Cancer Genes and Targets for Therapy

1. Cancer Genes and Targets for Therapy Centre for Molecular Oncology Institute of Cancer Charterhouse Square

2. Primary tumour - usually can be excised by the surgeon - then give chemo and/or radiotherapy to kill of any local or desseminated metastatic cells Tumours have gene alterations/lost tumour suppressor genes; gained mutations(oncogenes) I.e Cancer Genes and result frequently defective in their apoptotic pathways so are resistant to chemo- and/or radiotherapy. Hence cells that escaped from the primary tumour - lead to metastatic tumours: colon to liver; breast to lymph glands and bone. Idea is that by specifically targeting some of these cancer genes - re-sensitise tumours to conventional therapy.Primary tumour - usually can be excised by the surgeon - then give chemo and/or radiotherapy to kill of any local or desseminated metastatic cells Tumours have gene alterations/lost tumour suppressor genes; gained mutations(oncogenes) I.e Cancer Genes and result frequently defective in their apoptotic pathways so are resistant to chemo- and/or radiotherapy. Hence cells that escaped from the primary tumour - lead to metastatic tumours: colon to liver; breast to lymph glands and bone. Idea is that by specifically targeting some of these cancer genes - re-sensitise tumours to conventional therapy.

4. Signalling Pathways

5. Cancer progression in ductal carcinoma of the pancreas Hyperplasia through to DCIS then metastasisHyperplasia through to DCIS then metastasis

6. Proliferation and growth are different things and need both for cancer 80=90% of dry mass of a cell = protein so rate of protein accumulation dictates growth rate In most cell types the number of mRNA molecules > number of ribosomes therefore growth rates and increase in cancer cells if they produce more ribosomesProliferation and growth are different things and need both for cancer 80=90% of dry mass of a cell = protein so rate of protein accumulation dictates growth rate In most cell types the number of mRNA molecules > number of ribosomes therefore growth rates and increase in cancer cells if they produce more ribosomes

7. Examples of Targeted Therapies in Clinical Use Anti-endocrine therapies: Tamoxifen (anti-ER therapy) - breast cancer Anti-androgen therapy - prostate cancer Anti-Receptor tyrosine kinase 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

8. Oestrogen Receptor in Breast Cancer

9. The ER is a ligand dependent transcription factor

10. 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)

11. �. 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)

12. 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

13. 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 prevents local synthesis of oestrogens (adipose tissue)

14. Activation of other signal pathways lead to ER activation by alternative means e.g by being phosphorylated subsequent to kinase cascade activation - therefore presence or absence of oestrogen and oestrogen-like compounds becomes irrelevant One pathway activated = EGFR - will discuss later inhibitors of this pathway that may be used in combination with anti-oestrogen therapiesActivation of other signal pathways lead to ER activation by alternative means e.g by being phosphorylated subsequent to kinase cascade activation - therefore presence or absence of oestrogen and oestrogen-like compounds becomes irrelevant One pathway activated = EGFR - will discuss later inhibitors of this pathway that may be used in combination with anti-oestrogen therapies

16. What are Receptor Tyrosine Kinases? Trans-membrane glycoproteins with an extracellular ligand binding domain and an intracellular tyrosine kinase domain Several families of related proteins known e.g. EGFR or ErbB family Ligand binding ? receptor dimerisation, kinase activation, auto-phosphorylation (on Y) ? signalling cascade initiation Normal function ? mediate cell-cell interactions in organogenesis and during adulthood

17. The ErbB Network

18. Signalling by ErbB homodimers in comparison with ErbB2-containing heterodimers. Receptors are shown as two lobes connected by a transmembrane stretch. Binding of a ligand (EGF-like or NRG) to the extracellular lobe of ErbB1, ErbB3 (note inactive kinase, marked by a cross) or ErbB4 induces homodimer formation. When ErbB2 is overexpressed, heterodimers form preferentially. Unlike homodimers, which are either inactive (ErbB3 homodimers) or signal only weakly, ErbB2-containing heterodimers have attributes that prolong and enhance downstream signalling (blue box) and their outputs (orange box). Apparently, homodimers of ErbB2 are weaker signalling complexes than heterodimers containing ErbB2. (EGF, epidermal growth factor; NRG, neuregulin.)Signalling by ErbB homodimers in comparison with ErbB2-containing heterodimers. Receptors are shown as two lobes connected by a transmembrane stretch. Binding of a ligand (EGF-like or NRG) to the extracellular lobe of ErbB1, ErbB3 (note inactive kinase, marked by a cross) or ErbB4 induces homodimer formation. When ErbB2 is overexpressed, heterodimers form preferentially. Unlike homodimers, which are either inactive (ErbB3 homodimers) or signal only weakly, ErbB2-containing heterodimers have attributes that prolong and enhance downstream signalling (blue box) and their outputs (orange box). Apparently, homodimers of ErbB2 are weaker signalling complexes than heterodimers containing ErbB2. (EGF, epidermal growth factor; NRG, neuregulin.)

19. 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

20. �Humanising� an antibody

21. 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 In addition to down regulating surface ErbB2,Herceptin induces the cyclin-dependent kinase inhibitor p27Kip1 and the Rb related protein p130,which reduce the number of cells in S phase.The recruitment and activation of immune effector cells to the ErbB2-overexpressing tumour might also contribute to Herceptin�s mechanism of action. In addition to down regulating surface ErbB2,Herceptin induces the cyclin-dependent kinase inhibitor p27Kip1 and the Rb related protein p130,which reduce the number of cells in S phase.The recruitment and activation of immune effector cells to the ErbB2-overexpressing tumour might also contribute to Herceptin�s mechanism of action.

22. 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.

25. 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 Generic names: Gefitinib, Traz.. Trade names: Iressa, Herceptin, Gleevec Other related compounds also in trialsGeneric names: Gefitinib, Traz.. Trade names: Iressa, Herceptin, Gleevec Other related compounds also in trials

26. Mode of Action of Iressa

27. 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

28. Chronic Myeloid Leukaemia (CML) Accounts for 15-20% of all leukaemia cases Characterised by a massive clonal proliferation of myeloid cells, especially the granulocytic lineage Biphasic disease: chronic (or stable) ? blast phase Chronic phase: excess numbers of myeloid cells that still differentiate (i.e. cease dividing) as normal In 3-4 years accumulation of genetic and/or epigentic abnormalities ? block in cell differentiation ? disease progresses to blast crisis(30%+ myeloid or lymphoid blast cells in blood/bone marrow Transplantation a problem as not enough matching donors - and graft vs host disease means non-matching donors a risk Blast crisis phase - a few monthsTransplantation a problem as not enough matching donors - and graft vs host disease means non-matching donors a risk Blast crisis phase - a few months

30. Philadelphia chromosome = shortened chromosome 22 First discovered in the 1960sPhiladelphia chromosome = shortened chromosome 22 First discovered in the 1960s

31. The development of chronic myelogenous leukaemia. Chronic myelogenous leukaemia (CML) is a biphasic disease, initiated by expression of the BCR�ABL fusion gene product in self-renewing, haematopoietic stem cells (HSCs). HSCs can differentiate into common myeloid progenitors (CMPs), which then differentiate into granulocyte/macrophage progenitors (GMPs; progenitors of granulocytes (G) and macrophages (M)) and megakaryocyte/erythrocyte progenitors (MEPs; progenitors of red blood cells (RBCs) and megakaryocytes (MEGs), which produce platelets). HSCs can also differentiate into common lymphoid progenitors (CLPs), which are the progenitors of lymphocytes such as T cells and B cells. The initial chronic phase of CML (CML-CP) is characterized by a massive expansion of the granulocytic-cell series. Acquisition of additional genetic mutations beyond expression of BCR�ABL causes the progression of CML from chronic phase to blast phase (CML-BP), characterized by an accumulation of myeloid (in approximately two-thirds of patients) or lymphoid blast cells (in the other one-third of patients). Although the CML stem cell is multipotent, production of B cells from the neoplastic clone occurs only at low levels, and only rare T-cell precursors can be detected. This indicates that lymphopoiesis, particularly the development of T cells, is compromised by BCR�ABL expression The development of chronic myelogenous leukaemia. Chronic myelogenous leukaemia (CML) is a biphasic disease, initiated by expression of the BCR�ABL fusion gene product in self-renewing, haematopoietic stem cells (HSCs). HSCs can differentiate into common myeloid progenitors (CMPs), which then differentiate into granulocyte/macrophage progenitors (GMPs; progenitors of granulocytes (G) and macrophages (M)) and megakaryocyte/erythrocyte progenitors (MEPs; progenitors of red blood cells (RBCs) and megakaryocytes (MEGs), which produce platelets). HSCs can also differentiate into common lymphoid progenitors (CLPs), which are the progenitors of lymphocytes such as T cells and B cells. The initial chronic phase of CML (CML-CP) is characterized by a massive expansion of the granulocytic-cell series. Acquisition of additional genetic mutations beyond expression of BCR�ABL causes the progression of CML from chronic phase to blast phase (CML-BP), characterized by an accumulation of myeloid (in approximately two-thirds of patients) or lymphoid blast cells (in the other one-third of patients). Although the CML stem cell is multipotent, production of B cells from the neoplastic clone occurs only at low levels, and only rare T-cell precursors can be detected. This indicates that lymphopoiesis, particularly the development of T cells, is compromised by BCR�ABL expression The development of chronic myelogenous leukaemia. Chronic myelogenous leukaemia (CML) is a biphasic disease, initiated by expression of the BCR�ABL fusion gene product in self-renewing, haematopoietic stem cells (HSCs). HSCs can differentiate into common myeloid progenitors (CMPs), which then differentiate into granulocyte/macrophage progenitors (GMPs; progenitors of granulocytes (G) and macrophages (M)) and megakaryocyte/erythrocyte progenitors (MEPs; progenitors of red blood cells (RBCs) and megakaryocytes (MEGs), which produce platelets). HSCs can also differentiate into common lymphoid progenitors (CLPs), which are the progenitors of lymphocytes such as T cells and B cells. The initial chronic phase of CML (CML-CP) is characterized by a massive expansion of the granulocytic-cell series. Acquisition of additional genetic mutations beyond expression of BCR�ABL causes the progression of CML from chronic phase to blast phase (CML-BP), characterized by an accumulation of myeloid (in approximately two-thirds of patients) or lymphoid blast cells (in the other one-third of patients). Although the CML stem cell is multipotent, production of B cells from the neoplastic clone occurs only at low levels, and only rare T-cell precursors can be detected. This indicates that lymphopoiesis, particularly the development of T cells, is compromised by BCR�ABL expression The development of chronic myelogenous leukaemia. Chronic myelogenous leukaemia (CML) is a biphasic disease, initiated by expression of the BCR�ABL fusion gene product in self-renewing, haematopoietic stem cells (HSCs). HSCs can differentiate into common myeloid progenitors (CMPs), which then differentiate into granulocyte/macrophage progenitors (GMPs; progenitors of granulocytes (G) and macrophages (M)) and megakaryocyte/erythrocyte progenitors (MEPs; progenitors of red blood cells (RBCs) and megakaryocytes (MEGs), which produce platelets). HSCs can also differentiate into common lymphoid progenitors (CLPs), which are the progenitors of lymphocytes such as T cells and B cells. The initial chronic phase of CML (CML-CP) is characterized by a massive expansion of the granulocytic-cell series. Acquisition of additional genetic mutations beyond expression of BCR�ABL causes the progression of CML from chronic phase to blast phase (CML-BP), characterized by an accumulation of myeloid (in approximately two-thirds of patients) or lymphoid blast cells (in the other one-third of patients). Although the CML stem cell is multipotent, production of B cells from the neoplastic clone occurs only at low levels, and only rare T-cell precursors can be detected. This indicates that lymphopoiesis, particularly the development of T cells, is compromised by BCR�ABL expression

32. Treatment for CML Allogenic stem cell transplantation is only know curative therapy, however: CML occurs at all ages but majority of cases in 50s and 60s ? cannot tolerate side effects; Few suitable stem-cell donors ? less than 20% of cases can be cured this way

33. Regions that contribute to oncogenic properties of the fusion protein: *** = crucial role of tyrosine kinase encoded by src-homology (SH)1 domain * = important motifs of SH2 prot-interaction domain and the c-term actin binding domain ** = coiled-coil motif encoded by Bcr 1st exon which is responsible for dimerisation of the oncoprotein and constitutive activity of the abl tyrosine kinaseRegions that contribute to oncogenic properties of the fusion protein: *** = crucial role of tyrosine kinase encoded by src-homology (SH)1 domain * = important motifs of SH2 prot-interaction domain and the c-term actin binding domain ** = coiled-coil motif encoded by Bcr 1st exon which is responsible for dimerisation of the oncoprotein and constitutive activity of the abl tyrosine kinase

34. The enzymatic (tyrosine kinase) activity of the normal ABL protein (p145ABL), encoded by its SRC-homology 1 (SH1) domain, is kept under tight control, probably by the intramolecular binding of an N-terminal cap region encompassed by the first exon (1b or 1a) and the first part of exon a2. In the BCR-ABL fusion protein (p210BCR-ABL), lack of the ABL cap region and a dimerization domain encoded by the first exon of BCR are responsible for constitutive activation of the ABL SH1 domain, resulting in uncontrolled signal transduction and an abnormal cellular phenotype. The various functional domains of the ABL protein include the SRC-homology 3 and 2 regulatory domains (SH3 and SH2, respectively), the SH1 domain with its ATP-binding site, the nuclear-localization signal motif, the nuclear-export signal motif, the DNA-binding domain, and the G-actin and F-actin DNA-binding domains. The last two are important for the control of cytoskeletal organization, cell adherence, cell motility, and integrin receptor�mediated signal transduction.The enzymatic (tyrosine kinase) activity of the normal ABL protein (p145ABL), encoded by its SRC-homology 1 (SH1) domain, is kept under tight control, probably by the intramolecular binding of an N-terminal cap region encompassed by the first exon (1b or 1a) and the first part of exon a2. In the BCR-ABL fusion protein (p210BCR-ABL), lack of the ABL cap region and a dimerization domain encoded by the first exon of BCR are responsible for constitutive activation of the ABL SH1 domain, resulting in uncontrolled signal transduction and an abnormal cellular phenotype. The various functional domains of the ABL protein include the SRC-homology 3 and 2 regulatory domains (SH3 and SH2, respectively), the SH1 domain with its ATP-binding site, the nuclear-localization signal motif, the nuclear-export signal motif, the DNA-binding domain, and the G-actin and F-actin DNA-binding domains. The last two are important for the control of cytoskeletal organization, cell adherence, cell motility, and integrin receptor�mediated signal transduction.

35. 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 especially for early stages of CML inducing remission in 80% of patients Remission = complete cytogenetic response Approved in May 2001 < 3yrs after first Phase I study

36. The Downside Patients with more advanced CML respond less often and relapse more rapidly Presence of residual disease ? must give continued therapy ? develop resistance to Glivec Main mechanism: reactivation of Bcr-abl kinase via point mutations ? single aa changes ? alters structure of protein ? drug binding and sensitivity ? 3- to >100-fold Gleevec is a substrate for the multidrug resistance associated P-glycoprotein and could be a mech for resistance but not observed so far in patients BCR-ABL gene amplification and activation of alternative signalling pathways may also contribute to resistance in individual patients Interferon used to be the standard therapy for CML Glivec has rather poor binding affinity for Bcr-ablGleevec is a substrate for the multidrug resistance associated P-glycoprotein and could be a mech for resistance but not observed so far in patients BCR-ABL gene amplification and activation of alternative signalling pathways may also contribute to resistance in individual patients Interferon used to be the standard therapy for CML Glivec has rather poor binding affinity for Bcr-abl

38. Summary Targeted therapies can be more selective and show improved efficacy with minimal toxicity Almost invariably, initial response and latency are 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 Minimal toxicity = few adverse events (AEs)Minimal toxicity = few adverse events (AEs)

39. 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 �Mechanisms of BCR�ABL in the pathogenesis of chronic myelogenous leukaemia� Ren R (2005) Nat. Rev. Cancer 5, 172-183