richard.lock@unsw.edu.au

Tel: 9382-1846

Molecular mechanisms of drug resistance and new therapeutic approaches in childhood acute leukaemia

Despite dramatic improvements in therapy over the past 40 years, leukaemia remains one of the most common causes of death from disease in children and accounts for the greatest number of deaths from childhood cancer overall. We have recently developed a clinically relevant experimental model of childhood acute lymphoblastic leukaemia (ALL), by establishing xenografts of primary childhood ALL cells in immune-deficient (NOD/SCID) mice. Our research centres on the use of this model system to delineate clinically relevant mechanisms of drug resistance in childhood leukaemia. In addition, we are using these models to identify novel targets to facilitate the development of new therapies for this disease.

Project 1. Epigenetic regulation of gene expression in acute lymphoblastic leukaemia.

Dr Richard Lock
Tel:  +61 2 9382 8558
Email:  rlock@ccia.unsw.edu.au 

Acute lymphoblastic leukaemia (ALL) remains one of the most common causes of death from disease in children, and a significant number of patients relapse and succumb to their disease. Emerging data indicate that epigenetic repression of critical genes via modification of histone acetylation and DNA methylation confers a poor prognosis in paediatric ALL. This project will test the hypothesis that epigenetic silencing of pro-apoptotic genes is associated with poor treatment response. The aims will be to utilise primary ALL biopsy specimens and ALL cells established as xenografts in immune-deficient mice to: investigate genome-wide patterns of DNA methylation; analyse DNA methylation and histone acetylation status of candidate genes; develop strategies to reactivate silenced genes. A variety of cell and molecular biology techniques will be used to achieve these aims, as well as utilisation of a clinically relevant xenograft model. Success of the project will lead to the design of new therapeutic strategies to treat paediatric ALL.

Project 2. Overcoming glucocorticoid resistance in childhood acute lymphoblastic leukaemia.

Dr Richard Lock
Tel:  +61 2 9382 8558
Email:  rlock@ccia.unsw.edu.au 

Dr Barbara Szymanska
Tel:  +61 2 9382 1829
Email:  bszymanska@ccia.unsw.edu.au

Glucocorticoids are used effectively to treat childhood acute lymphoblastic leukaemia (ALL). They promote apoptosis in ALL cells by inducing the pro-apoptotic BH3-only protein Bim. Using a clinically relevant experimental model of ALL we have shown that resistance to the glucocorticoid dexamethasone is associated with over-expression of the anti-apoptotic Bcl-2 protein that neutralises Bim. This project will test the hypothesis that small molecule inhibitors of Bcl-2 can overcome dexamethasone resistance in childhood ALL. The techniques that will be used include in vitro culture of leukaemia cells, drug treatments, cell viability assays, flow cytometric analysis, RT-PCR, and Western blotting. This work will be carried out in parallel with in vivo xenograft studies in the lab to confirm the ability of small molecule Bcl-2 inhibitors to overcome glucocorticoid resistance in ALL cells both in vitro and in vivo. The success of the work has the potential to lead to clinical trials in ALL patients with drug-resistant disease.

Project 3. Testing anti-leukaemic natural products to improve the treatment of childhood acute lymphoblastic leukaemia.

Dr Richard Lock
Tel:  +61 2 9382 8558
Email:  rlock@ccia.unsw.edu.au 

Vinca alkaloids are a central component of all treatment regimens for ALL. Patellamide metabolites from the tropical Indo-Pacific acidian Lissoclinum patella display a variety of biological activities, the most striking of which is the reduction of drug resistance in human leukaemic lymphoblasts treated with the vinca alkaloid, vinblastine. This project will further characterise the activity of patellamides against a panel of ALL cell lines derived from patients with drug-resistant disease, and characterise the mechanisms by which the compounds kill ALL cells. The techniques to be used will include leukaemia cell culture, western blotting, and a variety of cell biological assays to assess cell death mechanisms. These experiments will prioritise the patellamides for future in vivo testing against drug-resistant ALL xenografts in immune-deficient mice to determine their potential for clinical trials.

Dr Richard Lock
Tel: +61 2 9382 8558
Email: rlock@ccia.unsw.edu.au

Dr Hernan Carol
Tel: +61 2 9382 0104
Email: hcarol@ccia.unsw.edu.au 

Our group has established a panel of childhood acute lymphoblastic leukaemia (ALL) xenografts in NOD/SCID mice. Mice from an alternative strain (“3KO”) are not receptive to engraftment with B-cell precursor (BCP) ALL cells. TNF-alpha, an early pro-inflammatory cytokine, has been linked to cancer progression and reported to support the in vitro survival and proliferation of ALL cells. Two soluble receptors, sTNF RI and RII bind to and inactivate TNF-alpha. We have found that sTNF RI and RII levels are higher in 3KO bone marrow culture supernatants compared to NOD/SCID mice. The hypothesis is that TNF-alpha contributes to ALL cell survival in the human bone marrow microenvironment. Blocking of the cytokine or each of the two receptors will be performed during the initial stages of in vivo engraftment of NOD/SCID and 3KO mice. Supplementing TNF-alpha at the time of engraftment (or inducing its production) will also be tested as a conditioning step to further increase engraftment rates. Cells responsible for both TNF production and soluble receptor production will be identified by flow cytometry and immunohistochemistry. This project will further define the role of TNF and its regulators in ALL, and lead to the design of novel therapeutic intervention strategies.

Dr Richard Lock
Tel:  +61 2 9382 8558
Email:  rlock@ccia.unsw.edu.au 

The mechanisms by which glucocorticoids induce apoptosis of leukaemia cells, and the lesions underlying resistance, remain poorly understood. A clinically relevant model of childhood acute lymphoblastic leukaemia (ALL) has been developed in our laboratory. In our model the response to glucocorticoids is associated with induction of the pro-apoptotic Bcl-2 family member, Bim, activation of the intrinsic apoptotic pathway, and cell death. The aim of this project is to characterise the molecular responses to the glucocorticoid, dexamethasone, in a series of cell lines to define mechanisms of glucocorticoid resistance. The project will involve determining the sensitivity of these cell lines to dexamethasone and investigating their molecular responses by RT-PCR and Western blotting of key genes and proteins involved in glucocorticoid signalling pathways. The epigenetic status of genes found to be down-regulated will be further investigated by chromatin immunoprecipitation and methylated DNA immunoprecipitation assays to determine the mechanism of gene silencing. The expected outcome is that epigenetic silencing of key genes involved in glucocorticoid-induced signalling pathways plays a key role in conferring drug resistance.

Dr Richard Lock
Tel:  +61 2 9382 8558
Email:  rlock@ccia.unsw.edu.au 

Despite dramatic improvements in combination chemotherapy, acute lymphoblastic leukaemia (ALL) remains one of the most common causes of death from disease in children. Mechanisms by which ALL cells develop drug resistance in patients are poorly understood. This project will use contemporary cell and molecular biological approaches, and a clinically relevant murine xenograft model, to identify novel mechanisms of drug resistance in paediatric ALL. ALL xenograft cells will be transduced with “bar-coded” lentiviral libraries expressing shRNA sequences for gene knockdown, or cDNA sequences for gene overexpression, following which cells will be inoculated into immune-deficient mice and selected in vivo for resistance to chemotherapeutic drugs. Knocked down shRNA targets and/or overexpressed genes associated with the drug resistant phenotype will be identified and functionally characterised using ALL cell lines, xenografts and primary biopsy specimens. In this fashion, novel mechanisms of drug resistance, as well as new targets for therapeutic exploitation, will be identified in paediatric ALL.

Dr Richard Lock
Tel:  +61 2 9382 8558
Email:  rlock@ccia.unsw.edu.au

Dr Amanda Philp
Tel:  +61 2 9382 8580
Email:  aphilp@ccia.unsw.edu.au

Acute promyelocytic leukaemia (APL) is a disease characterised by translocations involving the retinoic acid receptor alpha (RAR-alpha) gene, principally with the PML gene. The retinoid all-trans retinoic acid (ATRA), and the trivalent arsenical arsenic trioxide, are used successfully to treat APL, although severe toxic side effects against the normal cells of the body are frequently encountered. Several water-soluble derivatives of arsenic trioxide have been synthesised in an attempt to reduce these side effects. Arsenic trioxide treatment of APL cells leads to post-translational modification and degradation of the PML/RAR-alpha fusion protein. In addition, the class 1a PI3K subunits (p85 and p110) interact with RAR-alpha in the presence of ATRA. This study will compare the effects of arsenic trioxide and new derivatives on PML/RAR-alpha modifications and its interactions with PI3K subunits. The techniques to be used include cell culture, co-immunoprecipitation of proteins, western blotting and PI3Kinase assays. The outcome of the project will be to further define pathways for the selective eradication of APL cells.

Dr Richard Lock
Tel:  +61 2 9382 8558
Email:  rlock@ccia.unsw.edu.au

Dr Nicole Barber
Tel:  +61 2 9382 1829
Email:  nbarber@ccia.unsw.edu.au 

Treatment regimens for acute lymphoblastic leukaemia (ALL) include L-asparaginase, and there is some evidence that the topoisomerase I inhibitor, topotecan, has activity against the disease. The majority of patients are cured; however some patients will relapse, often developing resistance to one or more drugs. The mechanisms by which cell death occurs in ALL cells treated with these drugs are not fully elucidated; however there is evidence that the expression levels of anti- and pro-apoptotic proteins are involved. This project aims to investigate the regulation of these proteins in cell death induced by topotecan and L-asparaginase at a transcriptional and translational level as well as investigating the signalling pathways involved. The techniques used in this project will include the culture of leukaemia cells, western blotting, RT-PCR and flow cytometry. This work will increase our understanding of the action of chemotherapeutic drugs and aid in determining mechanisms of resistance to improve the outcome for leukaemia patients.

• Lock, RB, Liem, N, Farnsworth, ML, Milross, CG, Xue, C, Tajbakhsh, M, Haber, M, Norris, MD, Marshall, GM, and Rice, AM (2002) The non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biological characteristics at diagnosis and relapse. Blood 99: 4100-4108.

• Liem, NLM, Papa, RA, Milross, CG, Schmid, MA, Tajbakhsh, M, Choi, S, Ramirez, CD, Rice, AM, Haber, M, Norris, MD, MacKenzie, KL, and Lock, RB (2004) Characterization of childhood acute lymphoblastic leukemia xenograft models for the preclinical evaluation of new therapies. Blood 103: 3905-3914.

• Bachmann, PS, Gorman, R, MacKenzie, KL, Lutze-Mann, L, and Lock, RB (2005) Dexamethasone resistance in B-cell precursor childhood acute lymphoblastic leukemia occurs downstream of ligand-induced nuclear translocation of the glucocorticoid receptor. Blood 105: 2519-2526.

• Bachmann, PS, Gorman, R, Papa, RA, Bardell, JE, Ford, J, Kees, UR, Marshall, GM, and Lock, RB (2007) Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia. Cancer Research, 67: 4482-4490

• Kang, MH, Kang, YH, Szymanska, B, Wilczynska-Kalak, U, Sheard, MA, Harned, T, Lock, RB, and Reynolds, CP (2007) Activity of vincristine, L-ASP and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo. Blood In Press (accepted 18 May 2007)