The role of c-FLIP in regulating response to chemotherapy in Acute Myeloid Leukaemia

Lead Research Organisation: Queen's University of Belfast
Department Name: Centre for Cancer Res and Cell Biology

Abstract

Acute Myeloid Leukaemia (AML) is treated with combination chemotherapy to achieve remission. Many patients unfortunately do not respond to initial chemotherapy or relapse early. Our goal is to investigate whether the protein c-FLIP actually stops AML cells being killed by chemotherapy.

This research will be carried out by a PhD student at the Centre for Cancer Research and Cell Biology, Queen‘s University Belfast. Pioneering work into the role of c-FLIP in colonic cancer has been carried out at this institute by one of the supervisors, Dr DB Longley.

Initial work will involve biological models, known as cell lines, with further extension to clinical samples. Blocking the action of c-FLIP with targeted drugs could lead to a better response to chemotherapy and hence an overall improvement in patient survival. Finally, we also want to investigate if c-FLIP levels expressed on leukaemia cells are linked to overall patient prognosis.

Technical Summary

c-FLIP (Fas-associated death domain (FADD)-like interleukin-1? converting enzyme (FLICE) inhibitory protein) is a key regulator of death receptor-mediated apoptosis. c-FLIP potentially converts death receptor signalling from pro- to anti-apoptotic. We aim to investigate the biology of c-FLIP in AML in addition to its prognostic significance.

Design of Study/Methods

Preliminary experiments demonstrated that siRNA-mediated c-FLIP gene silencing not only sensitises U937 AML cells to death ligand-induced apoptosis, but also to apoptosis induced by the chemotherapeutic agent daunorubicin. These results suggest that c-FLIP may be a key regulator of chemoresistance in AML cells.

Relevant chemotherapies will be investigated including daunorubicin, cytarabine and etoposide. The effect of drug treatment on the expression of both c-FLIPS and c-FLIPL will be assessed by real-time RT-PCR and Western Blot techniques. Each cell line will be examined for constitutive and chemotherapy-induced cell surface expression of the death receptors Fas, DR4 and DR5 and the death ligands FasL and TRAIL using flow cytometry. We plan to examine the effects of c-FLIP on chemoresistance using a FLIP-targeted siRNA (FT siRNA) that down-regulates expression of both c-FLIP splice forms. We will assess the impact on cell viability and apoptosis of FT siRNA as a single agent and also examine its effect on drug induced apoptosis. Cell viability will be assessed by MTT assay, flow cytometry and Poly (ADP-ribose) polymerase (PARP), caspase 8 and caspase 3 cleavage assays. To investigate the mechanism by which c-FLIP regulates the response of AML cells to chemotherapy we will use siRNAs to silence expression of individual components of the death receptor pathway. The effect of overexpression of each splice form on chemosensitivity and death ligand-induced apoptosis will also be assessed.

We plan to utilise quantitative RT-PCR to examine c-FLIPS/L mRNA and Western blot techniques to examine c-FLIP protein expression in AML patients at time of diagnosis. We also intend to examine expression of the Fas and TRAIL death receptors and their ligands. The initial study will be carried out on locally obtained samples following appropriate consent/ ethical approval. The second part of the study will be carried out on samples from the AML Trial banks. This will permit the correlation of marker expression with clinical outcome and prognostic significance following statistical analysis.

In conclusion the importance of c-FLIP in AML and the potential prognostic significance of its expression will be investigated. This will be invaluable in determining if it represents a therapeutic target.

Publications

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McLornan D (2013) Prognostic and therapeutic relevance of c-FLIP in acute myeloid leukaemia. in British journal of haematology

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McLornan DP (2007) Molecular mechanisms of drug resistance in acute myeloid leukaemia. in Expert opinion on drug metabolism & toxicology

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McLornan DP (2010) Prognostic significance of TRAIL signaling molecules in stage II and III colorectal cancer. in Clinical cancer research : an official journal of the American Association for Cancer Research

 
Description CRUK audience on visit to CCRCB 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Highlighted findings and methodology of my MRC funded work tp the CRUK audience

Enjoyable experience for the participants to understand what work was involved in my project
Year(s) Of Engagement Activity 2007,2008,2009