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Dr Steven Whittaker, Molecular Drug Resistance team

Functional genomic screens to identify drug resistance mechanisms

Lentiviral shRNA expression libraries enable us to conduct pooled, genome-scale screens with cancer cell lines to identify loss of function events, which modulate the sensitivity cells to targeted therapeutics. For example, suppressor screens enable the identification of loss-of-function events that have the functional capacity to induce resistance to a particular drug.

Conversely, synthetic lethal screens permit the discovery of genes that when silenced, potentiate the effect of a drug and enhance its activity in an otherwise resistant, or drug-tolerant tumour model. These studies can nominate candidate resistance effectors, which once validated, can direct the selection of combination therapies using approved/emerging drugs or suggest novel targets for drug discovery activities.

Generation of cell line models of drug resistance

By prolonged exposure of cancer cell lines to growth-inhibitory concentrations of specific inhibitors we expect to generate drug-resistant cell line clones, harbouring mechanisms of resistance relevant to the cell lineage and genotype being studied.

Through genetic and functional characterization of these clones we will pinpoint the relevant driver(s) of resistance and validate their potential as clinically pertinent resistance effectors and as targets for therapeutic intervention.

The generation of tumour cell lines has typically been a challenging process with low success rates. However, recent advances have enabled rapid generation of viable primary tumour cell cultures by co-culturing freshly isolated tumour cells with J2 murine fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Rapid propagation of a viable cell culture has the likely advantage of being more representative of the original tumour.

Through genomic characterization (copy number analysis, whole exome sequencing, expression profiling), drug combination studies and pooled, shRNA screening we would seek to identify novel dependencies in this panel of primary cultures and explore the potential for therapeutic intervention using the approaches described above.

Through an integrated analysis of the genetic dependencies discovered in these lines and pharmacological profiling, we could potentially identify dependencies associated with either drug sensitivity or drug resistance and prioritize rational drug combinations for testing of targets and further validation.

Profiling of clinical resistance mechanisms by whole exome sequencing

In vitro models of drug resistance mechanisms have the potential to nominate the functional capacity of genes to drive intrinsic or acquired resistance to therapy. However, going forward it will be instrumental to establish parallel studies of clinical resistance mechanisms by analysis of tumour (both pre- treatment and drug-resistant biopsies) and matched normal samples.

The identification of drug-resistance associated alterations would not only credential candidate mechanisms of resistance identified in preclinical studies but would also nominate additional resistance effectors for functional validation using in vitro model systems. With ongoing advances in the analysis of circulating tumour cells or cell-free DNA and falling sequencing costs, minimally invasive biopsy protocols may markedly enhance our ability to obtain patient samples and monitor clinical resistance.

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