Malignancy and Therapy
Gefitinib-induced killing of non-small cell lung cancer cell lines expressing mutant EGFR requires the pro-apoptotic BH3-only protein Bim and can be enhanced by BH3 mimetics
MS Cragg, J Kuroda, H Puthalakath, CL Scott, A Strasser. Pub ref: 27
Abnormalities in epidermal growth factor (EGF) signalling, such as mutations that render the EGF receptor (EGFR) hyper-active or cause over-expression of the wild-type receptor, have been found in diverse cancers, including lung, breast or colon carcinoma. EGFR inhibitors such as gefitinib have proven successful in treatment of certain cancers, particularly, non-small cell lung cancers (NSCLC) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumour regression remain unknown.
We found that Gefitinib triggers “mitochondrial” apoptotic signalling, involving the activation of Bax and mitochondrial release of cytochrome c, and ultimately unleashes the caspase cascade. Gefitinib rapidly increased the pro-apoptotic BH3-only protein Bim through both transcriptional and post-translational mechanisms. Using RNA interference, we demonstrated that Bim is essential for Gefitinib-induced killing of NSCLC cells. Moreover, we found that Gefitinib-induced apoptosis is enhanced by addition of the BH3-mimetic ABT-737.
As we have previously shown that Bim also mediates Imatinib (Gleevec)-induced killing of BCR-ABL-transformed cells, such as chronic myelogenous leukemia, Bim may be generally required for apoptosis of tumour cells caused by shut down of the oncogenic kinases that provoked their neoplastic transformation.
EGFR inhibitors such as Gefitinib trigger apoptosis via the mitochondria by inhibiting the Ras-ERK pathway, thereby up-regulating the pro-apoptotic BH3-only protein Bim, which targets all its pro-survival relatives. Killing is enhanced by adding the BH3 mimetic ABT-737, which also targets Bcl-2 and Bcl-xL. Inactivation of all the pro-survival family members frees Bax to permeabilize the mitochondria. The inhibition of BCR-ABL by Imatinib has similar effects.
Tumorigenic impact of loss of the Myc antagonist Mnt
KJ Campbell, ML Bath, CL Scott, S Cory in collaboration with PJ Hurlin (Oregon Health & Science University, USA)
Reduced expression of Mnt, a widely expressed Myc antagonist, has been shown to induce mammary adenocarcinoma and T-cell lymphoma in mice. We have used Eµ-myc transgenic mice to investigate whether Mnt suppresses the lymphomagenic potential of Myc. Surprisingly, heterozygous loss of mnt did not accelerate Myc-induced lymphomagenesis. We have now generated mice with conditional homozygous deletion of Mnt in haemopoietic cells and are investigating the impact of complete loss of Mnt on haemopoiesis and Myc-driven lymphomagenesis.
Impact of Mcl-1 over-expression on haemopoiesis and tumour development
KJ Campbell, ML Bath, P Bouillet, S Cory
Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is essential for the survival of haemopoietic stem cells. To investigate the impact of over-expression of Mcl-1 on haemopoiesis, tumour development and resistance to cancer therapy, we generated four independent lines of VavP-mcl-1 transgenic mice. The Mcl-1 over-expression disrupted normal haemopoiesis in young mice and promoted lymphoma development as they aged. To further explore the oncogenic potential of Mcl-1, we are crossing VavP-mcl-1 mice with transgenic lines susceptible to diverse haematologic malignancies and will test the tumours that arise for responsiveness to different therapeutic regimes.
Impact of Polycomb Group (PcG) Genes on lymphomagenesis and responses to DNA-damaging drugs
A Adisa, L Happo, CL Scott
The Polycomb Group (PcG) of epigenetic regulators silences genes involved in many cellular processes, including apoptosis, proliferation and senescence. Two of these proteins, Bmi1 and Cbx7, promote lymphomagenesis in mice, and elevated CBX7 expression in human follicular lymphoma correlates with both high MYC expression and advanced tumour grade. Bmi1 and Cbx7 repress transcription from the Ink4a/Arf locus and act epistatically to the Arf-p53 pathway. As p53 is important in promoting apoptosis in response to anti-cancer drugs, we are investigating the ability of Cbx7 and Bmi1 to cause drug resistance in Myc-driven lymphomas and are searching for down-stream targets, including those independent of the Ink4a/Arf locus.
In vivo efficacy of the BH3-mimetic ABT-737 for Myc-driven lymphomas
KD Mason, CJ Vandenberg, CL Scott, AH Wei, S Cory, DCS Huang in collaboration with AW Roberts (Cancer and Haematology Division)
Deregulated MYC expression drives many human cancers, including Burkitt’s lymphoma and a highly aggressive subset of diffuse large cell lymphomas. These tumours often become chemoresistant due to mutations that impair apoptosis. Since pro-survival members of the Bcl-2 family probably mediate the chemoresistance in most tumours, there is now considerable interest in the prospect of targeting those proteins with the new class of anti-cancer agents termed “BH3 mimetics”. Particular promise has been shown in pre-clinical studies by ABT-737, a BH3 mimetic which binds to and neutralises pro-survival Bcl-2, Bcl-xL and Bcl-w but not Mcl-1 or A1. We have therefore assessed its potential for Myc-induced lymphomas.
As a single agent, ABT-737 significantly prolonged the survival of mice transplanted with lymphomas derived from mice co-expressing Myc and Bcl-2 transgenes, but was ineffective for lymphomas expressing Myc alone, probably because they express more pro-survival Mcl-1. Strikingly, a combination of low dose cyclophosphamide with ABT-737 produced complete remission and extended disease-free survival for two of three Myc/Bcl-2 lymphomas.
Thus, ABT-737 is a promising agent for treating lymphomas whose survival relies upon one or more of the pro-survival proteins that it targets.
Improved survival of lymphoma-bearing mice treated with ABT-737. (A) Kaplan-Meier survival plots indicate that mice transplanted with Myc/Bcl-2 lymphoma cells survive significantly better if treated with ABTâ€‘737 in combination with low-dose cyclophosphamide (blue) than with a high-dose cyclophosphamide alone (red). When mice surviving at day 150 were culled for autopsy, the six that had received combination therapy had normal peripheral blood films (B) and no macroscopic evidence of disease. In contrast, the single mouse that survived after high-dose cyclophosphamide was relapsing, with tumour cells prominent in the peripheral blood (C), spleen, liver and peritoneal cavity.
Role of BH3-only proteins in genotoxic chemotherapy
MS Cragg, L Happo, E Michalak, E Jansen, S Cory, A Strasser, CL Scott
DNA-damaging chemotherapeutics induce apoptosis. Treatment failure often reflects acquisition of mutations conferring resistance to apoptosis. Genotoxic damage induces expression of the p53-responsive BH3-only proteins Noxa and Puma. Using lymphoma cells derived from Eµ-myc transgenic mice lacking expression of individual BH3-only proteins, we found that loss of Puma increased resistance of most lymphomas to the genotoxic drugs etoposide and cyclophosphamide, but loss of Noxa alone had little impact. Surprisingly, loss of both Puma and Noxa seems to increase drug sensitivity in vivo instead of increasing resistance, and we are currently exploring the mechanism.
Mouse models for multiple myeloma
CJ Vandenberg, KJ Campbell, S Cory
Eµ-v-abl mice develop plasma cell tumours (plasmacytomas) that represent a model of human multiple myeloma. We have found that disruption of the gene encoding the BH3-only protein Bim accelerates plasmacytoma development in Eµ-v-abl mice, suggesting that Bim is an important regulator of apoptosis in plasma cells. Since multiple myelomas often over-express the Bcl-2 homologue Mcl-1, Eµ-v-abl mice will be crossed with newly generated transgenic mice having either pan-haemopoietic or plasma-cell specific expression of Mcl-1.
Developing lead compounds that target breast cancer by inhibiting the LMO4 oncogene
M-L Asselin-Labat, S Holroyd, H Barker, G Lindeman, J Visvader with I Street, K Lackovic (Structural Biology)
The transcriptional regulator LMO4 appears an appropriate target for intervention in breast cancer, because we have shown that its expression is elevated in breast cancer and that its over-expression promotes both cell proliferation and invasion and is oncogenic in transgenic mice. To search for compounds that reduce LMO4 expression in breast cancer cells, we performed a high throughput screen of 100,000 compounds using cells having LMO4 expression linked to a GFP or luciferase reporter. The 48 hits that we identified are now being selected for ability to reduce LMO4 levels and to inhibit cancer cell growth. Promising leads will be modified by medicinal chemists to generate derivatives that could form the basis of a drug development program.