Acute Lymphoblastic Leukemia (work group 1)

Understanding chemotherapy resistance – developing conceptionally new leukaemia therapies

Resistance to anticancer therapy means an inefficient or ineffective response to treatment. We aim to elucidate the mechanisms underlying resistance to a group of substances called glucocorticoids which constitute a major component in leukaemia treatment.

Our aim is to understand the effects of glucocorticoids on leukemia cells on a molecular level and to identify the resistance mechanism. This knowledge should allow us to develop concepts for new therapies.

Our investigation is to accomplish our aim, we follow 4 major research strategies:

1) Identification of candidate genes for the anti-leukemic glucocorticoid effects. Glucocorticoids trigger apoptosis in leukemia cells by influencing which genes are active in these cells (i.e., the “expression profile” of a cell). To identify these genes, we determined all genes regulated by GC in patients with acute lymphoblastic leukaemia (collaboration with the Departments of Paediatrics and Internal Medicine of the Innsbruck Medical University) before and during therapy with glucocorticoids using whole genome microarray-based expression profiling. The latter was performed in the "Expression Profiling Core Facility" of our University situated at the TCRI and run by our lab. For comparisons, we generate expression profiles from other biologic systems in which GC induces or does not induce, apoptosis such as peripheral blood lymphocytes from GC-exposed healthy donors, GC-sensitive and resistant ALL cell lines and mouse thymocytes treated with GC in vivo and in vitro. This generated an essentially complete list of GC-regulated candidate genes in clinical settings and experimental systems, allowing immediate analysis of any gene for its potential significance to GC-induced apoptosis.

2) Verification of regulation and functional analysis of candidate genes. Regulation of the above genes on the RNA and protein level has been analyzed (or is currently being analyzed) by real time RT-PCR and Western blotting technologies. A possible function of the GC-regulated genes in cell death induction (or cell cycle arrest, another anti-leukemic GC effect) is currently being analyzed by regulated expression and/or suppression of these genes in leukaemia model systems and cells from patients. For this purpose, we use lentiviral, conditional systems for gene over-expression and RNA interference based systems for gene knock-down.

3) Analyses of model systems of GC resistance. To understand how leukaemia cells escape cell death induced by GC, we have generated a large number of GC-resistant leukaemia cell lines that have been, and are still being, investigated with respect to the mechanism(s) of resistance development. Work with GC-resistant primary leukaemia cells from patients is performed in collaboration (A. Hall and J. Irving, Northern Institute for Cancer Research, Newcastle Upon Tyne) Analyses include determination of GC-receptor structure and expression, DNA finger printing, expression profiling, and several fluorescence activated cell sorter (FACS) – based analyses.

4) Novel therapy combinations in preclinical models. The knowledge obtained from the above studies is used to develop concepts for novel therapy combinations to treat acute lymphoblastic leukaemia. For example, based on our extensive expression profiling analyses, we have combined GC with an inhibitor of glucolysis (2-deoxyglucose) and observed a remarkable combinatorial effect on cell survival in an in vitro model for acute lymphoblastic leukaemia.

Current results:
Comparing gene expression in patient samples and in model systems clearly demonstrated the relevance of research done on patient material: In several instances, we could not verify the assumed significance of genes identified in model systems in previous studies. In contrast our study revealed some genes so far not implied in any hypothesis on glucocorticoid induced apoptosis. Among those are genes involved in energy-metabolism and a potentially new member of the apoptosis related molecule family of BH3-only molecules, which mediates death signals.

General involvement of energy-metabolism and BH3-only molecules in apoptosis in some model systems had previously been investigated in our laboratory in the Division of Molecular Pathology at the Innsbruck Medical University. However, the new and comprehensive gene expression analysis allows us to focus research on genes and pathways that are in fact involved in glucocorticoid triggered cell death in tumor cells of leukaemia patients.