Dr Keith Giles

Laboratory for Cancer Medicine, Western Australian Institute for Medical Research, School of Medicine and Pharmacology,University of Western Australia

kgiles@waimr.uwa.edu.auGenetics of brain tumours

Overview1. Glioblastoma - the most common & lethal form of adult primary brain tumour

2. What we know about the molecular biology of glioblastoma

3. Targeted therapy of glioblastoma

4. New advances in understanding glioblastoma genetics

5. Research into microRNAs and glioblastoma

Glioblastoma Most common & lethal primary brain tumour in adults

Highly resistant to therapy (surgery, radiation therapy & chemotherapy)

Disease recurrence is common following surgery

Life expectancy of glioblastoma multiforme patients (GBM; Grade IV) is ~14 months

Urgent need for new treatment options

organismorgancellMolecular biologymolecules:DNA, RNA, proteins

What cells make up a tumour?

DNA(genes)RNAProteinStructure & function of cellsDiseaseHow genes cause disease

Genetic basis of cancerCancers originate as the result of hereditary or accumulated changes (mutations) in genes that control critical processes in cellsDNAsequenceG A C T A A T C G GNormal gene G A C T A G T C G GSingle base changeG A C T A A C C A T C G GInsertionG A C T C G GDeletion

Genetic basis of cancerMutations can activate oncogenes or silence tumour suppressor genesoncogenes(bad)tumour suppressor genes (good)

Genetic basis of cancerThere is increased or decreased expression of specific genes in cancerNormal cellCancer cellGene AGene BGene AGene B

Genetic basis of cancerThese changes (mutations) can be studied in the laboratory using sophisticated genetic analysis methods

The hallmarks of cancer

How does glioblastoma arise?

Two main pathways by which glioblastomas develop (primary vs secondary)

Primary and secondary glioblastomas can arise via different mutations

3. Mutations between primary or secondary glioblastomas can differMolecular development of glioblastoma

Increased EGFR expression and signaling in glioblastoma

How can understanding the genetics of cancer cells (glioblastoma) help us to develop new treatments for the disease?

Understand what has gone wrong in glioblastoma cellsDesign a drug to correct what has gone wrong

Targeted cancer therapyFind & understand mutation/alteration that drives cancer cell growth (choosing the right target)Design & develop drug that specifically targets this mutation/alterationNormal cells lack the mutation & should be relatively unaffected; side effects should be minimised

Gleevec & chronic myelogenous leukaemia (CML)(TIME magazine,May 2001)

(TyrosinekinaseInhibitor)

Is there a gleevec for glioblastoma?

Not yet

There may never be one drug that works on all glioblastomas

Why?

Heterogeneity(no single mutation causes all glioblastomas)

Redundancy(a glioblastoma is not dependent on one mutation; other mutations can compensate)

Resistance(by targeting one mutation, new mutations can arise that allow glioblastoma cells to escape this targeting)

Case study of a targeted glioblastoma drug

Erlotinib (Tarceva)

(A small molecule tyrosine kinase inhibitor of the epidermal growth factor receptor [EGFR])

Epidermal growth factor receptor (EGFR) as a therapeutic target in glioblastomaERK1/2PI3K/Akt

The rationale for using erlotinib to treat glioblastoma About half of glioblastomas have high expression of EGFR

Blocking EGFR should block glioblastoma growth & invasion

Promising results in other cancer with high expression of EGFR (eg. lung)

Small molecule tyrosine kinase inhibitor (TKI) - crosses blood-brain barrier

Erlotinib and glioblastoma Unfortunately, few patients (~10-20%) respond to erlotinib and survival benefit is small

Need to identify what determines whether a patient will respond/not respond to erlotinib

Combine erlotinib with other treatments (chemotherapy, other targeted agents, radiation therapy) to improve responses and increase patient survival

Mutations downstream of EGFR render glioblastoma cells resistant to erlotinib

New advances in understanding of glioblastoma

Brain tumour stem cells Cancer stem cell hypothesis: tumours are dependent on a small population of cancer stem cells that are distinct from the more abundant tumour cells.

Cancer stem cells are highly resistant to conventional cancer therapies

Express specific cell surface markers (eg. CD133).

Molecular characterisation has identified possible drug targets for brain tumour stem cells.

Targeting brain tumour stem cells

The Cancer Genome Project Human Genome Project: database of a complete genome of a normal human

Cancer Genome Project: established in 2006; to characterise >10,000 tumours at a molecular level from at least 20 tumour types (incl. glioblastoma) by 2015.

Will identify many more mutations responsible for glioblastomas - new treatment targets?

Made possible by rapid development of high throughput techniques - researchers can screen millions of DNA bases quickly and cheaply. This has only been feasible in the last few years.

The Cancer Genome ProjectSome achievements to date in understanding glioblastoma:

Discovery that patients with an unmethylated version ofMGMT gene respond better to temozolomide. Patient selection?

(2) Discovery that a subset of glioblastoma patients that live anaverage of three years have different gene mutations to regularglioblastoma patients. What do these do?

(3) Identification of at least four glioblastoma subtypes, based ontheir DNA signatures. Survival, response to aggressivechemotherapy & radiotherapy differed according to subtype.

microRNAs and glioblastoma

microRNAs (miRNAs) miRNAs are short, endogenous, non-coding RNAs- >900 miRNAs, many are conserved, cell & tissue-specific expression

miRNAs negatively-regulate gene expression- bind to specific target mRNAs, predicted to regulate 1/3 of all genes

miRNAs have important functions in normal cells- development, differentiation, angiogenesis, proliferation, apoptosis

miRNA expression is altered in disease states- cancer - oncogenes & tumour suppressor genes

microRNAs block expression of specific target genes

microRNA expression is altered in cancer cells vs normal cells Cancer miRNA signature - classify tumours

Biomarkers?

Strategies to use microRNAs as therapeuticsAB

A role for microRNAs in glioblastoma?Normal cellGlioblastoma cellMicroRNA 1MicroRNA 2MicroRNA 3highlowlowlowlowhigh

Levels of miR-7 microRNA are significantly reduced in glioblastoma patient tissues vs normal brain

Culture of glioblastoma cell lines in the laboratoryglioblastoma cell lineglioblastoma tumour Study gene mutations/alterations Study new treatments

Levels of miR-7 microRNA are significantly reduced in glioblastoma tumour cell lines vs normal brain

Glioblastoma cell lines can be transfected with microRNAglioblastoma cell linemicroRNA (eg. miR-7) Determine effects on other genes involved in glioblastoma (eg. EGFR) Measure effects on glioblastoma cell growth

EGFR protein expression is decreased by miR-7 microRNA in human cancer cell lines(Webster et al 2009 JBC)

miR-7 microRNA reduces glioblastoma cell growth

SummaryGlioblastomas are different & often arise via different mutations. This might explain why they can respond differently to treatment.

First generation of targeted agents have yielded disappointing results, but research can explain why this has been the case and improvements made to future drug design.

Understanding all of the important mutations in glioblastoma (eg. via large scale research efforts such as the Cancer Genome Project) should allow the development of new drugs that are effective in patients with the correct mutation.

More work is needed but progress is being made

Ive been treating glioblastoma for about 22 years. Ivetaken care of more than 20,000 patients. The kinds ofthings weve seen in the clinic in the last four yearsblows away anything I saw in the previous 18 years ofmy career.

Howard Fine, MD - Chief, Neuro-oncology, Centre for CancerResearch, National National Cancer Institute, commenting in Jan 2010 on a report estimating that the percentage ofglioblastoma patients who survive two years from diagnosis hasmore than tripled in the last five years as a result of newtreatment regimens.

AcknowledgementsRebecca Webster, Priscilla Zhang, Karina Price, Michael Epis, Andrew Barker, Felicity Kalinowski

The Leedman Lab

Western Australian Institute for Medical Research

Terry Johns (Monash), Kerrie McDonald (Lowy), Greg Goodall (Hanson), John Mattick (UQ)

Cancer Council WA & Pearl Bethel Allan Research Grant Endowment

National Health and Medical Research Council

miRNAs may act as tumour suppressors or oncogenes

miRNAs bind to specific target mRNAs, regulate 1/3 of all genes

One miRNA can have 100s of mRNA targetsmicroRNAs block expression of target genes

Glioblastomas arise from glial cells Glioblastomas are a group of low-grade and high-grade brain tumours that originate from glia (Greek for glue)

Normally, glial cells (eg. astrocytes) provide support to neurons (nerve cells): nutrients, mechanical support, development, immune function

Genetic alterations occur in glial cellsglioblastoma