A Research Project on the Autophagy as Therapeutic Target and Progression Marker in Relapsed/Refractory Human Lymphomas
Project location: ITALY, Rome
Project start date: June 2011 - Project end date: October 2012
Project number: 2011-20
Beneficiary: Arcobaleno ONLUS
The present project is aimed at investigating the role autophagy in affecting the therapeutic response of patients with relapsed/refractory lymphoma to molecularly targeted agents, in order to clarify the relevance of autophagy as well as to develop new therapeutic strategies exploiting the modulation of autophagy as a therapeutic challenge. The detection of autophagy determinants of possible use in the clinical practice as progression markers or drug response bioindicators will also be a key point of the Project.
Lymphoma is the third most common childhood cancer. Approximately 60% of paediatric lymphomas are non-Hodgkin's lymphomas, with the remainder being Hodgkin's lymphomas. In the adults NHL represents 89% of all lymphomas while HL represents 11% of all lymphoma diagnoses. Both non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL) will be considered in this study.
NHL and HL are genetically heterogeneous diseases with a remarkable ability to develop adaptive strategies to cytotoxic or pro-apoptotic challenges. Thus, molecular abnormalities with a key role in the pathogenesis and progression of lymphoma represent attractive targets for exploiting new therapeutic strategies.
Despites therapeutic improvements achieved in the last two decades, significant proportions of lymphomas are not cured with currently available therapeutic strategies, suggesting that new treatments are required particularly for high-risk, relapsed or refractory patients.
Based on clinical results obtained by the group of Prof. C. Carlo-Stella at the University of Milan (Italy) and on in vitro and ex vivo preliminary results of the Applicant, the project will refer to two main tasks to be performed in collaboration with University of Milan:
TASK 1. In vitro modulation of autophagy by molecularly targeted agents in human lymphoma-derived cell lines.
Targeted agents used in the proposed project will include the PI3K/Akt/mTOR inhibitor perifosine, the Raf/MEK/Erk inhibitor sorafenib, and the histone deacetylase inhibitor (HDAC) givinostat, used alone or in combination with each other.
Initial studies of different human lymphoma-derived cell lines will be carried out in order:
• to characterize the modulation of cell death processes (apoptosis, autophagy, the shift autophagy/apoptosis) by the compounds under investigation;
• to verify whether autophagy plays a role in the survival of treated cells.
• to explore the relationship between molecular features of tumor cells, detected using gene expression profiling by microarray analysis, and responses to in vitro treatments.
In this context, the ability of autophagy inhibitors (e.g., 3-methyladenine, chloroquine, bafilomycin A1) to enhance anticancer efficacy of the tested drugs, by sensitizing lymphoma cell lines to therapy-induced cell death, will be studied. Additionally, RNA interference-mediated depletion of autophagy regulators (e.g. Beclin-1 and Atg5) will be also carried out.
TASK 2A. Blood sampling from relapsed/refractory lymphoma patients enrolled in phase ii studies.
After informed consent, blood samples will be obtained for in vitro studies from patients with relapsed/refractory lymphoma enrolled in phase II studies with sorafenib alone (30 patients) or in combination with perifosine (36 patients). For both phase II trials, sample size was calculated according to a 2-stage Simon study design for determination of response rates based on a single treatment group. The study protocols have been approved by the Institutional Review Board and the Ethical Committee of the Fondazione IRCCS Istituto Nazionale Tumori, Milan. According to study designs, blood samples will be harvested prior to therapy administration, and monthly for 3 months thereafter. This follow up schedule will allow the early identification of biomarkers predictive of therapeutic response. At each time-point, both heparizined and non-anticoagulated blood samples will be collected in order to cryopreserve lymphocytes and freeze fresh serum. Patient data will be transferred as confidential and tagged with random numbers to protect privacy.
TASK 2B. Evaluation of autophagy in relapsed/refractory lymphoma patients treated with molecularly targeted agents.
Recent developments reveal that autophagy has a crucial role in maintaining peripheral lymphocyte homeostasis. Moreover, an imbalance of lymphocyte homeostasis has been found in the peripheral blood of lymphoma patients. Therefore, the presence of autophagy in peripheral blood lymphocytes from lymphoma patients, enrolled in the above mentioned studies, will be evaluated, prior to therapy administration, and monthly for 3 months thereafter. On the basis of previous data by Corcelle et al., who provided evidence that ERK and p38 displayed a tight control of the autophagy process at the maturation step, the activation state of these protein kinases will be also evaluated.