechanisms underlying this redistribution of lymphoid cells nor its immunological effects have been completely characterized. A Minority of Patients Do Not Show Other groups have claimed many different reasons to explain Increased Absolute Lymphocyte Counts the lymphocytosis occurring during dasatinib therapy including After Dasatinib Intake viral reactivations and genetic mechanisms (Ilander et al., 2014; The composition of the main lymphoid subsets also changed Paydas, 2014; Schiffer et al., 2016). However, to our knowledge, from the first intake, with a significant increase of absolute counts this is the first report associating the effects of dasatinib on T-cell of B-cells, NK-cells, and T-cells (CD4+ and CD8+) 2 h after the migration in CML patients with lymphocytosis. Interestingly, the intake (Figure 4A, left panel). Furthermore, the proportion of total reduction of the migratory capacity of lymphocytes in response T-cells and the CD4+ T-cells subset in total lymphocytes decreased to CCR7 ligands in the post samples of patients is associated in the postintake samples, whereas the proportions of CD8+ with an accumulation of CCR7 negative effector T lymphocytes T-cells, B-cells, and NK-cells increased (Figure 4A, right panel). into PB but not with direct effects of dasatinib on the migration Accordingly, absolute lymphocyte counts in blood increased toward CCL19 or CCL21, as we could observe in the in vitro significantly after the first dasatinib intake in most patients. experiments. Conversely, we found a significant reduction of However, we observed that 3 out of 17 CML patients did not lymphocyte spontaneous migration. have changes in absolute lymphocyte counts in the postintake In order to confirm whether all these changes underlie the acute samples. Therefore, we next divided the patients into "mobilizers" lymphocytosis caused by dasatinib, we separately analyzed three (n = 14) and "non mobilizers" (n = 3) (Figure 4B). non mobilizers patients who did not have changes in the absolute The mobilizers had, in addition to increased absolute counts lymphocyte count in the postintake samples and compared them of all lymphocyte subsets in postintake samples (Figure 4C, with the mobilizer group of patients, which included most subjects left panel), also increased percentage of total lymphocytes, of this study. Interestingly, these non mobilizers did not undergo as well as NK-cells and B-cells (Figure 4C, right panel). any of the changes described here, including the redistribution of Conversely, a significant reduction of the proportion of CD3+ chemokine receptor expression or the change in the percentage of T-cells was observed in the postintake samples. Furthermore, migration. A limitation of these results is the low number of non the proportion of CD4+ T-cells in total T-cells significantly mobilizers. The differences are clear and our data are potentially decreased whereas CD8+ T-cells increased. As expected, interesting. However, to confirm these results, further studies with no differences in the proportion or absolute numbers of the a higher number of patients would be required. different populations were observed in those three patients who Our results therefore suggest that dasatinib-mediated did not have changes in lymphocyte counts between preintake lymphocytosis is partly due to a redistribution and accumulation and postintake samples (Figure 4C). of terminally differentiated CCR7 negative lymphocytes, both Frontiers in Pharmacology | www.frontiersin.org 7 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes FIGURE 4 | Non mobilizers do not show changes in lymphocyte subsets, chemokine receptor expression and migratory capacity. (A) Absolute counts (left panel) and percentages (right panel) of T-, CD4-, CD8-, B- and NK-cells in total lymphocytes are compared between paired preintake and postintake samples collected before (gray bars) and 2 h after (black bars) the first dasatinib intake from DASAPOST patients (n = 17). (B) Patients from DASAPOST study were classified as mobilizers (n = 14) or non mobilizers (n = 3) depending on whether their absolute lymphocyte counts increased after the first dasatinib intake or not. (C) Absolute counts (left panel) and proportions (right panel) of total lymphocytes, T-cells, NK-cells, B-cells, CD4+ and CD8+ T-cells are compared between paired preintake and postintake samples from mobilizers (n = 14) and non mobilizers (n = 3). (D) Expression of CCR7 on CD4+ and CD8+ T-cells is compared between paired preintake and postintake samples from mobilizers (n = 9) and non mobilizers (n = 3). (E) Migration of CD4+ and CD8+ T-cells toward CCL19+21 or CXCL12 is compared between paired preintake and postintake samples from mobilizers (n = 9) and nonmobilizers (n = 3). Data are represented as medians+SEM. Statistically significant p-adj values are shown on the graphs. ns: not significant. Red bars, mobilizers; Blue bars: non mobilizers; light colors: preintake samples; dark colors: postintake samples CD4+ and CD8+ T-cells, as well as memory B-cells, in the positive cells would eventually leave the bloodstream following a bloodstream. Moreover, we favor the hypothesis that dasatinib chemotactic gradient whereas CCR7 negative lymphocytes would recruits lymphocytes from SLO. Alternatively, this TKI may remain temporally "blocked" in PB due to yet undefined effects affect the capacity of terminally differentiated lymphocytes of of dasatinib on motility, adhesion and/or deformability of CCR7 exiting to the peripheral tissues, causing a lymphocyte blockage negative lymphocytes. on the bloodstream translated into higher absolute lymphocyte Still an alternative or additional in vivo effect of dasatinib could counts. The inhibition of spontaneous migration in the in vitro be the inhibition of chemokine signals required for access of experiments supports this last possibility in a scenario where CCR7 lymphocytes to tissues from the bloodstream, which would lead Frontiers in Pharmacology | www.frontiersin.org 8 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes to a transient accumulation of those lymphocytes and the typical From a clinical perspective, the positive association between lymphocytosis mediated by this TKI. In addition, dasatinib lymphocytosis and clinical response (Lee et al., 2011; Paydas, might block the intimate molecular connection between antigen 2014; Schiffer et al., 2016) would be justified not only by the receptor activation, both in B- and T-cells, and regulation of mobilization and accumulation of cytotoxic lymphocytes but cell adhesion to other immune cells or to stromal cells through also by effector CD4+ T-cells and memory B lymphocytes that molecules including integrins and chemokine receptors we have demonstrated here. It is important to highlight that this (Carrasco and Batista, 2006; de Gorter et al., 2007; Arana et al., rapid mobilization of effector lymphocytes may have prognostic 2008). This is quite intuitive since the immune synapse requires significance and occurs in the majority of patients, whereas a a temporary but stable interaction which is guaranteed by maintained lymphocytosis (defined as 3.6x109/L on at least two the adhesive connections between the cells involved and the consecutive occasions after at least four weeks of treatment) occurs surrounding microenvironment (Martín-Cófreces et al., 2018). in a moderate percentage of patients treated with dasatinib (Lee In this regard, dasatinib inhibits kinases that are essential in both et al., 2011; Mustjoki et al., 2013; Schiffer et al., 2016). In addition, the B-cell receptor and T-cell receptor signalosomes like LYN, we have shown for the first time that this rapid mobilization can SYK, and BTK, and LCK, ITK, and ZAP-70, respectively (Berg be already seen the day the patients begin the treatment and it is et al., 2005; Chakraborty and Weiss, 2014; Seda and Mraz, 2015). likely to occur daily, as indicated by the results obtained in the Moreover, it is important to note that some of these signaling same patient on the day the treatment begins and several months modules, including LCK and ZAP-70 are shared by chemokine later. A chronic daily mobilization of effector CD4+ T-helper, receptors and the T-cell receptor and that some of the dasatinib cytotoxic CD8+ T-cells, and memory B lymphocytes into tissues targets, e.g., LYN or ITK phosphorylate actin-regulatory proteins infiltrated by the CML, such as PB and bone marrow, may account like the hematopoietic linage cell-specific protein 1 (HS1). HS1 for the beneficial immunomodulatory effects of dasatinib. plays a crucial role as an actin-regulatory protein and, therefore, One possible downside of this effect is that lymphocytosis in lymphocyte cytoarchitecture (Carrizosa et al., 2009). It is likely may be associated with both beneficial effects and toxicity of that the inhibition of all these pathways by dasatinib results in dasatinib. Thus, the incidence of pleural effusion seems to be detachment and egress of lymphocytes from the SLO into PB. higher in patients who develop lymphocytosis, although it is This explanation has already been suggested for the mobilization only statistically significant in patients with advanced disease of chronic lymphocytic leukemia or mantle lymphoma cells (Paydas, 2014; Schiffer et al., 2016; Hughes et al., 2019). A possible that occurs with B-cell receptor inhibitors like ibrutinib and explanation to this association is that the extent of disruption of fostamatinib (Buchner et al., 2010; de Rooij et al., 2012; McCaig the endothelial cell monolayer (or other stromal cells of the SLO) et al., 2012; Chang et al., 2013; Purroy et al., 2017). In addition to is related to the magnitude of detachment and egress of effector these effects, dasatinib also inhibits the metastatic dissemination lymphocytes; either because detachment is a consequence of the of solid tumors, an effect which seems to be mediated mainly disassembly of endothelial cell-cell contacts, or because both through the inhibition of SFK (Montero et al., 2011). effects are caused by the action of the drug on the same target. Finally, we have recently demonstrated that dasatinib The increase in permeability would therefore be associated with disrupts the homotypic interaction of endothelial cells through a greater number of effector lymphocytes in peripheral blood, and phosphorylation of the myosin light chain which, in turn, is both circumstances would favor a pleural effusion with effector probably dependent on the inhibition of SFK (Kreutzman et al., lymphocytes, which may explain the exudative nature of the 2017). This effect is reversible but the temporary loss of integrity pleural effusion. It should be mentioned that in the largest study of the endothelial surface where lymphocytes roll could interfere of patients receiving dasatinib therapy, advanced age and dose with their extravasation and explain, at least in part, the temporal and longevity of treatment were found to be the only risk factors accumulation of lymphocytes in the bloodstream. associated to pleural effusion (Hughes et al., 2019). In that sense, As we have discussed above, dasatinib could be affecting the prevalence of effusion with the current 100-mg QD scheme different signal transduction pathways. At this stage it is unclear has been considerably reduced with respect to previous schedules, which molecular target/s are responsible for the effects of while the mobilization that we have described here seems to occur dasatinib described here. However, the identification of these in the majority of patients with that same scheme. In addition, targets is beyond the scope of our study. the lymphocyte mobilization induced by dasatinib seems to be Previous reports suggested that dasatinib induces a preferential controllable by drug dose (Mustjoki et al., 2013) and recent sub mobilization of cytotoxic lymphocytes (Mustjoki et al., 2009; Nagata analyses of DASISION trial showed that dose reductions for adverse et al., 2010; Kreutzman et al., 2011; Mustjoki et al., 2013). Our results effects did not affect efficacy. This fact would offer us a therapeutic extend those observations and further demonstrate that effector window to avoid toxic effects on the vascular endothelium but to CD4+ T-cells and memory B lymphocytes accumulate after dasatinib take clinical advantage of the immediate mobilization of circulating intake. These findings suggest that dasatinib is somehow affecting effector and memory cells from all lymphoid subsets. effector/memory lymphocytes independently of their linage. This connects with the interesting field of the different contractile, motile, adhesive, and migratory properties of lymphocytes depending on DATA AVAILABILITY STATEMENT their maturation stage and/or homing patterns (Jacobelli et al., 2013; Mueller et al., 2013; Martín-Cófreces et al., 2018). All datasets generated for this study are included in the article. Frontiers in Pharmacology | www.frontiersin.org 9 November 2019 | Volume 10 | Article 1340 Colom-Fernández et al. Dasatinib Redistributes Naïve and Memory Lymphocytes ETHICS STATEMENT RP, GS, and JS provided study material and patients. CM-C, BC-F, and AK drafted the manuscript. CM-C, BC-F, AK, This study was carried out in accordance with the recommendations VG-G, and JS edited and revised the manuscript. All authors of Clinical Research Ethics Committee of Hospital Universitari approved the final version of manuscript. Germans Trias i Pujol (Badalona, Barcelona, Spain) and Clinical Research Ethics Committee of Hospital Universitario La Princesa (Madrid, Spain) (Reference number PI-561). All subjects gave written FUNDING informed consent in accordance with the Declaration of Helsinki. This work was supported by Bristol Myers Squibb and Grants PI18/01163 from Fondo de Investigaciones Sanitarias to CM-C. AUTHOR CONTRIBUTIONS CM-C was co-financed by FEDER funds. CM-C, BC-F, AK, VG-G, and JS conceived and designed research. BC-F, AK, AM-J, YP-G, and IP-S performed ACKNOWLEDGMENTS experiments. 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K., Moscinski, L., Zhang, L., and Copyright © 2019 Colom-Fernández, Kreutzman, Marcos-Jiménez, García-Gutiérrez, Mustjoki, S. (2011). A molecular and functional analysis of large granular Cuesta-Mateos, Portero-Sainz, Pérez-García, Casado, Sánchez-Guijo, Martínez-López, lymphocyte expansions in patients with chronic myelogenous leukemia Ayala, Boqué, Xicoy, Montero, Soto, Paz, Silva, Vega-Piris, Steegmann and treated with tyrosine kinase inhibitors. Leukemia Lymphoma 52, 668–679. doi: Muñoz-Calleja. This is an open-access article distributed under the terms of 10.3109/10428194.2010.550074 the Creative Commons Attribution License (CC BY). The use, distribution or Purroy, N., Carabia, J., Abrisqueta, P., Egia, L., Aguiló, M., and Carpio, C. (2017). reproduction in other forums is permitted, provided the original author(s) and the Inhibition of BCR signaling using the Syk inhibitor TAK-659 prevents stroma- copyright owner(s) are credited and that the original publication in this journal mediated signaling in chronic lymphocytic leukemia cells. Oncotarget 8, 742– is cited, in accordance with accepted academic practice. No use, distribution or 756. doi: 10.18632/oncotarget.13557 reproduction is permitted which does not comply with these terms. Frontiers in Pharmacology | www.frontiersin.org 11 November 2019 | Volume 10 | Article 1340