Anticancer - oncogene specific therapies

Designing drugs to target cancer

• Identify the difference between cancer cells and normal cells:   

  • Molecular events  

  • Phenotypic changes, as a consequence of molecular events  

• Current drugs we have  target these 2 areas:  

  • Cytotoxic drugs usually target the faster-growing nature/phenotypically different cells  

  • Molecular target drug targets the changes that take place leading to cancer and tend to be more specific 

• Cancer cells rely on oncogenes and the production of their “surviving signals” —> known as oncogene addiction  

• if we remove these surviving signals, this is one of the methods to selectively treat cancer  

• But cancer cells will try and reestablish and use other surviving signals, known as resistance 

• For some patients, we don’t know the molecular mechanism that leads to their cancer, so treatment can be less specific and more challenging 

Cell culture 

• Use 3D chromatogel culture to mimic the cell in vivo better compared to 2D culture 

• Breast epithelial cells in culture usually look round and symmetrical (top right); however, the cancerous cells are asymmetrical with lots of protrusions (bottom left)  

• The other two samples are not normal either: the one on the top left has a tumour suppressor gene knockout while the bottom right has an activation of an oncogene 

• Shows that there is more than one types of mutations needed to induce cancers and more than one type of cancer 

• For example: If you wanted to treat the bottom left sample you could simply knock out the oncogenes as they’re reliant on this  

Oncogenes and Tumou supressor genes

• Two types of cancerous genes  

  • Oncogenes – genes which drive the tumour phenotype expression that the cells are reliant on  

  • Tumour suppressor genes – prevent the formation of tumours  

• To fully transform into a cancerous cell, you need two events to fully transform cells from normal to cancer cells 

• This also allows for selective targeting of cancer cells  

Selective Toxicity 

• Drugs of diseased cells have similar properties to normal cells (especially those in bone marrow, hair, GI mucosa and skin)  

• If we target these fast-growing sites we will have consequences in the form of side effects:  

  • If we target bone marrow cells will impact the immune system —> anaemia or susceptibility to infection 

  • If we target hair cells will cause hair loss  

• We need to look for “windows of opportunity” where cancer cells are especially vulnerable to avoid damage to our cells 

Precision medicine 

• Instead of grouping the patients by the organ of their cancer, can group patients by their molecular signature, indicative of the driving events leading to the cancer  

• Can create more efficacy and selective toxicity  

• Can also apply this to other diseases, such as diabetes  

How can we selectively target cancer cells

• PTEN tumour suppressor genes  

• PTEN+ cells normal cells with tumour suppressor, PTEN- cancerous cells with no tumour suppressor  

• Mixed in a 1:1 ratio and can knock down genes individually, can treat the cells with a drug or siRNA library and observe the outcome  

• The siRNA targets specific mRNA and degrades it creating a knockout of each gene 

• Can see if the specific gene knockout affects the cancer cells, the normal cells or both 

• Additionally, you can do this with a drug library instead of knocking out the genes with siRNA libraries 

• Through this technique was able to identify the gene WDHD1 if knocked out, it selectively inhibits or kills PTEN. Targeting these genes can selectively target this type of breast cancer 

Transcriptomic Profiling

• Another technique of categorising patients for selective treatment, but instead of wet lab can use bioinformatics  

• Can group cells from their molecular signatures  

• Examined neuroblastoma patients and examined the overall survival rate 

  • Can help you categorise the molecular markers into high or low risk cancers 

  • The green profile patients had a high overall survival rate without any treatment —> suggests that their cancer type is low risk 

• Can also create maps which hotspot the molecular events which occur, leading to that cancer. 

• Red signals indicate oncogenic signals, and were associated with the aggressive, high mortality rate cancers, supporting the idea that we can group patients based off their gene expression profiles 

• Can then create therapeutic targets which help to specifically target these molecular events that lead to cancer  

Success of current treatments

• Surgery is not likely a viable treatment method for melanoma as it spreads all over the body in many tumours  

• Can understand the oncogene activation and block the oncogene pathway to target the cancer cells using a drug  

• However, after a few rounds of treatment, the cancer returns due to their ability to reestablish surviving treatments (called drug resistance)  

Molecular basis of cancer

1. Normal cell growth and cancer  

2. Cancer cells have altered genomes  

3. Mutations  

4. Oncogenes and Tumour supressing genes  

 

1. Normal cell growth and cancer

•  Usually, there's a fine balance between apoptosis and proliferation for normal cell growth  

• In cancer the events of oncogene activation result in more proliferation or less apoptosis  

• The removal of the tumour suppressor genes prevent these cells from being targeted  

Classification of cancers

Neoplasia – excess proliferation without relation to normal growth or repair. Growth may be fast but rarely exceeds that in the fastest growing tissues  

Benign – called a tumour rather than cancer. Proliferate locally and retain characteristics, have a defined boundary.  

Malignant – Not encapsulated, ill defined edge, projections extend into surrounding tissues, less well differentiated than the cells of origin. Spread to other sites by invading surrounding tissues 

Can also develop from benign to malignant:  

Altered genome of cancer cells

• Karyotypes can detect the changes in genomes that occur in cancer  

• Karyotypes of cancer genome have:  

  • Different number of chromosomes —> more or less copies  

  • Translocated DNA across the chromosomes causing mutated chromosome shape  

  • Lost DNA 

• Chromosomes might start with small chromosome altering events but as the cancer develops accumulate changes leading to chromosome instability  

3. Mutations

• Germline mutations  

  • A change in the DNA sequence that can be inherited from either parent  

  • Occurs in all the cells 

• Somatic mutation  

  • A change in the DNA sequence in cells other than sperm or egg  

  • The mutation is present in the cancer cells and its offspring but not in the patient's healthy cells 

  • Occurs only in the tumour cells 

• Most mutations causing oncogene activation will be point mutations, while  in tumour supressor genes will be frameshift  

Hereditary Predisposition

• If generations before you in your family had cancer doesn’t mean you’ll inherit it —> some familial mutations that you will inherit, but only 5% of cancers are hereditary  

• Loss of tumour suppressor genes will result in a higher chance of cancer —> For cancer to occur you need multiple events and this is one of them 

• Don’t necessarily have cancer, but they have a higher chance of having cancer later in life as we have 2 tumour suppressor genes, it's likely that you’ll have at least one healthy copy.  

Transformation of cells to cancer cells

• For normal cells to transform into cancer cells 2 events need to happen (hallmarks of cancer cells) 

  • Activation of oncogene 

  • Inactivation of tumour suppressor gene  

• This offers the cancer growth advantages compared to other cells  

• Also creates therapeutic targets —> once we understand these therapeutic events we can target these mechanisms  

• We won’t target normal cells because they’re not addicted to the oncogenes  

Events leading to transformation of cells BRCA

• Somatic or germline mutations in the tumour suppressor genes  

• BRCA important for damage repair in the DNA, therefore loss of BRCA results in high chance of breast or ovarian cancer  

• Once the tumour cells have lost BRCA they can rely on POP to repair the damages —> we can target POP with AntiPOP inhibitors  

• In most cases those with BRCA mutations have no family history —> developed over their lifetime.  

Origins of cancer

• Most tumours require multiple mutations, but this can vary  

• E.g: white blood cell cancers only need one mutation whereas epithelial cell cancers need multiple —> this is because for an epithelial cell they need multiple mutations to develop the properties to proliferate, then grow out of the space etc. however, white blood cells don’t need many events as they’re already highly proliferative and are already circulated around the body 

Car Crash Theory

• Like engines which drive the cell cycle (the car) 

• Tumour suppressor genes are like brakes that prevent the crash – in the absence, these mutations are not repaired and allow them to be passed onto other cells and accumulate 

• For cancer you need both events

Events causing colon cancer (adenocarcinoma)

• Loss of APC (tumour suppressor) involved in Wnt signalling activates the oncogenic pathway  

• At this point is not a cancer, only a tumour as its not yet invasive  

• Next is the activation of KRAS is the activation of the oncogene and drives the progression from an early adenoma to a late adenoma 

• Next is the loss of p53 which allows the tumour cell to acquire multiple mutations to drive the cancer 

• Progression of late adenoma to adenocarcinoma which has invasive properties 

• We need the loss of the tumour suppressor early on to allow the mutations to accumulate 

p53

• Can decide whether damage can be reversed or repaired —> whether the cell survives or dies  

• Most cancers actually have increased rates of apoptosis but due to the sheer amount of proliferation the net result is growth 

• Loss of function p53 results in more cell survival, whereas overactive p53 results in unnecessary cell death, resulting in ageing, neurodegenerative disease or radiation sickness 

• Elephants have 40 copies of p53 while we only have 2, so despite having more cells can better control the progression of cancer 

Examples of key oncogenes - Ras 

• Mutations in Ras responsible for a majority of cancers 

• 3 isoforms of ras – k,h,m Ras  

• Ras activity is controlled by GDP or GTP bound  

• When GTP bound it will be able to bind to Raf or PI3K  

  • PI3K causes activation of AKT  

  • While activation of Raf causes activation of ERC signalling pathways 

• Ras can also be activated by upstream factors like EGFR  

• Very regularly mutated pathway in cancer  

  • EGFR highly mutated in lung cancer  

  • Raf oncogene for melanoma  

  • K Ras oncogene in pancreatic cancer  

Identifying mutations using sequencing

• Each colour represents a nucleotide, can compare healthy DNA to mutated DNA 

• Can then figure out which amino acids in the proteins will be changed  

  • For example at position 12 a glycine is changed to a valine, which will lock the Ras into the GTP format due to a higher affinity –L> doesn’t require upstream signalling for activation  

  • Called a G12V mutation as its G—>V at position 12  

Heterozyous muations

• As we have two copies of the genes, we can have a mutated gene and a normal one which looks like this: representing a normal and mutated allele 

How common are Kras mutations

• There are specific hotspot mutations which are more common  

• These are in position 12, 13 and 61  

• The particular mutations will create a unique binding site in the tumour KRAS which we can target 

• Therefore, the wild-type Ras will be unaffected —>selectivity  

Impact of Kras mutations

• Growth signals, released when cells are damaged stimulate Kras 

• Kras activity is important for the proliferation of cells when they’re damaged 

• Ras has GAP, an enzyme which can inactivate it by exchanging GTP for GDP 

Comparison of Kras and RB1 tumour supressor gene

• Mutations in the RB1 (tumour supressor) are a lot more evenly dispersed, don’t necessarily have specific hotspots  

• Also deletion mutations a lot more common in the tumour suppressor genes 

• On the right is RB1 and left is Kras mutations 

• For the tumour suppressor genes, as long as there is a loss of function of the tumour suppressor gene, it will be inactive. However, mutations in the oncogene must be more specific as it must affect the GTP binding site. 

Carcinogens 

• Introduce mutations to key genes 

• Can activate oncogenes 

Herb treatments  

• Some can be carcinogens 

• Unique mutation patterns in Asian cohorts linked to carcinogens in herbal medicines 

UV light  

• Linked to malignant melanoma  

• Signature C—>T or G—>A mutations  

• If these DNA changes occur in critical genes like BRAF this can lead to inappropriate cell growth and melanoma  

Tobacco smoke  

• Tobacco smoke carcinogens such as PAH (polycyclic aromatic hydrocarbons)  

• Signature G—>T mutations  

HPV  

• Linked to cervical cancer  

• The most high risk (HPV16) the viral protein can activate oncogenes and cause activation of tumour suppressor genes  

• Oncoproteins integrated into the cells 

• Now a vaccine to reduce cases of cervical cancer  

Diet and obesity  

• Linked to bowel and stomach cancer  

• Inflammation from the diet linked to damage to the cells lining the stomach/bowel  

Viral and cellular Oncogenes  

• Some cancers linked to viruses, were previously thought that it was caused by the oncoproteins from the virus causing cancer  

• Cellular oncogene will only be activated when necessary but when infected with viral infection the virus controls the expression of oncogene  

Viral and cellular oncogenes

• Some cancers linked to viruses, were previously thought that it was caused by the oncoproteins from the virus causing cancer  

• Cellular oncogene will only be activated when necessary but when infected with viral infection the virus controls the expression of oncogene  

Translocation - Example C-Myc

• CMyc an important oncogene which is controlled by its own promoter  

• Will only be expressed when it needs to proliferate 

• A single translocation event can cause Myc to be under control of IgG promoter rather than its own promoter 

• IgG more regularly produced than Myc 

Targeting cancers

• Loss of tumour suppressors gives cancer cells growth advantages: 

  • Sustaining proliferative signals 

  • Evading growth suppressors 

• Depending on which molecules provide these advantageous signals, we can create selective targets  

• Very challenging for us to restore tumour suppressor functions, as there are often more mutations in the tumour suppressor gene and you don’t know which mutation is causing loss of function  

• Oncogenes are good targets as they often have mutation hotspots —> relatively easy to design a drug to stop oncogene signalling  

• These targets can be applied for different types of cancer —> targeting based off the driving mutation not location  

Specific examples of gene mutations 

1. BCR-ABL  

2. EGFR  

3. BRAF 

4. PD-1/PDPL-1 

5. VEGF 

6. Precision medicine  

1. BCR-ABL oncogene

• Results in a Philadelphia chromosome eliciting a fused protein  

• BCR gene is usually present in the q11 region of chromosome 22, ABL gene usually present on q34 region of chromosome 9  

• This translocation is written as t(9:22)(q34,q11)  

  • T before brackets indicates a translocation  

  • Q indicated that the mutation is on the long arm, p indicated the short arm  

• forms a fused protein which drives chronic myeloid leukaemia in all cases and acute lymphocytic leukaemia in 10% of cases 

• When you have the fused protein present there’s a coiled-coil domain which is always dimerised, leading to constant activation of the cell cycle 

• The wild type proteins are essential for the normal function in the cell but the difference is that signalling is only activated when required.  

• Most effective drugs against this mutation is the tyrosine kinase inhibitors —> this inhibits the ABL protein allowing for selective destruction of the cancer cell  

Visualisation of fused proteins using FISH

• These fused proteins can be visualised by FISH (fluorescent in situ hybridisation) 

• The green tag indicated the ABL protein, and the red indicates the BCR  

• In the normal situation, the two colours are distinct, but when there’s a translocation, the colours are mixed 

• Useful for diagnosis/ identifying mutation occurring in the cancer 

Targeting these proteins

• Imatinib is a tyrosine kinase inhibitor  

  • Any drugs which end in -ib means it’s a small molecule inhibitor  

• Holds the activation loop in the inactive conformation which stops signalling of both w/t and cancerous ABL 

• Kinases are relatively easy to inhibit so tend to be most successful anticancer agents 

Mutations and persistence of cancer

• However, cancer cells have an unstable genome so can relatively easily acquire mutations  

• If a mutation occurs in the binding site for the imatinib, then this drug is no longer effective, and the cancer will persist  

• After a few rounds of treatment around 1/3 of patients will develop a mutation.  Common mutations which change the structure include: T315I (most common), E255K, H396P  

• Other mechanisms of resistance include 

  • Use of an alternative pathway to create the survival signals 

  • Use a different mechanism to activate downstream events 

  • Amplify the ABL to overcome the inhibition  

2nd generation drugs

• Bind more tightly to the binding site  

• Can't cope with the T315I mutation —> both 1^st and 2^nd generation are not successful at all  

• All of them successfully bind to wild-type ABL 

• Examples: Dasatinib, nilotinib 

3rd generation

• SGX393 a 3^rd Gen BRC-ABL inhibitor  

• We can see from Western blots that this is the only drug which can inhibit mutant T315I 

• Since this is the only drug which works against this specific mutation, it’s essential to know which kind of mutation is causing the patients cancer.  

EGFR as target

• Epidermal Growth Factor Receptor  

• Important signal for tissue repair and wound healing  

• Controlled by ligand binding to the receptor, causing a dimerisation which activates the Ras signalling pathway (which goes onto activate Raf and Erg) 

• Unlike BCR-ABL, EGFR mutation is mainly driven by point mutations  

• Illicits lung cancer  

• Cetuximab prevents the ligand from binding to EGFR, however, this will not work in cancer types where the mutation is in the EGFR causing it to be constantly dimerised  

• Useful in cancers which are caused by amplification of ligand (EGF) or overexpression of the receptor 

• Gefitinib is another drug which binds to the centre of EGFR to prevent the signalling —> successful in cancers which are caused by mutations in EGFR which result in constant dimerisation 

 

Monoclonal antibodies as targeted therapy

• Can be used in some cases E.g: EGFR 

• Need to humanise the antibodies so they’re more stable

 

• However, again, when we tray and treat the mutation the cancer will try to develop other mechanisms to overcome this 

• T790M mutation accounts for 55% of mutations resulting in the re-establishment of this pathway.  

• Other mechanisms to re-establish this pathway 

• BRAF: Overexpression of Raf (downstream) 

• MAPK1: downstream of the Ras 

• PI3K: downstream of EGFR 

• MET: upstream of the Ras pathway  

• HER2: mechanism to activate Ras signalling pathway (upstream) 

EGFR mutations

• Big protein, mutations frequently occurring in exons 18,19, 20,21  

• examining where the mutation lies is important for determination of treatment —> sequencing of EGFR necessary for this 

• Below the diagram is the index of mutations associated with drug sensitivity so can give anti-EGFR treatment  

  • L858R mutation In exon 21 is an activation mutation  

• Above the diagram is the index of drug resistance mutations, which usually happens after several cycles of treatment.  

  • T790M mutation is a drug resistance mutation  

How can we treat patients with resistance induced by first gen treatments?

• In EGFR mutations causing cancer L858R and E746-A750 mutations are oncogene activation mutations while T790M is a resistance mutations  

• First-generation treatment causes this mutation to arise, second generatuion drugs such as Afatinib and Neratinib are also ineffective against this type of cancer.  

• Typically second generation EGFR TKIs form irreversible covalent binds with the ATP binding site of EGFR as well as other members of the HER family of receptors 

• Third-generation drugs on the other hand, such as AZD99291, HM61713 and Co-1686 will be effective in approximately half of cases.  

Vermurafenib

• Small inhibitor targeting BRAF V600E mutation  

• Can no longer activate the MEK/ERK pathway 

• However, the cancer cells can reestablish the ERK surviving signals by: 

  •  mutations in MEK1 and MEK2 

  • Upregulation of genes that can activate MEK 

  • Overexpress another isoform of BRAF 

  • Increase the expression of another RAF called CRAF 

  • Introduce mutations in RAF genes/other upstream events 

• Once we understand these alternative mechanisms we can come up with another drug to treat them  

• Unlikely that we can treat the cancer completely but can keep the cancer at a low level, so is less of a burden to the patient —> cancer now considered a chronic disease 

Immunotherapy

• 2 major checkpoints in our body 

  • CTLA-4 in the lymph node 

  • PD-1 in the tumour 

• These two signals act as surviving signals for cells and confer protection against the immune system 

• This interaction between PD-1 on the T cells and PDL-1 on the tumour cells protects the tumour against the immune system 

• However, we can create anti PD-1 antibodies to block this signal and selectively kill the tumour cells. 

• Anti-PD01 treatment shown better efficacy than the chemotherapeutics.  

VEGFR

• Tumour outgrowths creates a hypoxic environment which triggers the expression of VEGF (vascular endothelial growth factor) which will stimulate the creation of new blood vessels to supply nutrients and oxygen 

• VEGF antibodies removes the nutrient supply to the new cancer cells 

Precision medicine

• Treating the patient based off the driving mutation  

• When we first diagnose the patient with cancer must decide if their cancer is squamous or non-squamous 

• In most cases squamous wont have a driving mutation and so will have to use traditional cytotoxic or chemotherapeutics 

• If the patient has a non-squamous cancer, such as EGFR mutation, can give erlotinib or afatinib —> if unsuccessful can use chemotherapy  

• If the patient has ALK translocation we give them crizotinib and if that doesn’t work certinib  

• If the patient is EGFR and ALK negative give them platinum based chemotherapeutics.  

Potential algorithm for incorporating chemotherapies, immunotherapy and targeted therapies