Cancer Genes and Targets for Therapy

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Cancer Genes and Targets for Therapy
Helen C Hurst

• Centre for Tumour Biology
• Institute of Cancer
• Charterhouse Square

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Cancer Treatment
Surgery
Chemotherapy Radiotherapy
Apoptosis
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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
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Cancer progression in ductal carcinoma of the
pancreas
.progressive mutation/activation of cancer
genes
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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
• Its 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

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Signalling Pathways
Proliferation/survival
GFs
2nd messenger cascade
Small Molecules
Growth Arrest/apoptosis
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Cancer Genes and Targets for Therapy

• Target specific molecules or genetic defects
  found only or mostly in tumour cells
• Therefore reduce the toxicity commonly found for
  non-targeted chemo- and radio-therapy
• Ultimate goal is to replace toxic therapies with
  better tolerated AND effective targeted therapies
  - improve patient tolerance, response and survival

So what are these targeted therapies?
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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 - monoclonal antibody 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

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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
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The ER is a ligand dependent transcription factor
Anti-oestrogens

Proteins that ? proliferation/survival
ERE (oestrogen response element)
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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 Tamoxifen to early stage, ER ve patients
  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...
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. 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.
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Pluses and minuses

• Selective estrogen receptor modulators (SERMs)
  such as 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

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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 (AIs) which prevent local synthesis of
  oestrogens (adipose tissue)
• Clinical trials have shown AIs to be effective
  and well-
• tolerated and resistance is slower to develop
• Treatment Sequencing of anti-oestrogens and AIs
  prolongs clinical usefulness

however, resistance to all these agents is an
issue and develops for largely similar reasons
as Tamoxifen resistance
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Receptor Tyrosine Kinase
Signal pathway cross-talk ? oestrogen-independence
? target 1 or more of these pathways in addition
(combination therapy)
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• 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

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Anti Receptor Tyrosine Kinase therapies - what
are RTKs?

• 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

Docking sites for signalling proteins
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The ErbB Network
Drug Resistance
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IHC

FISH
ERBB2 overexpressed in many solid tumours e.g.
25 breast carcinomas ? correlates with
ER negativity and poor prognosis
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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 -
  immunogenic in humans
• ? Recombinant, humanised antibody created
• Herceptin has a higher affinity for ErbB2 than
  4D5 and has a cytostatic growth inhibitory effect
  against ErbB2ve breast cancer lines

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Humanising an antibody
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Herceptin in the clinic

• In Phase I trials was well-tolerated had
  anti-tumour activity
• In randomised trials - improved survival in
  patients with amplification of the ERBB2 gene
• Approved for use in metastatic ErbB2ve breast
  tumours (1998)
• Largely used in combination with chemotherapy
  drugs (taxol, cisplatin cardiac side-effects
  with dox)
• Mode of action not totally clear but can
  downregulate ErbB2 prevent cleavage of
  extracellular domain (causes activation)
  activate patients own immune response

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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 as Pertuzumab being tested
  in combination with other drugs
• May be useful in a wide range of ErbB2 ve solid
  tumours and can synergise with Herceptin

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No signalling
Proliferation/Survival
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Other ways to target RTKs
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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

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Mode of Action of Iressa
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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
• Who will benefit? In NSCLC - responding patients
  carry somatic mutations in the EGFR gene not
  true in breast cancer patients where sensitivity
  influenced by activity of pathways downstream of
  EGFR
• Combination therapies being optimised

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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

What mutations cause this?
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Chromosome 1
Gene A
Gene B
Chromosome 2
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
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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(922)(q34q11) reciprocal translocation
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Self-renewing haematopoietic stem cells
Neoplastic transformation
Myeloid
Lymphoid
2/3 patients
1/3 patients
Chronic Phase (3-4 yrs) ? Blast Phase
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Treatment for CML

• Allogenic stem cell transplantation is only known
  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

Can the Bcr-abl fusion protein be targeted?
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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 especially for early stages of
  CML inducing remission in 80 of patients
• Remission complete cytogenetic response
• Approved in May 2001 lt 3yrs after first Phase I
  study

but
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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 gt100-fold

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Possible Solutions

• Combination therapy using Glivec with cytotoxic
  agents and/or interferon
• Rational drug design ? make similar molecules
  that bind more avidly e.g. AMN107 (nilotinib)
  with gt20-fold higher affinity for wt and mutant
  Bcr-abl shows early clinical promise
• Dasatinib (BMS-354825) - inhibits Abl and Src
  family kinases - another promising new clinical
  candidate
• Target the additional mutations found in blast
  phase patients e.g TP53 mutations further
  chromosomal translocations

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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

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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
• Advances in targeting human epidermal growth
  factor receptor-2 signaling for cancer therapy
  Meric-Bernstein F Hung M-C (2006) Clin. Cancer
  Res. 12, 6326-30
• Molecular Mechanisms of Epidermal Growth Factor
  Receptor (EGFR) Activation and Response to
  Gefitinib and Other EGFR-Targeting Drugs Mayumi
  Ono Michihiko Kuwano (2006) Clin. Cancer Res.
  12, 7242-51
• Mechanisms of BCRABL in the pathogenesis of
  chronic myelogenous leukaemia
• Ren R (2005) Nat. Rev. Cancer 5, 172-183