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Cancer Genes and Targets for Therapy. Helen C Hurst. Molecular Oncology Unit Charterhouse Square. Primary Tumour. Metastasising cells. Metastatic Tumours. Targeted Therapy. “Cancer Genes”. Cancer Treatment. Surgery. Chemotherapy Radiotherapy. Apoptosis.
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Cancer Genes and Targets for Therapy Helen C Hurst Molecular Oncology Unit Charterhouse Square
Primary Tumour Metastasising cells Metastatic Tumours Targeted Therapy “Cancer Genes” Cancer Treatment Surgery Chemotherapy Radiotherapy Apoptosis
Cells in multicellular organisms are continually receiving signals from each other and their environment This leads to proliferation, differentiation or even cell death (apoptosis) as appropriate to the needs of the organism as a whole In cancer, this normal balance goes awry Cancer Genes
Cancer progression in ductal carcinoma of the pancreas ….progressive mutation/activation of “cancer genes”
What is a “Cancer Gene”? • Proliferation: Oncogenes and Tumour suppressor genes • Cell survival: Apoptosis vs DNA repair • Epithelial-stromal interactions: Angiogenesis, Invasion and Metastasis • Cell surface markers: Immune Evasion • Membrane pumps: Drug resistance and response to therapy • Metabolism: allow more rapid growth (e.g. ribogenesis) virtually any gene product may be a target for therapy as long as: • It’s expression level/structure/activity is sufficiently different between normal and tumour cells • It is required for continued growth/survival of the tumour cells • Many are involved in cellular signalling pathways:
Gene expression/ Signalling Pathways Growth/survival GFs 2nd messenger cascade Small Molecules Arrest/apoptosis
Examples of Targeted Therapies in Clinical Use • Anti-endocrine therapies: • Tamoxifen (anti-ER therapy) - breast cancer • Anti-androgen therapy - prostate cancer • Anti-ErbB therapies: • Herceptin - immunotherapy against HER2/ ERBB2 in breast cancer • Iressa - small molecule tyrosine kinase inhibitor against EGFR for solid tumours • Glivec - small molecule tyrosine kinase inhibitor against Bcr-abl for CML
Oestrogen Receptor in Breast Cancer Normal - only a few cells express ER ER +ve tumour ~65% of breast tumours are ER +ve show proliferative response to oestrogens (ovaries) benefit from anti-oestrogen therapy
oestrogen The ER is a ligand dependent transcription factor Proteins that Growth/survival ++++ ERE (oestrogen response element)
Use of anti-oestrogens in treating breast cancer • Anti-oestrogens block the binding of oestrogen to the ER proliferative gene expression and signalling are blocked • Giving early stage, ER+ve patients Tamoxifen for 5 years immediately after surgery has mortality by 28% • Tamoxifen use in early stage disease UK annual breast cancer mortality rate fell from ~16,000 to 12,800 in 12 years (1988-2000) But…...
…. there are problems: • Tamoxifen is associated with a ~2-fold risk of blood clot formation (thromboembolism) • Tamoxifen is linked to a ~2.5-fold risk of endometrial cancer • Significant numbers of ER +ve patients never respond to Tamoxifen (de novo resistance) • Those that do respond initially, can relapse with resistant disease (acquired resistance) because oestrogen has a bad and a good side….
Pluses and minuses • Anti-oestrogens like Tamoxifen and Raloxifene are “partial agonists” block oestrogen action in breast; allow some signalling in other organs • This has consequences that are both positive and negative: • Tamoxifen and Raloxifene are both agonists in bone protect against osteoporosis • In the endometrium Tamoxifen (but not Raloxifene) is an agonist, hence endometrial cancer
Alternative strategies • Use total oestrogen agonists like Faslodex that block all oestrogenic activity and result in down-regulation of the ER • Remove oestrogens altogether using aromatase inhibitors which prevent synthesis of oestrogens Clinical trials have shown aromatse inhibitors to be effective and well-tolerated and resistance is slower to develop…. …however, resistance to these agents is an issue and develops for largely similar reasons as Tamoxifen resistance:
Signal pathway cross-talk oestrogen-independence target 1 or more of these pathways in addition (combination therapy)
AR in Prostate Cancer • All PC initially respond • to anti-androgen therapy • After 2-5 years tumours • become resistant • Various mechanisms e.g. • mutation of AR and/or • gene amplification • Increased signalling via • other pathways (as in • breast cancer) also • important
Ligand P P What are ErbB proteins? • ErbB = family of trans-membrane glycoproteins with an extracellular ligand binding domain and an intracellular tyrosine kinase domain • Referred to as “receptor tyrosine kinases” • Ligand binding receptor dimerisation, kinase activation, auto-phosphorylation (on Y) signalling cascade initiation • Normal function mediate cell-cell interactions in organogenesis and during adulthood Docking sites for signalling proteins
Slow ligand dissociation • Relaxed ligand specificty • Slow endocytosis • Rapid cycling • Prolonged “firing” • proliferation • migration • resistance • to apotptosis IHC FISH ERBB2 overexpressed in many solid tumours e.g. 25% breast carcinomas correlates with ER negativity and poor prognosis
The development of Herceptin®(Trastuzumab) • Researchers at Genentech raised mouse monoclonal antibodies against the extra-cellular domain of ErbB2 • One of these, 4D5, potently inhibited growth of ErbB2 overexpressing cultured human breast tumour cells • Murine antibodies are limited clinically due to being immunogenic • Recombinant, humanised antibody created: • Herceptin has a higher affinity for ErbB2 than 4D5 and has a cytostatic growth inhibitory effect against ErbB2+ve breast cancer lines
Mouse hybridoma specificity determining (variable) regions Consensus human IgG1 framework Genetic engineering V gene cloning CDR grafting Eucaryotic expression “Humanising” an antibody
Herceptin in the clinic • Shown to: be well-tolerated; have anti-tumour activity • In randomised trials - improved survival in patients with amplification of the ERBB2 gene • Approved for use in metastatic ErbB2+ve breast tumours (1998) • Largely used in combination with chemotherapy drugs (taxol, cisplatin; cardiac side-effects with dox) • Mode of action: ErbB2 downregulation; prevents cleavage of extracellular domain (causes activation); activates patient’s own immune response
Future improvements • Herceptin has no activity on tumours that express moderate levels of ErbB2 limits its use • 2C4 binds a different epitope blocks ErbB2 dimerisation with other ErbB receptors prevents signalling in low- and high-expressing lines • Anti-tumour effects in xenografts of breast and prostatic tumours • Shown to be safe (Phase I); now in Phase II (efficacy) trials • May be useful in a wide range of ErbB2 +ve solid tumours.
No signalling Proliferation/Survival
Iressa®(Gefitinib ; ZD1839) • Selective and reversible small molecule inhibitor of EGFR tyrosine kinase activity (from AstraZeneca) • Also inhibits signalling via EGFR dimerisation with other ErbB family members • Preclinical studies - inhibited growth of various tumour lines and xenografts • Synergised with cytotoxic chemotherapy agents (e.g. paclitaxel) and radiation therapy in sensitive lines • Paradox: senisitive lines could not be predicted from their level of EGFR expression
Iressa in the clinic • Good oral bio-availability and well-tolerated can be taken once daily (Phase I) • Good anti-tumour responses in mono- and combination therapy in a variety of solid tumours: NSCLC, colorectal, breast, head & neck (Phase II/III) • Approved for use in patients with advanced, chemo-resistant NSCLC • Assays to determine which patients (NSCLC and other) will benefit most being developed • Combination therapies being optimised
Chronic Myeloid Leukaemia (CML) • Characterised by a massive clonal proliferation of myeloid cells • Accounts for 15-20% of all leukaemia cases • Has 3 phases: chronic (or stable); accelerated; blast • Chronic phase: excess numbers of myeloid cells that still differentiate (i.e. cease dividing) as normal • In 4-6 years disease progresses to blast crisis accumulated mutations ability to differentiate is lost • Transplantation can cure (but problematic) less than 20% of cases can be cured What mutations cause this?
Chromosome 1 Gene A Chromosome 2 Gene B Fusion Gene Primary transcript Fusion mRNA Unique Properties Altered Pattern of gene expression Chimaeric protein Acts as an oncogene Differentiation Blocked 65% of leukaemias are characterised by particular somatically acquired chromosome translocations Continued self-renewal
Bcr-abl = constitutively active tyrosine kinase (The protein product from this fusion gene only found in ~70% of patients) Chronic myeloid leukaemia (CML) is characterised by the t(9;22)(q34;q11) reciprocal translocation
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Bcr-abl inhibitor, Glivec®(Gleevec; Imatinib; ST1571) • Rationally designed small molecule that binds to an inactive form of Bcr-abl and prevents ATP recruitment tyrosine kinase activation is blocked • Pre-clinical studies growth inhibition and induction of apoptosis specifically in Bcr-abl expressing cells • Shown to be orally active and well tolerated • Effective therapy for all stages of CML inducing remission in 80% of patients • Approved in May 2001 < 3yrs after first Phase I study • >95% patients with chronic phase (stable) disease durable response …but
The downside • ~all patients with advanced disease will relapse develop resistance to Glivec • Main mechanism: reactivation of Bcr-abl kinase via point mutations that drug sensitivity 3- to >100-fold The solution • Combination therapy using Glivec with cytotoxic agents and/or interferon • Use rational drug design to make similar drug that binds more avidly: AMN107 with >20-fold higher affinity for wt and mutant Bcr-abl, published Feb 2005
Summary • Targeted therapies can be more selective and show improved efficacy with minimal toxicity • Almost invariably, initial response and latency followed by disease resistance • inherent weakness of monotherapy • Combination therapy with cytotoxic drugs is being assessed but the mutagenic nature of these may accelerate the development of resistance • Simultaneous use of multiple targeted agents may faster responses and more durable remissions • Need yet more detailed knowledge of the molecular changes during cancer progression TARGETS
Suggested Reading • “Tamoxifen: a most unlikely pioneering medicine” Jordan VC (2003) Nat. Rev. Cancer 2, 205-13 • “Aromatase Inhibitors for breast cancer: lessons from the laboratory” Johnston SRD & Dowsett M (2003) Nat. Rev. Cancer 3, 821-31 • “Untangling the ErbB signalling network” Yarden Y & Sliwkowski MX (2001) Nat. Rev. Cancer 2, 127-37 • “STI571 (Gleevec) as a paradigm for cancer therapy” BJ Drucker (2002) Trends Mol. Medicine 8, S14-18