Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia

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1 Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia Stefania Trino, Luciana De Luca, Vittorio Simeon, Ilaria Laurenzana, Annalisa Morano, Antonella Caivano, Francesco La Rocca, et al. Tumor Biology Tumor Markers, Tumor Targeting and Translational Cancer Research ISSN DOI /s

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3 DOI /s ORIGINAL ARTICLE Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia Stefania Trino 1 & Luciana De Luca 1 & Vittorio Simeon 1 & Ilaria Laurenzana 1 & Annalisa Morano 1 & Antonella Caivano 1 & Francesco La Rocca 1 & Giuseppe Pietrantuono 2 & Gabriella Bianchino 3 & Vitina Grieco 3 & Elisabetta Signorino 4 & Alberto Fragasso 5 & Maria Teresa Bochicchio 6 & Claudia Venturi 6 & Gianantonio Rosti 6 & Giovanni Martinelli 6 & Luigi Del Vecchio 7,8 & Daniela Cilloni 4 & Pellegrino Musto 9 Received: 30 April 2015 /Accepted: 7 July 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015 Abstract Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, Stefania Trino, Luciana De Luca, Daniela Cilloni and Pellegrino Musto contributed equally to this work. * Stefania Trino stefania.trino@crob.it Laboratory of Pre-clinical and Translational Research, IRCCS Referral Cancer Center of Basilicata, Rionero in Vulture, PZ, Italy Department of Onco-Hematology, IRCCS Referral Cancer Center of Basilicata, Rionero in Vulture, PZ, Italy Laboratory of Clinical Research and Advanced Diagnostics, IRCCS Referral Cancer Center of Basilicata, Rionero in Vulture, PZ, Italy Department of Clinical and Biological Sciences, University of Turin, Turin, Italy Hematology Unit, Ospedale Madonna delle Grazie, ASM, Matera, Italy Institute of Hematology BL. e A. Seràgnoli^, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy Biotecnologie Avanzate, CEINGE, Naples, Italy Department of Molecular Medicine and Medical Biotechnologies, BFederico II^ University of Naples, Naples, Italy Scientific Direction, IRCCS Referral Cancer Center of Basilicata, Rionero in Vulture, PZ, Italy along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease. Keywords Chronic myeloid leukemia. BCR-ABL1. Bridging integrator 1. Rab interactor 1. AXL Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder carrying the so-called Philadelphia chromosome (Ph + ), which is due to translocation t(9;22) (q34;11). This chromosomal alteration generates the BCR-ABL1 fusion gene producing a protein with a constitutive tyrosine kinase activity. BCR-ABL1 is necessary and sufficient for leukemic transformation [1] and is the specific target for current therapeutic approaches with tyrosine kinase inhibitors (TKIs) [2]. Although the role of BCR-ABL1 tyrosine kinase in CML is well established, possible additional mechanisms involved in CML progression remain poorly understood [3]. In particular, we were interested to investigate the putative interaction of BCR-ABL1 with proteins regulating endocytosis, according to the dataset previously published by Bruennert and

4 colleagues [4], showing a cluster of genes specifically involved in this process (bridging integrator 1, CBL, and RAB family members). Bridging integrator 1 (BIN1) proteins are members of the Bin/amphiphysin/RVS (BAR) family and contain three functional domains: BAR domain, Myc-binding domain, and Src homology (SH) 3 [5]. The BAR domain is a sensor of membrane curvature and acts as small GTPase effector in endocytotic complexes [6, 7]. BIN1 maps to long arm of human chromosome 2 (2q14) encoding at least seven different splice variants [5]. BIN1 function is complex and regulated by alternative splice isoforms. Splice isoform that interacts with c- Myc has a tumor suppressor role and is missing or inactivated in breast carcinoma, metastatic prostate cancer, gastric cancer, and hepatocellular carcinoma [8 11]. Tumor suppressor function of BIN1 is due to activation of caspase-independent apoptosis [12, 13]. During the first stage of tyrosine kinase receptor (RTK) endocytosis, BIN1 is recruited from Ras and Rab interactor 1 (RIN1) to aid the formation or resolution of early endosome structure [14]. Prolin-rich domain of RIN1 interacts directly with SH3 domain of BIN1; analogous interaction was proposed with RIN3 implying a link between RIN family members and membrane bendingbybin1[14, 15]. The RAS effector RIN1 activates RAB5 GTPase to promote early endosome fusion and RTK degradation. In addition, RIN1 activates ABL proteins to regulate actin remodeling. RIN1 coordinates the activation of RAB5 and ABL1 to determine the type of receptor endocytosis, the rate of receptor ubiquitination, and degradation or recycling. In particular, an excess of RAB5 over ABL1 signaling triggers membrane ruffling and macropinocytosis with increased RTK downregulation, while an excess of ABL1 over RAB5 induces an RTK recycling [14, 16, 17]. AXL is a member of the TAM family RTKs, which include Tyro3 (or Sky) and Mer receptors. AXL was for the first time identified in patients with chronic myeloid leukemia cells in 1991 [18]. The biological ligand for AXL receptor is the product of growth arrest-specific gene 6 (Gas6). AXL activation, by Gas6 binding, induces different mechanisms like cell survival, proliferation [19], and migration [20, 21]. Recent data reported AXL overexpression in several type of tumors [22 24] and its involvement in cancer progression [25] and resistance to therapies [22, 26]. In this paper, we aimed to investigate BIN1 and RIN1 expression in CML and their relationship with BCR-ABL1, focusing on their involvement in RTK endocytosis. AXL internalization studies were used as a proof of endocytosis function of the axis BCR-ABL1/BIN1 in K562 cell line following to treatment with Imatinib. Material and methods Patients Bone marrow (BM) and peripheral blood (PB) mononuclear cells from 14 control donors (6 BM and 8 PB) and 91 different Ph + CML patients (46 BM and 45 PB) were collected. All patients gave written informed consent according to the Declaration of Helsinki. Blood was drawn into EDTA tubes. Thirty-four CML patients were analyzed at diagnosis in chronic phase and 57 during the TKI treatment. Molecular response of CML patients was assessed according to the International Scale (IS) as the ratio of BCR-ABL1 transcripts to ABL1 transcripts, and it was expressed and reported as BCR-ABL1 % on a log scale [2]. Major molecular response (MMR) was defined as BCR-ABL1 expression <0.1 % and complete molecular response (CMR) as BCR-ABL1 <0.01 % (>4-log reduction from IRIS baseline) [2, 27]. Among treated patients, 17 did not achieve MMR (they are defined in this paper as <MMR), 9 were in MMR, 19 were in CMR, and 12 were TKIs resistant. Cell lines and treatment Human CML cell line, K562, and human embryonic kidney (HEK) 293 cell line were acquired from American Type Culture Collection (Rockville, MD, USA). K562 cells were maintained in RPMI 1640 medium (Gibco, Life technologies, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS) (Gibco), 1 % of penicillin-streptomycin (Gibco), and 4 mm of L-glutamine (Gibco). HEK-293 cells were maintained in DMEM medium (Gibco) supplemented with 10 % FBS, 1 % of penicillin-streptomycin (Gibco), and 2 mm of L-glutamine (Gibco). Cells were grown at 37 C in 5%CO 2. K562 and HEK-293 cells were treated with 1 μmof Imatinib for 24 and 48 h. RNA isolation and quantitative real-time-pcr (qrt-pcr) Mononuclear cells were obtained by Ficoll-Paque gradient centrifugation. Total RNA was extracted using Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer s instructions. Reverse transcription was performed using 1 μg of total RNA from each sample by Transcriptor First Strand cdna Synthesis kit (Roche, Indianapolis, IN, USA). qrt-pcr was performed to quantify BCR-ABL1, BIN1, and RIN1 mrna expression using the LightCycler 480 II (Roche). BCR-ABL1 transcripts were detected and quantified using Fast Start DNA Master Hybridization Probes (Roche Molecular Biochemicals) as BCR-ABL1/ABL1 Ratio (%). BIN1 and RIN1 mrna relative expression values were

5 detected using intercalating dye SYBR Green using Light Cycler 480 SYBR Green I Master (Roche) with 50 ng of cdna for each sample. BIN1 and RIN1 mrna relative expression values were normalized using ABL1 as reference gene and Universal Human Reference RNA (Agilent Technologies, Santa Clara, CA, USA) as calibrator and calculatedonthebasisofthee ΔΔCp method. The primer sequences of human ABL1, BIN1, and RIN1 mrna were as follows: ABL1 forward, 5 -CTTCTCGCTGGACCCAGTGA -3, ABL1 reverse, 5 -TGTGATTATAGCCTAAGACCCG GAG-3 ; BIN1 forward, 5 -ATGAGCAGTTTGAGCAGT GC-3, BIN1 reverse, 5 -CCAGCTTCTGGTGGTAATCC-3 ; RIN1 forward, 5 -GGCAGCAGAGGAGTAGCTTGA-3, RIN1 reverse, 5 -GCTTGCTGGCGCTAAAAGG-3. The amplicon lengths were 127 bp for ABL1, 234 bp for BIN1, and 91 bp for RIN1. Thermal cycling conditions were as follows: denaturation at 95 C for 10 min, and then 45 cycles of amplification for 10 s at 95 C and 10 s at 60 or 62 C for RIN1 and BIN1, respectively, followed by a final extension stage of 25 s at 72 C and then cooling to 40 C. PCR specificity was confirmed by examining the melting curves. Primer efficiency was 1.98, 1.84, and 2 for ABL1, BIN1,andRIN1,respectively. Each sample was analyzed in triplicate. Western blotting (WB) Total proteins were extracted from K562 and HEK-293 cell lines and PB of CML patients. Equal amount of protein extract (60 μg) was transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA, USA). The membranes were blocked with 5 % milk (Sigma-Aldrich, St Louis, MO, USA) at room temperature for 1 h and then incubated with primary antibodies directed toward GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ABL1 (Santa Cruz Biotechnology), P-Tyr (Santa Cruz Biotechnology), BIN1 (Santa Cruz Biotechnology), and RIN1 (Santa Cruz Biotechnology), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (BioRad). Protein bands were visualized using a chemiluminescent detection system and scanned with ChemiDoc system (BioRad). The bands were quantified by obtaining optical density (O.D) values using Image Lab software (BioRad). Flow cytometry K562 and PB mononuclear cells from 11 healthy subjects and 3 CML patients resistant to TKIs were analyzed by FACS Calibur cytometer using CellQuest software. Myeloid cells were gated from PB mononuclear cells. Cells were labeled with antihuman AXL-Alexa Fluor 488 mouse monoclonal antibody (IgG1, clone ; R&D Systems, Minneapolis, MN, USA) and with the specific isotypic control (IgG1, clone 11711; R&D Systems). To analyze total AXL protein levels, K562 cells were fixed and permeabilized with IntraCell kit (Immunostep, Salamanca, Spain) and subsequently labeled with AXL antibody or its isotypic control. Statistical analysis Statistical analysis was performed after evaluating normal distribution by Kolmogorov-Smirnov test. Differences in BIN1 and RIN1 expression among healthy donors and CML samples were estimated as appropriate using ANOVA followed by Bonferroni s post test or the nonparametric Kruskal-Wallis followed by Dunn s post test for multiple comparisons. Correlation analysis of BIN1/RIN1 and BCR-ABL1 mrna expression levels was performed using Spearman rank test. Student s t test was used to assess differences between Imatinib-treated cell lines and their control, and among healthy donors and CML-resistant patients. A p value <0.05 was considered statistically significant. Results BCR-ABL1 modulates BIN1 mrna expression in CML patients To evaluate the putative correlation of BCR-ABL1 and BIN1, we first assessed both transcript levels in PB and BM mononuclear cells from CML patients at diagnosis and during TKI therapy of CML. As expected, the high BCR-ABL1 transcript levels were progressively reduced during TKI treatment up to >4-log in CMR. Drug-resistant patients retained high levels of BCR-ABL1 transcript (Fig. 1a). BIN1 transcript levels were significantly lower at diagnosis compared to healthy controls (p<0.001), while they showed a significant increment during TKI treatment in <MMR (p<0.001), MMR (p<0.001), and CMR (p<0.001) compared with diagnosis (Fig. 1b). The same trend of upregulation in CML samples after TKI treatment respect to diagnosis patients was observed from BM or PB samples (data not shown). Spearman rank test demonstrated a strong inverse correlation between BIN1 and BCR-ABL1 mrna expression in TKI responsive CML patients at diagnosis and during treatment (r= 0.69, p=0.001). Notably, in TKI-resistant patients, we observed no difference of BIN1 expression as compared to diagnosis (Fig. 1b). The same result was obtained stratifying BM and PB samples (data not shown). BCR-ABL1 modulates RIN1 mrna expression in CML patients To assess if RIN1 was correlated with BIN1 in CML patients, we evaluated RIN1 transcript levels in the same cohort of subjects previously analyzed for BIN1. LikeBIN1, RIN1

6 Fig. 1 BIN1 modulation in CML patients at different stages of disease. a BCR-ABL1 mrna expression was evaluated as BCR-ABL1/ABL1 ratio (%) in BM and PB CML patients (diagnosis n=34, <MMR n=17, MMR n=9, CMR n=19, and resistant n=12) by qrt-pcr assay. b BIN1 mrna relative expression in BM and PB samples of healthy donors (control n=10) and CML patients (diagnosis n=34, <MMR n=17, MMR n=9, CMR n=19, and resistant n=12) was determined by qrt- PCR assay. Each data samples were normalized to the endogenous reference ABL1 by use of the E ΔΔCp. The relative expression values were used to design box and whisker plots. Kruskal-Wallis analysis assessed that BIN1 was differentially expressed between diagnosis and control and from the diagnosis to<mmr, MMR, and CMR. Statistically significant analyses are indicated by asterisks: ***p<0.001 transcript levels were significantly lower at diagnosis compared to healthy controls (p<0.05) and underwent a significant increment during TKI treatment in <MMR (p<0.01), MMR (p<0.01), and CMR (p<0.001) compared with diagnosis (Fig. 2). The same RIN1 increment in CML samples after TKI treatment respect to diagnosis patients was observed analyzing separately BM or PB samples (data not shown). In addition, RIN1 mrna was negatively correlated with BCR- Fig. 2 RIN1 modulation in CML patients at different stages of disease. RIN1 mrna relative expression in BM and PB samples of healthy donors (control n=10) and CML patients (diagnosis n=34, <MMR n= 17, MMR n=9,cmr n=19, and resistant n=12) was determined by qrt- PCR assay. Each data samples were normalized to the endogenous reference ABL1 by use of the E ΔΔCp. The relative expression values were used to design box and whisker plots. Kruskal-Wallis analysis assessed that RIN1 was differentially expressed between control and diagnosis and from the diagnosis to<mmr, MMR, and CMR. Statistically significant analyses are indicated by asterisks: *p<0.05, **p<0.01, ***p<0.001 ABL1 (r= 0.53, p=0.002). Again, in TKI-resistant patients, RIN1 expression was not significantly different from diagnosis (Fig. 2), also evaluating BM or PB samples (data not shown). BIN1 and RIN1 protein levels in CML patients We analyzed the protein levels of BIN1 and RIN1 in PB mononuclear cells of healthy donors and CML samples. BIN1 protein expression was significantly lower in CML patients at diagnosis compared to controls (p<0.05) and was significantly increased after TKI treatment in patients achieving MMR (p<0.001) and CMR (p<0.001), compared to diagnosis. RIN1 protein expression was lower (but without reaching statistical significance) in CML patients at diagnosis, compared to controls; RIN1 protein levels were found increased in MMR (p<0.001) and CMR (p<0.01) patients, compared to diagnosis (Fig. 3a, b). Therefore, BIN1 and RIN1 protein expression reflected both transcript levels. Moreover, comparing BIN1 and RIN1 protein expression in TKI-resistant patients to healthy controls, we observed a significant downregulation of both proteins (p<0.01) in CML-resistant patients (Fig. 3c, d). Pharmacological inhibition of BCR-ABL1 enhances BIN1 and RIN1 expression To better elucidate the inverse correlation between BCR-ABL1 and BIN1/RIN1, we performed in vitro experiments in K562 and HEK-293 cell lines. Both cell lines were treated for 24 and

7 Fig. 3 BIN1 and RIN1 protein levels in CML patients. a WB analysis of RIN1, BIN1, and GAPDH in PB samples of healthy donors (control) and CML patients at different phase of disease (diagnosis, MMR, and CMR). Two representative cases of each group are shown. b Absolute O.D. values of a were normalized to GAPDH and shown as mean+s.e.m. from two independent experiments (control n=4, diagnosis n=4, MMR n=4, and CMR n=4). c WB analysis of RIN1, BIN1, and GAPDH in 5 PB samples of resistant patients and 4 healthy donors (control). d Absolute O.D. values of c were normalized to GAPDH. Statistically significant analysis is indicated by asterisks: *p<0.05, **p<0.01, ***p< h with Imatinib to selectively inhibit the BCR-ABL1 tyrosine kinase activity. BCR-ABL1 inhibition by treatment in K562 was confirmed by a significant reduction of fusion protein expression (p<0.05) and its tyrosine auto-phosphorylation (P-Tyr) activity by WB experiments (Fig. 4a, b). A timedependent increase of BIN1 was observed at 24 and 48 h of Imatinib treatment (p<0.05). Likewise, RIN1 protein increased in response to Imatinib (p<0.05) (Fig. 4a, b). Moreover, BCR-ABL1 inhibition also induced an increase of BIN1 (p<0.05) and RIN1 mrna expression levels in K562 at 48h(Fig.4c). Steady levels of BIN1 and RIN1 proteins (Fig. 4d, e) and transcripts (Fig. 4f) were instead found in a nontumorigenic cell line HEK-293 after Imatinib treatment. Thus, we confirmed the inverse correlation between BCR- ABL1 tyrosine kinase activity and BIN1/RIN1 expression in CML. AXL RTK in CML-resistant patients and in K562 after in vitro treatment with Imatinib Altered endocytosis could be an interesting mechanism in TKI-resistant patients. Therefore, we evaluated cell surface AXL expression in PB myeloid cells in this subset of CML patients demonstrating a significant AXL overexpression in TKI nonresponder patients compared to control subjects (p<0.05) (Fig. 5a, b). In order to indicate that AXL endocytosis was associated to BCR-ABL1, we analyzed the expression of AXL receptor in K562 cells after in vitro treatment with Imatinib. We observed that AXL cell surface expression was significantly reduced in K562 after treatment (p<0.05) (Fig. 5c). Furthermore, AXL total expression levels remained comparable to untreated control (Fig. 5d) demonstrating that AXL was internalized into cytoplasm following BCR-ABL1 inhibition (Fig. 5c). Thus, AXL internalization paralleled BIN1/RIN1 upregulation (Fig. 4a, b, c), which is consistent with an impaired RTK endocytosis in CML. Discussion Different proteins involved in RTK endocytosis are altered in CML. For example, disabled-2 (DAB-2), an endocytic adaptor protein [21], and RAB5, a small GTPase, are involved in early endosome formation [28] and downregulated in CML patients at diagnosis. Their expression is further decreased in CML blast crisis and returns comparable to control subjects in remission phase [28, 29]. It has also been reported that the E3 ubiquitin ligase CBL, a key negative regulator of RTKs, is abnormally phosphorylated

8 Fig. 4 In vitro pharmacological inhibition of BCR-ABL1 by Imatinib. a WB analysis of P-Tyr, BCR-ABL1, RIN1, BIN1, and GAPDH in K562 treated with 1 μm of Imatinib for 24 and 48 h. b Relative O.D values of a were normalized to GAPDH and shown as mean+s.e.m. from three independent experiments. c BIN1 and RIN1 mrna relative expression wasdeterminedbyqrt-pcrassayink562treatedwith1μm of Imatinib for 24 and 48 h. Each data samples were normalized to the endogenous reference ABL1 by use of the E ΔΔCp.Thebar graphs represented mean+s.e.m. from three independent experiments. d WB analysis of RIN1, BIN1, and GAPDH in HEK-293 treated with 1 μm of Imatinib for 24 and 48 h. e Relative O.D values of d were normalized to GAPDH and shown as mean+s.e.m. from three independent experiments. f BIN1 and RIN1 mrna relative expression was determined by qrt-pcr assay in HEK-293 treated with 1 μm of Imatinib for 24 and 48 h. Each data samples were normalized to the endogenous reference ABL1 by use of the E ΔΔCp.Thebar graphs represented mean+s.e.m. from three independent experiments. Statistically significant analyses are indicated by asterisks: *p<0.05

9 Fig. 5 AXL expression in resistant patients and in K562 treated with Imatinib. a Overlay FACS histogram of surface AXL expression in a control subject (black lane) and a CML-resistant patient (gray lane). b Mean fluorescence intensity of AXL cell surface expression in control subjects and CML-resistant patients (control n=11, resistant n=3). Cytofluorimetric analysis of AXL cell surface (c) and total (d) expression in K562 treated with 500 nm of Imatinib for 24 h. The bar graphs represented mean+s.e.m. from three independent experiments. Statistically significant analyses are indicated by asterisks: *p<0.05 by BCR-ABL1 in CML cells, contributing to oncogenesis [30 32]. Here, we showed, for the first time, an inverse regulation of BCR-ABL1 and BIN1/RIN1, two key partners involved in endocytosis process [14, 15]. In particular, we demonstrated that these proteins are downregulated in CML patients at diagnosis and that the initial inhibition of BCR-ABL1 in response to TKIs was associated with significant increase of BIN1 and RIN1 expression, displaying an inverse correlation with BCR- ABL1 levels. This finding was highlighted in CML-resistant patients, who showed high expression of BCR-ABL1 due to therapy failure and downregulation of both BIN1 and RIN1 proteins. The inverse correlation between BIN1/RIN1 and BCR-ABL1 was further supported by in vitro experiments carried out on K562 cell line, where BIN1 and RIN1 expression were increased after Imatinib treatment. Furthermore, no variations were observed in a nontumorigenic cell line HEK- 293, thus confirming the relationship between BIN1/RIN1 and BCR-ABL1 fusion protein in CML. Overall, downregulation of RTK endocytosis inhibits the receptor degradation causing an increase of RTK levels at cell surface and leading to an uncontrolled receptor activation [30, 33]. In addition, downregulation of RAB5 [29] might contribute to the RTK degradation in CML [14]. Emerging evidence reported that AXL is involved in TKI resistance, resulting overexpressed in Imatinib and PD resistant cells and preventing Imatinib-mediated apoptosis [22, 34]; AXL expression was found also to increase in Ponatinib-resistant CML cell lines [35] and in nilotinibresistant CML cell line and patients [36]. We demonstrated higher expression of AXL receptor in TKI-resistant patients than control subjects. Furthermore, we showed that AXL receptor was internalized into cytoplasmatic compartment

10 following K562 Imatinib treatment, supporting the BCR- ABL1 link with endocytosis. In this setting, downregulation of BIN1 and RIN1 in CMLresistant patients could deregulate AXL endocytosis, causing an aberrant and constitutive receptor signaling. In conclusion, our data suggest that BCR-ABL1 regulates endocytosis (directly or in an indirect manner), causing the maintenance of RTK aberrant signaling. Since downregulation of RTK endocytosis could have an important role in tumor progression and proliferation, endocytosis reactivation could be considered a new potential therapeutic target for CML-resistant patients. Acknowledgments We are grateful to Prof. Maria Alessandra Santucci and Dr. Enrico Bracco for technical and scientific advice. This paper was supported by Italian Ministry of Health, Current Research Funds for IRCCS, CUP E66J DC was funded by grants from the following: AIRC B5 per 1000^ and AIL. Conflicts of interest References None 1. Cilloni D, Saglio G. 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