Cancer 6.

Targeted Cancer Therapy

Professor Lenka Munoz
School of Medical Sciences
 Cancer module
• 6 lectures
•Cancer 1. Definition, nomenclature and characteristics
•Cancer 2. Metastasis and epidemiology of cancer
•Cancer 3. Etiology, clinical features and diagnosis
•Cancer 4. Hallmarks of cancer and oncogenic drivers
•Cancer 5. Tumour suppressors, apoptosis and DNA damage
•Cancer 6. Targeted cancer therapy
• 3 Pracs
– Microscopy Practical 1: Benign tumours (4 May 2022)
– Microscopy Practical 2: Malignant tumours (11 May 2022)
– Museum tutorial: Macrospecimens of tumours (18 May 2022)
 Learning objectives
• Understand the mechanism of activation/deactivation of cancer genes
• Understand the principle of cancer therapy
• Understand the role and integration of pathology in the cancer drug discovery process
• Be familiar with case studies on clinical cancer drugs
Increased expression/activity of oncogenes
1. Growth Factors: VEGF amplification
2. Growth Factors Receptors (RTK): EGFR ampl/mutations
3. Signal Transducing G-proteins: Ras mutations
4. Intracellular Kinases: B-Raf, Jak mutations, Abl-Bcr chimera
5. Transcription Factors: Myc amplification
6. Cyclins and CDKs: Cyclin/CDK amplification

Up-regulation of anti-apoptotic
proteins; Bcl2, Bcl-XL Loss of Tumour Suppressors
Rb, NF1, APC, PTEN deletion/mutations
p53 Deletions/mutations

VEGF amplification
VEGFR ampl/mutations

Upregulation/ re-activation of telomerases

 Activation of Cancer Genes
Five primary mechanisms
1. Point mutations
• In the proliferative signals: increase activity (e.g.; EGFR T790M, H-Ras G12V, B-Raf V600E, JAK V617F)
• In the tumour suppressor genes: loss-of-function mutations (e.g.; p53 R175, G245, R248)

2. Chromosomal translocation
• Translocation leading to over-expression of a proto-oncogene: in Burkitt's lymphoma c-myc from Chr. 8 translocated to
Chr. 14 close to the IgH gene, a region with hectic transcriptional activity, hence overexpression of normal Myc protein
• Translocation and genetic alteration of a proto-oncogene: Philadelphia chromosome in CML: translocation of the
abl gene (encoding a kinase) on Chr. 9 to form a chimeric protein with the bcr gene on Chr. 22 => Abl-Bcr protein with
potent kinase activity

3. Activation by gene amplification

• Reduplication of proto-oncogenes up to several hundred times over on their chromosome
• Results in the appearance of "homogeneous staining regions" on the chromosomes, and/or the presence of small
lumps of DNA called "double minutes"
• Amplification of a proto-oncogene results in increased expression of its protein product, predisposing to neoplastic
transformation.
• Myc amplification in neuroblastoma, EGFR amplification in glioblastoma
 1. Point mutations: JAK mutations
• Janus kinases: located on the cytoplasmic surface of the cell membrane, with no cell surface receptor element
• JAK2: tyrosine kinase that engages with cytokine receptors (e.g.; EPO receptor), becomes activated upon
ligand binding => in cell proliferation
• JAK2 with an activating point mutation is associated with some myelo-proliferative neoplasms (MPNs)
• MPNs are ABL-BCR negative haematological malignancies: e.g.; polycythemia vera, essential
thrombocythaemia, primary myelofibrosis
• JAK2 mutation results in cytokine independent proliferation of tumour cells
• valine-to-phenylalanine substitution at codon 617 – JAK2V617F - found in significant proportion of patients with
MPNs

Mutant JAK2 V617F: kinase activity

permanently switched on

Nat Rev Drug Discov 2011, 10: 1

2. Chromosomal translocation: ABL-BCR Chimera
• ABL gene encoding ABL tyrosine kinase is translocated from its normal abode on the chromosome 9 to
chromosome 22 where it forms a hybrid with the BCR (breakpoint cluster region) gene

• The ABL-BCR chimera aka Philadelphia chromosome encodes for ABL-BCR kinase that has potent kinase
activity

• Philadelphia chromosome occurs in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia
(ALL)
9 22 9 22 Philadelphia
bcr chromosome
locus
bcr
locus
abl-bcr
+ hybrid
gene
abl
oncogene

Potent
tyrosine kinase
chimeric protein

abl
oncogene Chromosomal
Translocation
 Deregulated ABL-BCR Signalling
• CML cells require BCR-ABL tyrosine kinase to proliferate and survive, hence the inhibition is highly effective
- oncogene addiction
• CML can be treated with ABL-BCR inhibitors: imatinib “cures” CML: ~97% response versus 70% response
with standard IFN-α/cytarabine therapy
• Not complete cure, therapy must be continued indefinitely: stem cells with BCR-ABL persist and could re-
initiate full leukemia if treatment stopped
• Acquired resistance to therapy

Clin Cancer Res 2011;17:212-221

 Activation of cancer genes
4. Epigenetic changes
• Epigenetics refers to reversible, heritable changes in gene expression that occur without mutation and involve
post-translational modifications of histones and DNA methylation, both of which affect gene expression

• In normal differentiated cells, the majority of the genome is not expressed, due to silencing by DNA methylation
and histone modifications, that lead to the compaction of DNA into heterochromatin

• Cancer cells are characterized by a global DNA hypomethylation (“switch on”) and selective promoter-
localized hypermethylation (“switch off”)

• e.g.; hypermethylation of CDKi loci for p14, involved in the Rb and p53 signalling (colon and gastric
cancers)

• e.g.; hypermethylation of BRCA1 in breast cancer, VHL in renal cell carcinomas

• Cancer cells also characterized by altered post-translational modifications of histones

 Activation of Cancer Genes
5. miRNA

• miRNAs are noncoding, single-stranded RNAs, ~22 nucleotides, that are incorporated into the RNA-induced
silencing complex. They mediate sequence-specific recognition of mRNAs and posttranscriptional gene
silencing.

• miRNAs control cell growth, differentiation and cell survival.

• miRNAs undergo changes in expression in cancer cells, via amplifications and deletions of miRNA loci, which
can increase oncogenes expression or reduce tumor suppressor gene expression

• e.g.; down-regulation or deletion of miRNAs in some leukemias and lymphomas increases expression of
BCL2, the anti-apoptotic protein

• e.g.; miRNA-mediated upregulation of oncogenes RAS in lung tumors and MYC in B-cell leukemias
 Cancer Therapy
• In the majority of cases, therapy follows surgery

• Chemotherapeutic agents: drugs intended to be toxic for malignant (fast growing) cells =>
predominantly target rapidly proliferating cancer cells
– DNA-alkylating agents: temozolomide, cisplatin
– Anti-mitotic drugs: paclitaxel, vincristine
– Inhibitors of RNA synthesis: doxorubicin, etoposide
– Inhibitors of nucleotide interconversion: 6-mercaptopurine, 6-tioguanine

• Radiotherapy is the use of precisely targeted x-rays to destroy cancer cells while reducing the
impact of radiation on healthy cells

• Identification of molecular pathways driving carcinogenesis (i.e.; hallmarks of cancer) enabled

discovery of tumour specific therapy known as Molecularly Targeted Therapy
 Molecularly Targeted Therapy
• Molecularly-targeted drugs specifically target proteins involved in the neoplastic process => targeting cancer hallmarks
• Each hallmark is driven by complex signalling networks of interacting proteins
• Drugs that interfere with each of the hallmark necessary for tumour growth have been developed
• Many are approved for clinical use in treating certain forms of human cancer
• Thousands of candidate drugs with different modes of action currently in development for all hallmarks

Increased expression/activity of oncogenes

1. Growth Factors: VEGF amplification
2. Growth Factors Receptors (RTK): EGFR ampl/mutations
3. Signal Transducing G-proteins: Ras mutations
4. Intracellular Kinases: B-Raf, Jak mutations, Abl-Bcr chimera
5. Transcription Factors: Myc amplification
6. Cyclins and CDKs: Cyclin/CDK amplification

Up-regulation of anti-apoptotic
proteins; Bcl2, Bcl-XL
p53 Deletions/mutations

Loss of Tumour Suppressors

Rb, NF1, APC, PTEN deletion/mutations

VEGF amplification
VEGFR ampl/mutations

Upregulation/ re-activation of telomerases

 Molecular Players of Oncogenesis
• Oncogenes: PDGFR, EGFR, ALK, Ras, B-RAF, JAK2, MYC, CDK4, BCR-ABL fusion
• Tumour-suppressor genes: PTEN, NF1, RB, BRCA1/2, TP53 deletion/mutation
• Metabolic alterations: PKM2
• Evasion of apoptosis: over-expression of BCL-2 proteins
• Limitless replicative potential: telomerase expression and activity
• Angiogenesis: VEGF amplification, VEGFR activation
• Metastatic oncogenes: SNAIL and TWIST promoting EMT
• Escape of host defense: TGFβ, PD-1 ligand
• Cancer-enabling inflammation: IL-1, IL-4, IL-13
• Epigenetic changes: MLL1, SNF5

• obvious question is which one of these proteins to target….

– Validated target
– Druggable target
 Druggable targets
• Druggability is a term used in drug discovery to describe a biological target (e.g.; protein) that is known, or
is predicted, to bind with high affinity to a chemical molecule

• Consequence of the “disease-first, drug-second” strategy is that many predicted drug targets in cancer are
non-enzymes, such as scaffolding, regulatory or structural proteins, and their activities are often dependent
on protein-protein interactions => drug design challenging => considered non-druggable targets

• Enzymes are druggable: they contain deep grooves that are amenable to binding by low molecular weight
“drug-like” molecules

• The binding of the drug to a druggable target must alter the function of the target to a therapeutic benefit

• Druggability is most often restricted to small molecules = low MW organic compounds = drugs; but also to
biologics such as monoclonal antibodies
 Druggable vs Non-Druggable Targets
Druggable targets contain deep grooves (pockets) that are amenable to binding by
molecules and this binding will alter their function.

Druggable: RTKs (growth factor receptors), Non-druggable: Ras, PTEN, Myc, p53, cyclins
intracellular kinases (RAF, PDK, Akt, mTOR)
and enzymes (MDM2)
 From Genes to Mechanisms to Medicines

Genetics and genomics Molecular pathways Drug development Precision medicines:

of human disease and target validation in to target the pathway Match the right patient
relevant models with the right drug

Molecular pathways: Delineation of the mechanism by which gene/protein of interest (target) initiates and/or
drives the course of the disease.
Target validation: Evaluation of whether gene/protein of interest (target) has a key role in a disease process
and whether pharmacological modulation of the target could be effective in a defined patient population.
Genetics and genomics
Target validation
of human disease
Target validation in relevant models

Pathology component: Phenotype upon target silencing; phenotype

upon target over-expression, mechanism by which target drives
carcinogenesis; expression in human tissues etc.

Pharmacology component: partial/full/allosteric agonist/antagonist for

the receptor; reversible/irreversible/allosteric inhibitors or false
substrates for enzymes etc.
 Validation of B-RAF as a Cancer Target
• B-RAF gene found to be mutated in many cancers (50% of melanomas, thyroid tumours)
• most common: valine-to-glutamic acid exchange at codon 600 (V600E) => increased kinase activity driving cell proliferation
• B-RAF is a kinase hence druggable
• B-RAF inhibitor hypothesized to inhibit the sustained proliferative signalling hallmark
• B-RAF validation
– In vitro: proliferation of melanoma cells stimulated by B-RAFV600E, blocked by genetic ablation of B-RAFV600E
– In vivo: B-RAFV600E gene conditionally suppressed resulted in regression of established tumours

Nat Rev Drug Discov 2012, 11: 873

 B-RAF inhibitor Vemurafenib
COLO205 xenografts

• Drug discovery campaign at Plexxikon led to B-RAF inhibitor vemurafenib

• Confirmation in cells: Vemurafenib inhibits B-RAF kinase activity => MEK and
ERK not phosphorylated => decreased cancer cell proliferation

• Confirmation in animal models (xenografts): Vemurafenib leads to tumour

regression in a dose-dependent manner
B-RAFV600E melanoma patients

• Validation in clinical trials: Vemurafenib caused substantial tumour regression

in melanoma patients, but not in colorectal cancer patients (both BRAFV600E)

• Vemurafenib (Trade name: Zelboraf) approved in 2011/12 for treatment of B-RAFV600E colorectal patients
metastatic melanoma with B-RAFV600E mutation

Nat Rev Drug Discov 2012, 11: 873

 B-RAFV600E Diagnostic Development
• Molecularly-targeted therapy = personalised therapy => only patients with BRAFV600E should be treated
with vemurafenib

• Accurate assay able to determine which melanomas harbour BRAFV600E developed by Roche Molecular
Systems, approved concurrently with vemurafenib

• Real-time PCR assays able to detect B-RAFV600E mutation: directly in formalin-fixed paraffin-embedded
tissue (FFPET) or in tumour sample (needs 125 ng of genomic DNA from 5 μm FFPET)

• Vemurafenib gives app. 3 months survival advantage over the chemotherapeutic dacarbazine

• Relapse occurs within a year of vemurafenib treatment: resistance due to alternative signalling pathways
(bypass signalling)
 Targeting EGFR in Cancer
• Excessive EGFR activity (due to gene amplification and mutation) leads to uncontrolled cell proliferation
• EGFR (and all other growth factor receptors or RTKs) can be targeted with antibodies (drug names will end with -
mab) or small-molecule kinase inhibitors (drugs name will end with -nib)
• Intracellular kinases (e.g.; B-RAF, JAK2) are targeted only with small-molecule kinase inhibitors (drug ending -nib)

EGFR mutations => constitutive EGFR activity

EGF EGFRvI (NH2-terminal del)
glioblastoma
EGFRvII (exons 14-15 del)
EGFRvIII (exons 2-7 del)

R84K, A265V, P545L

P P
EGF
R T790M
G-protein L858R
G791S
in-frame del in exon 19 NSCLC

Kinases

Cyclins
TF
CDKs
Proliferation
 Targeting EGFR in Cancer
• EGFR monoclonal antibodies: directed at the extracellular portion of EGFR (ligand-binding domain) and
inhibit ligand binding to the receptor: cetuximab, panitumumab => EGFR cannot be activated
• EGFR kinase inhibitors: bind to the ATP pocket in the kinase domain (intracellular), inhibit EGFR kinase
activity => EGFR cannot phosphorylate down-stream substrates => decreased/blocked proliferation
– reversible EGFR inhibitors erlotinib, gefitinib, lapatinib
– irreversible (covalent) EGFR inhibitors: irreversible (covalent) EGFR inhibitors afatinib, dacomitinib

cetuximab, panitumumab
EGF EGF lapatinib, erlotinib,
gefitinib, erlotinib

P P P P
EGFR EGFR

G-protein G-protein

Kinases Kinases

Cyclins Proliferation Cyclins Proliferation

TF CDKs TF CDKs
 Erlotinib (Tarceva) in lung cancer therapy
glioblastoma

• EGFR kinase domain mutations common in lung cancer

• point mutations in exon 19 and 21: EGFRG719S, EGFRL858R, EGFRL861Q
• rare exon 22 mutation recently reported: EGFRE884K
• mutations lead to constitutive kinase activity => proliferation NSCLC

• Selected clinical trials with Erlotinib (Tarceva)

• EURTAC study: Tarceva as 1st line treatment for patient with metastatic NSCLC with EGFR mutations
which must be detected with a diagnostic test
1st line Tarceva: Progression Free Survival (PFS) = 10.4 months vs 5.2 months with chemotherapy

• BR.21 Study: Tarceva as 2nd/3rd line treatment for metastatic NSCLC that progressed after chemotherapy
2nd/3rd line Tarceva; median Overall Survival (mOS) = 6.7 months vs 4.7 months over placebo

• SATURN Study: Tarceva as 1st line maintenance treatment for metastatic NSCLC that did not progress
during 1st line platinum-based chemotherapy
maintenance Tarceva; median Overall Survival (mOS) = 12 months vs 11 months over placebo
 glioblastoma

Targeting EGFR in glioblastoma

• 60% of glioblastomas present EGFR amplification
• Of these 60%, half also presents ligand binding domain mutations NSCLC

• Most common mutation: deletion of exons 2-7 => EGFRvIII => incapable of binding EGF, but displaying
constitutive kinase activity => excessive proliferation
• Rare EGFRA289V kinase-domain auto-activating mutation

• EGFR amplification and/or mutation drives several cancer hallmarks in glioblastoma: sustained
proliferative signalling, resistance to cell death, deregulating cellular energetics

• HOWEVER, all EGFR inhibitors failed in glioblastoma clinical trials

• Resistance due to alternative signalling pathways (by-pass signalling), blood-brain barrier, presence of
aggressive glioblastoma stem cells and extreme invasiveness
 Multi-kinase inhibitors
Advantages of polypharmacology in cancer
• targeting several pathways
• less probability for drug resistance
• combination therapy with one drug
Selectivity
• required for treatment of non-malignant diseases
• off-target activity often results in side effects
Nat Rev Drug Disc (2006) 5, 835
Multi-kinase inhibitors more effective in cancer therapy
• Sorafenib (Trade name: Nexavar)
• Raf1, BRAF, BRAFV600E, VEGFR1/2, Flt-3, p38, c-Kit
• advanced renal cell carcinoma
• Regorafenib (Trade name: Stivagra)
• RET, VEGFR1/2/3, KIT
• metastatic CRC
• Cabozantinib (Trade name: Cometriq)
• RET, MET, VEGFR1/2/3, KIT, FLT3
• metastatic medullary thyroid cancer
• Ponatinib (Trade name: Inclusig)
• BCR-ABL, KIT, RET, FLT3
• chronic myeloid leukaemia
 Angiogenesis and VEGFR
• Angiogenesis or neovascularisation: formation of new blood vessels from pre-existing ones
– essential for tumours to grow beyond 1-2 mm3
– switch from local vascular supply to novel micro-capillary formation
– supply of nutrients and oxygen to the tumour
– allows tumours cells to enter circulation (metastasis)

• Hypoxia (low oxygen levels esp. in the core of the tumour) triggers angiogenesis through HIF-1α (oxygen
sensitive transcription factor) => HIF-1α activates transcription vascular endothelial growth factor (VEGF)
=> pro-angiogenic factor => activate formation of blood vessels

• VEGF binds to VEGFR => VEGFR dimerises and auto-phosphorylates => active VEGFR initiates
intracellular signalling cascades => neovascularisation

• VEGFR is a kinase, hence druggable

• Drugs targeting VEGFR are clinically used anti-cancer drugs (not curative though)
 Anti-angiogenesis agents
• Monoclonal antibodies: Bevacizumab (Trade name: Avastin) binds to the extracellular portion of VEGFR
and prevent VEGF from binding to VEFGR => VEGFR cannot get activated
1st line in mCRC in combination with 5-FU chemotherapy, NSCLC with carboplatin and paclitaxel; metastatic
breast cancer with paclitaxel
2nd line in glioblastoma as monotherapy

• Kinase inhibitors: small molecule drugs binding to the ATP pocket of VEGFR => VEGFR cannot activate
down-stream signalling pathways
– Sunitinib (Trade name: Sutent): inhibiting VEGFR, and PDGRs, c-kit, Flt3, Ret
1st line in renal cancer as monotherapy
2nd line in gastrointestinal tumours as monotherapy
– Sorafenib (Trade name: Nexavar): inhibiting VEGFR and PDGFRs, Kit, FLT3
1st line in renal and hepatocellular cancer as monotherapy
Non-kinase Molecularly Targeted Therapy
• Understanding cancer at the molecular level led to discovery and development of numerous cancer drugs that
improve survival of cancer patients

• As our understanding of cancer at the molecular level improves, more molecularly targeted therapies are in
development and clinical trials

• Personalised therapy, multimodal approach and tissue-agnostic approach

Examples of non-kinase molecularly-targeted cancer drugs

• Carfilzomib (Kyprolis): 20S proteosome inhibitor, multiple myeloma
• Omacetaxine (Synribo): moA unknown, chronic myeloid leukeamia
• Pomalidomide (Pomalyst): immunomodulatory anti-caner agent, multiple myeloma
• Belinostat (Beleodaq): histone-deacetylase inhibitor, T-cell lymphoma
• Pembolizumab (Keytruda): PD-1 specific antibody, metastatic melanoma
• Nivolumab (Opdivo): PD-1 inhibitor, unresectable or metastatic melanoma
• Blinatumomab (Blincyto): CD19 and CD3-specific antibody, ALL
• Olaparib (Lynparza): PARP inhibitor, advanced BRCA-mutated ovarian cancer
 What do I need to know
• Describe mechanisms by which oncogenes are activated
• Understand epigenetic changes in cancer
• Describe role of miRNA in cancer
• Understand and briefly describe the concept of target validation
• Examples of cancer drugs targeting growth-factor receptors (-mabs & -nibs)