ALBERT

Description: Imatinib treatment has radically changed the prognosis of patients with CML. However, around 23-32% of patients discontinue this therapy due to lack of efficacy. Second generation TKI are available, which exhibit greater potency, so there is scope to further improve the strategy of selection of the appropriate TKI in the first line setting. Measurement of PTCH1 expression at diagnosis has been proposed as a useful strategy to tailor first line therapy as patients with low PTCH1 expression showed a worse outcome. Signalling via SMO is inhibited by non-Hedgehog ligated PTCH1 in Hedgehog pathway. SMO and PTCH1/SMO expression ratio has also been related to response to imatinib. Our aim was to corroborate imatinib outcome prediction in a different cohort and compare the prognostic power of PTCH1, SMO and PTCH1/SMO. We have retrospectively studied 101 pre-treatment samples of patients who received first-line imatinib from 14 Spanish centres. Clinical data were recorded in the Spanish CML Registry (RELMC). Informed consent was signed by every patient. Predesigned assays for PTCH1, SMO and GUSB (control gene) were used in single qPCR reactions in duplicates and run in an ABI 7900. Receiver operating characteristic (ROC) curves were plotted for PTCH1, SMO and PTCH1/SMO expression ratio and the area under curve (AUC) was used to compare its capacity to predict imatinib failure free survival (IFFS). For the measurement with higher AUC a threshold was set to divide patients with high and low expression. TKI failure was defined as loss of CCyR, progression to advanced phase disease, death or change in treatment from imatinib due to lack of efficacy. Secondary endpoints were: probability of achieving

Description: Background: Finding the best therapy option to treat myelofibrosis (MF) represents a huge challenge. Ruxolitinib (R) is a potent JAK1/2 inhibitor that has demonstrated improved survival and symptomatology in MF patients. There are a lot researches looking for the best combination of JAK inhibitor to improve its efficacy. In a previous work, we screened different drugs to evaluate if they were synergistic with ruxolitinib, we found that ruxolitinib was synergistic with nilotinib (N) and Prednisolone (P) as well as other drugs including bortezomib and HSP-90 inhibitors. Aim: To study the synergistic behavior and mechanism of actions of ruxolitinib in combination with nilotinib and prednisolone both in patient samples and cell lines. Methods: We have studied 20 secondary or primary MF patients and cell line: BA/F3 transfected with mutated JAK2 V617F (BA/F3 JAK2V617F). Classical assay was performed for patient samples: mononuclear cells isolated from peripheral blood were cultured during 2 weeks in Methocult TM GF_H4535 with 20 ng/ml IL-3, and 50 ng/ml SCF, in presence of increasing concentrations of R, N or P or their combinations. Also, mononuclear cells were cultured as described above, during 2 weeks but without drugs. Then, cells were washed with PBS and cultured 72 hours in RPMI 10% FBS and plated at 15,000 per well in 96-well plates with increasing concentrations of drugs. After both assay, cells were labeled with Annexin V and CD13 and analyzed in the Exvitech platform, an automated multiparametric flow cytometry platform. For cell lines, these were cultured in RPMI 10% FBS in the presence of increasing concentration of drugs. After 48 h of incubation, we performed a wst-8 assay to evaluate cell viability. To analyzed effect of treatment on survival and proliferation signaling pathways, cell lines were treated for 30 min with R 0.032 µM, N 1.6 µM and P 0.8 µM or combinations and total lysates were collected. To perform western blot, we use phospho-STAT5 (Tyr 964) and STAT5 after stripping as primary antibody and β-actin as load control. Graphpad Prism or XLFit was used to analyze dose-response curves. Synergism will be evaluated by the Median Effect methods described by T-C Chou and P. Talalay. Results: In patient samples when performed the classical assay, we found synergistic interaction in all combination in most of the patients (Table 1). Briefly, all combinations showed synergetic behavior (C

Description: Abstract 5054 Objectives: In the present study we have determined the antitumor effects, molecular mechanisms of action and potential synergies between Ruxolitinib and the molecular targeted drugs Sorafenib, KNK437, Dasatinib and Perifosine, in Philadelphia negative chronic myeloproliferative neoplasms (MPN). Materials and methods: The cytotoxic and cytostatic effects of the different compounds were determined in the JAK2V617F positive cell lines HEL and Ba/F3 JAK2V617F EPOR, and in mononuclear and bone marrow CD34 positive cells from 19 MPN patients. The effects of different drugs at the molecular level were analyzed by flow cytometry and western blot. The IC50 and the synergy combination index (CI) were determined with GrafPath Prism software and Calcusyn software respectively. Results: Ruxolitinib (IC50PV=15nM), as well as Sorafenib (IC50PV=8uM), KNK437 (IC50PV=100uM) and Perifosine (IC50PV=15uM), was able to inhibit proliferation in cell line models and in cells from MPN patients. Dasatinib (IC50PV=12nM), however, was only effective in patient samples, with similar effects in both MPN patients and control donors. Moreover, Dasatinib, KNK437 and Sorafenib showed a strong synergistic effect in combination with Ruxolitinib (CIPV

Description: Today, cardiovascular disease has a great impact on the global population due to its high prevalence. One challenge that cardiovascular patients face to achieve a better prognosis is to follow a healthy diet. This study focused on psychological factors linked to adaptation to a healthy diet in these patients. The main objective was to analyze the interrelationship between motivation to follow a healthy diet and self-efficacy to adhere to the Mediterranean diet with life satisfaction over time. The sample consisted of cardiovascular patients who were assessed at three measurement moments (NT1 = 755; NT2 = 593; NT3 = 323, average interval time: nine months). Correlation analyses showed that self-efficacy, motivation, and life satisfaction followed a pattern of positive relations across the three measurements. A time effect over the study variables was also observed. The results of path analyses showed that self-efficacy positively predicted autonomous motivation, which in turn was associated with patients’ life satisfaction. This interrelation was stable over a period of 18 months. Moreover, life satisfaction predicted self-efficacy nine months later. Psychological interventions might be a positive resource for cardiovascular patients, since psychological variables influence their life satisfaction and their subsequent quality of life in their new health condition.

Description: Myelofibrosis (MP) is characterized by stem cell-derived clonal myeloproliferation and a reactive cytokine-driven inflammatory bone marrow fibrosis (BMF). BMF plays an important role in MF symptom such us extramedullary hematopoiesis that implies hepatosplenomegaly and bone marrow failure with anemia and thrombocytopenia. Ruxolitinib is the only drug approved for MF therapy. It was associated with significant reduction in symptomatic splenomegaly and improved constitutional symptoms (Harrison et al. N Engl J Med. 2012 Mar 1;366(9):787-98; Verstovsek et al. Haematologica2015 Apr;100(4):479-88). Recently, Masarova and co-workers reported a case a MF patient who achieved a complete remission, including the complete disappearance of BMF (Masarova et al. Blood. 2016 Jun 20). In a previous work (Arenas et al. Blood Volume 122, Issue 21 (ASH Annual Meeting Abstrat)), we tried to identify the most promising synergistic drugs combinations for a Ruxolitinib. Nilotinib was selected between a set of synergistic drug. It is a BCR-ABL inhibitor but also is a potent PDGF-R inhibitor. Interestingly, different studies reported an anti-fibrotic activity in liver (Liu et al. J Hepatol. 2011 Sep;55(3):612-25.) and skin (Akhmetshina et al. FASEB J. 2008;22(7):2214-22). For these reasons we decided study the effect of the combination of Ruxolitinib and Nilotinib in BMF. To determine the ideal concentration of TGF-β to induce collagen mRNA expression in HS27a, we treated the culture of HSP27a with increasing concentration of TGF-β (0.625-10 ng/mL) during 24h. The best TGF-β concentration was 2ng/mL, with a fold change of 2.74 (SD=0.89) (p

Description: Background Identifying the most promising synergistic drugs combinations for a drug is a challenge for researchers. Identification of optimal combinations are key and should be translated in better designed clinical trials with fewer patients and ultimately more effective treatments. Ruxolitinib is a potent JAK1/JAK2 inhibitor that has demonstrated improved survival, rapid and durable improvements in splenomegaly, in patients with myelofibrosis (MF), however although improve survival in most of patients it does not change the natural history of the disease. There is however always a drive to improve outcomes and we hypothesize that treatment with a synergistic drug could enhance the activity in MF. Aim To design an ex-vivo model, based on flow cytometry, to identify the most synergistic drugs with Ruxolitinib in cell lines and primary samples from MF patients. Methods We have studied five secondary or primary MF patients (n = 5) and one cell line, BA/F3 transfected with mutated JAK2 V617F (BA/F3 JAK2V617F). We combined Ruxolitinib with a panel of 30 drugs whose mechanism of action is implicated in proliferation, differentiation and survival, cell-cycle inhibition, protein stabilisation, epigenetic, immune response. Briefly, mononuclear cells from peripheral blood, isolated, was cultured in Methocult TM GF_H4535 supplemented with 20 ng/ml interleukin (IL)-3, and 50 ng/ml stem-cell factor (SCF). After 2 weeks incubation, viable cells were plated at 15.000 per well in 96-well plates in increasing concentrations of each drug, alone or in combination with Ruxolitinib, in 8 or 5 point dose response curve. After 72 hr incubation, we performed a multiparametric flow cytometry, using Annexin V-fluorescein isothiocyanate (FITC) and CD13 to monitoring drug sensibility of myeloid lineage, in the ExviTech platform for screening by flow cytometry. Synergism will be evaluated by the Median Effect methods described by T-C Chou and P. Talalay. Regarding the cell line model, it confirms the results obtained in patients samples: Panobinostat was the most potent drugs tested in the assay with an IC50 of 86 nM and the most synergistic drugs with Ruxolitinib was Everolimus (CI = 0.613 when Ruxolitinib and Everolimus were 370 nM and 7.41 μM respectively). Conclusions This Vivia Ex vivo platform is highly efficient to study multiple synergisms of drugs in myeloproliferative diseases. We can test 30 drugs, inhibitors of multiple signaling pathways, epigenetics and immune response, alone or in combination with Ruxolitinib and test its activity and its potential synergy with Ruxolitinib. Based in these results, clinical trials combination Ruxolitinib with BKM120 (ongoing), Everolimus and LDE225 (ongoing) could potentially be explored in phase I clinical trials. Disclosures: Hernandez-Campo: Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Equity Ownership. Martínez-López:Vivia Biotech: Honoraria; Novartis: Research Funding.

Description: Acute Myeloid Leukemia (AML) is the most common type of acute leukemia in adults and the second in children. The overall survival is less than 35% and 60% for adults and children respectively. Activating mutations of FLT3 are now recognized as the most common molecular abnormality in this disease, and the poor prognosis of patients harboring these mutations renders FLT3 an obvious target of therapy. Although different tyrosine kinase inhibitors (TKI) have been used for this purpose, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. This strategy could be improved by rationally combining TKIs with other agents. In this work, we have explored by phosphoproteomics the alternative pathways activated after TKI treatment in vitro and ex vivo. The phosphoproteome profile of the bone marrow from a FLT3-AML patient before and after TKI treatment, studied by LC-MSMS after IMAC enrichment, suggested the activation of Ras-Raf-MEK-ERK1/2 pathway as a possible mechanism for TKI resistance, which could be avoided by dual inhibition using the MEK inhibitor trametinib. Therefore, we characterized the effect of trametinib in combination with the TIK pazopanib and sorafenib by the in vitro cell viability assay using WST8in the FLT3-ITD AML cell line MOLM13. As it is presented in figure 1a, trametinib showed an IC50 value in the low-nanomolar range (5.4 nM) and this MEKI produced a strong synergy (0.5

Description: Introduction Acute myeloid leukemia (AML) is a clonal disease with a reduced life expectancy due to a high relapse rate. One explanation is that leukemic stem cells (LSC) evade the action of conventional chemotherapy due to their quiescent state. Several mechanisms have been proposed that regulate their quiescence, however, by analogy with normal hematopoietic stem cells, a key role may be carried out by the signaling pathways Notch and Hedgehog (Hh). The objectives of this study are to analyze the role of Notch and Hh pathways in the quiescence of LSC and to verify if the pharmacological inhibition of the Notch and Hh pathways decreases the percentage of quiescent LSC. In this way, LSCs would be sensitized to chemotherapy treatments and the high relapse rate of AML could be reduced. Methods Expression of GLI1, a transcription factor of the Hh signaling pathway and NOTCH Internal Cleaved Domain (NICD) were analyzed in the hematopoietic stem and progenitor cells of four patients diagnosed with AML. The selection of quiescent fraction was performed by flow cytometry using anti-CD34-FITC, anti-CD117-PerCP, anti-CD45PE-Cy7, anti-CD38-APC-Cy7 and anti-KI67-BV510 antibodies. KI67 negative cells were considered quiescent. The activation of NOTCH and Hh pathways was studied using rabbit anti-GLI, anti-NICD primary antibodies and anti-rabbit BV421 secondary antibodies. Results were expressed in median (range) or mean ± standard deviation. Dose-response curves of inhibitors of the Notch pathway (BMS-906024, inhibitor of γ-secretase), Hh pathway (BMS-833923, SMO inhibitor and GANT61, GLI inhibitor), and cytarabine (AraC) were made to study drug potency in the OCI-AML3 cell line. We also analyzed its synergistic behavior in combination with Arac by calculating the combination index (CI) of each of them. These experiments were conducted in triplicate and values were expressed as the mean ± standard deviation. Finally, the effect of BMS-833923 and BMS-906024 on the quiescence of the CD34+CD38- cells of two patients diagnosed with AML was studied by flow cytometry. The paired samples t-test was used in the statistical analysis of GLI and NICD expression between G0 and proliferating cells and in the statistical analysis of the decrease of quiescent cells due to Notch and Hh inhibitors. Results First of all, hematopoietic and progenitor cells were quantified in four AML patients: the median of the percentage of CD34+CD38- cells with respect to total cells in the bone marrow of the AML patient studied was 1.1% (range: 0.12%-9.05%), within which 71.50% (range: 64.30%-88.43%) are quiescent. Interestingly, we found a trend for a higher expression of NOTCH signaling pathway in the proliferating CD34+CD38- cells (relative median fluorescent intensity (MFI) = 1.91 (range: 1.51-3.34)) compared to the quiescent fraction (relative MFI=1.55 (range: 1.18-1.94); p=0.105). But no differences were found in expression of GLI1. Before studying the effect on cellular quiescence of Notch and Hh inhibitors in monotherapy and in combination with AraC, we evaluated their effect on cell viability. The most potent drug studied was AraC (IC50 = 4.055 μM), followed by inhibitors of the Hh pathway (IC50 BMS-833923 = 5.041 μM; IC50 GANT61 = 7.042 μM) on the OCI-AML3 cell line. In contrast, the γ-secretase inhibitor (BMS-906024) showed no effect. Moreover, it was found that the combination 0.8 μM AraC plus 8 μM BMS-833923 was the most synergistic (CI = 0.53, 15% viability with respect to DMSO control). Subsequently, the effect of the SMO inhibitor on the quiescent CD34+CD38- cells of two patients diagnosed with CD34+ AML was analyzed: BMS-833923 decreased the percentage of quiescent CD34+CD38- cells by 88.5±16.3% in monotherapy (p=0.083) and in presence of AraC by 85.8 ± 21.2% (p=0.113) (figure 1). Conclusion The use of SMO inhibitors for the treatment of AML is promising because it increases the sensitivity of leukemic cells to chemotherapy and facilitates their action by reducing the percentage of quiescent LSC. This could mean a decrease in the probability of relapse in patients with AML treated with Hh inhibitors. These results were derived from an ongoing project and more patients are being studied in order to confirm the explained results. This work is partially funded by the Madrid Association of Hematology and Hemotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Control of oxidative stress in the bone marrow (BM) is key for maintaining the interplay between self-renewal, proliferation, and differentiation of hematopoietic cells. Breakdown of this regulation can lead to diseases characterized by BM failure such as the myelodysplastic syndromes (MDS). To better understand the role of oxidative stress in MDS development, we compared protein carbonylation as an indicator of oxidative stress in the BM of patients with MDS and control subjects, and also patients with MDS under treatment with the iron chelator deferasirox (DFX). As expected, differences in the pattern of protein carbonylation were observed in BM samples between MDS patients and controls, with an increase in protein carbonylation in the former. Strikingly, patients under DFX treatment had lower levels of protein carbonylation in BM with respect to untreated patients. Proteomic analysis identified four proteins with high carbonylation levels in MDS BM cells. Finally, as oxidative stress-related signaling pathways can modulate the cell cycle through p53, we analyzed the expression of the p53 target gene p21 in BM cells, finding that it was significantly upregulated in patients with MDS and was significantly downregulated after DFX treatment. Overall, our results suggest that the fine-tuning of oxidative stress levels in the BM of patients with MDS might control malignant progression.