Cancer Flashcards
PI3K/Akt/mTOR Pathway
PI3K catalyses the conversion of PIP2 into PIP3.
PIP3 recruits Akt to the membrane, and PDK1 and mTORC2 then activate it.
Activated Akt promotes cell growth and survival via mTORC1.
In cancer, mutations in the catalytic subunit cause PI3K to be hyperactive, enhancing the activation of mTORC1.
This can cause can cause as well as resistance to cancer treatment, such as the resistance to Herceptin for HER2-positive breast cancer.
The tumour is more likely to metastasise. Enhanced activation of the PI3K pathway is seen in 70% of patients with brain metastases as a result of breast cancer.
PI3K/Akt/mTOR Pathway inhibitors
PI3K inhibitors include:
- Idelalisib – approved for blood cancers including chronic lymphocytic leukaemia (CLL). It selectively inhibits the delta isoform of PI3K.
- Copanlisib – approved for the treatment of relapsed or refractory follicular lymphoma.
- Alpelisib – approved for use in combination with fulvestrant for the treatment of hormone receptor-positive, HER2-negative advanced breast cancer with PIK3CA mutations.
- Pictilisib – inhibitor in phase II clinical trial
mTORC1 inhibitors include:
- Everolimus – approved for the treatment of advanced RCC and pancreatic neuroendocrine tumors.
Temsirolimus – approved for the treatment of advanced RCC.
Dual PI3K/mTOR inhibitors include the investigational drugs:
- Bupalisib – in clinical trials for various cancer types including breast and endometrial cancers.
- Dactolisib – has shown activity in preclinical studies and early-phase clinical trials for several cancers.
- Paxalisib - brain penetrant - combination theraphy with Trastuzumab (Herceptin) reduces brain metastases in preclinical studies and is currently in pase II clinical trials.
Capivasertib is an Akt inhibitor approved last year as a breast cancer treatment.
KRAS Pathway
KRAS mutations are the most common oncogenic drivers in human cancers.
The KRAS pathway is implicated in:
- ∼ 90% of pancreatic ductal adenocarcinomas (PDAC)
- 43% of colorectal cancers (CRC)
- 25–30% of lung adenocarcinoma
- 30–35% of non-small cell lung cancers (NSCLC)
The KRAS protein activates multiple signalling pathways, including ERK signalling and the PI3K/Akt/mTOR signalling pathway.
These pathways mediate cell proliferation and survival downstream of growth factor receptors and tyrosine kinase receptors.
The KRAS G12C mutation, a substitution of glycine by cysteine, results in constitutive activity, which drives cancer.
The incidence of KRAS mutations is 25-35% in smokers and 5% in non-smokers.
KRAS mutants were previously considered ‘undruggable’ as there is no binding pocket on this protein, but it can now be targeted by small molecular inhibitors which covalently bind to the cysteine residue.
KRAS can also drive “immune escape” in cancer cells by:
- Increasing the expression of PD-L1. PD-L1 binds to PD-1, a protein on T cells. This prevents T cells from killing the cells that have PD-L1, helping cancer cells hide from the immune system.
- Decreasing MHC expression
- Producing immunosuppressive factors like IL-10 to reduce immune cells function
- Suppressing the expression of T cells and myeloid-derived suppressor cells (MDSCs).
Cancer cells can therefore escape the immune system.
KRAS inhibitors
KRAS G12C inhibitors covalently bind to the cysteine residue at position 12 of mutant KRAS to block KRAS activity and downstream signalling.
Inhibitors can be used to treat cancers where this mutation is present.
Sotorasib was licensed in May 2021 for the treatment of NSCLC.
Adagrasib is a potent, orally available inhibitor, approved in December 2022 by the FDA for NSCLC.
These covalent inhibitors irreversibly lock KRAS G12C in the inactive GDP-bound state.
Proteolysis-targeting chimeras (PROTACs) could also be used against the mutant KRAS.
PROTACs are bivalent molecules that link a small molecular inhibitor to a degrader that binds a ubiquitin E3 ligase, hijacking the ubiquitin-proteosome system.
The aim is to enhance ubiquitination and proteasomal degradation of the protein, as well as pharmacological inhibition.
Adagrasib was also found to act as PROTAC, marking the KRAS protein for degradation.
Despite the encouraging initial results with KRAS G12C inhibitors, resistance mechanisms have emerged. These mechanisms can include secondary KRAS mutations (e.g., Y96D) that affect drug binding, as well as the activation of bypass signalling pathways involving other RAS isoforms and RTKs.
Cyclin-dependent kinases (CDKs)
The PI3K/Akt/mTOR and ERK pathways downstream of KRAS lead to the activation of cyclin-dependent kinases (CDKs). These control the cell cycle and hence cell proliferation, so dysregulation causes uncontrolled growth and division.
CDK 4 and 6 are involved in promoting entry of the cell into the cell cycle.
The RB protein, part of the retinoblastoma family of proteins, actively suppressed cell division in the absence of appropriate signalling—it is a tumour suppressor.
RB is sequentially phosphorylated by CDK4/6–cyclin-D and CDK2–cyclin-E complexes.
RB holds the E2F transcription factor in place. Its phosphorylation allows the release of the transcription factor and initiation of DNA transcription, allowing cells to progress through G1 into the S phase.
There is a loss of control of the G1 phase in cancer. This can be due to CDK 4/6 or cyclin overexpression or a loss of RB activity.
The expression of D-type cyclins is increased by the Ras/Raf/MAPK pathway as this pathway targets the c-Myc transcription factor, which regulates expression of cyclin D.
Cyclin D1 is a key regulator of cell cycle progression. It is exported from the nucleus by glycogen synthase kinase 3-β (GSKβ) to prevent excess cyclin D1.
GSKβ is inhibited by the PI3K/AKT pathway, so this pathway prevents cyclin D1 export out of the nucleus.
Therefore, the Ras/Raf/MAPK pathway increases expression of cyclin D1 and the PI3K/AKT pathway keeps it in the nucleus. This results in excess cyclin D1 in the nucleus and therefore excess cell proliferation.
Overactivity of these pathways, or of KRAS upstream of both, can result in increased cell proliferation and therefore cancer.
CDK inhibitors
Ribociclib and Palbociclib are inhibitors of CDKs 4 and 6, used for the treatment of hormone receptor (HR) positive and HER2 negative tumours that have metastasized.
They sustain the active RB protein. Active RB suppresses E2F transcription factors and prevents transcription of genes required for cell cycle progression.
This leads to G1 cell cycle arrest and so reduces cancer cell proliferation.
EGFR-targeted therapy
Cetuximab (Erbitux) is an antagonist at the EGF receptor, limiting EGF receptor-dependent proliferation of tumour cells.
Cetuximab binds to the extracellular domain of EGFR with high affinity at the ligand binding site, which prevents endogenous agonists from binding.
EGFR activation initiates downstream signalling cascades, including the Ras/Raf/MAPK pathway and PI3K/Akt/mTOR pathway, which promote cell proliferation, survival and migration. By blocking ligand binding, cetuximab inhibits EGFR activation and downstream signalling, leading to reduced cell proliferation and survival.
Cetuximab is also capable of triggering antibody-dependent cellular cytotoxicity (ADCC), a process in which immune cells, such as NK cells, recognise and kill target cells bound to the antibody.
Cetuximab-bound cancer cells are recognised by immune cells expressing Fc receptors, leading to the destruction of the cancer cells.
It is licensed for head, neck, colon and lung cancers.
Panitumumab binds to HER1/EGFR and prevents its activation. Interestingly, it doesn’t lead to immune-mediated cytotoxicity due to the difference in the Ig antibody backbone used.
HER2 receptor signalling
HER2 receptors have no known ligand, so it is naturally found in its active conformation, ready to dimerise with another HER/ErbB receptor, particularly HER3.
Dysregulation of HER2 signalling is implicated in the development and progression of various cancers, particularly breast cancer and gastric cancer.
Activation of HER2 receptors leads to the activation of downstream signalling pathways, including Ras/Raf/MAPK and PI3K/Akt/mTOR pathways. These pathways regulate cell proliferation, survival and metabolism, contributing to tumour growth and progression.
When HER2 depression is high, dimerisation is more likely to occur.
HER2-targeted therapies are designed to inhibit HER2 signalling and suppress tumour growth by targeting overexpressed or dysregulated HER2 receptors in cancer cells.
HER2-targeted therapies
Trastuzumab (Herceptin) is the first HER2-targeted therapy approved by the FDA for treatment of HER2-positive breast cancer.
It is a humanised monoclonal antibody against the extracellular cysteine-rich subdomain IV of HER2, preventing HER2 receptors from forming homodimers and activating signalling.
It can also stimulate immune-mediated mechanisms to target and destroy cancer cells indirectly. Trastuzumab leads to endocytosis and degradation of HER2 receptors by NK cells through ADCC.
Pertuzumab is directed against the extracellular dimerization domain (subdomain II) of HER2, preventing the receptor from forming heterodimers with HER3 or other receptors. It also causes degradation by NK cells.
Bevacizumab
VEGF is a growth factor involved in angiogenesis.
Angiogenesis is required for tumour growth and metastasis, as blood vessels supply oxygenated blood and nutrients to the rapidly dividing tumour cells.
Bevacizumab binds to VEGF and prevents it from activating its receptor on endothelial cell surfaces, thereby inhibiting downstream signalling and angiogenesis.
Nanobodies
Nanobodies are much smaller than conventional antibodies, typically ranging in size from 12 to 15 kDa while antibodies are ~150 kDa.
Their compact size allows them to access and bind to epitopes that may be inaccessible to larger antibodies, such as grooves on protein surfaces. This confers high specificity and affinity.
Conventional antibodies that are made in humans and other animals contain both heavy and light chains.
Nanobodies are derived from single-domain or heavy chain-only antibodies. These are made by cartilaginous fish like sharks as well as members of the Camelot family like alpacas and llamas.
Nanobodies have a highly stabilised structure.
We can string nanobodies together, combining them in various formats. This is known as multimerization.
Dual specificity therapeutics can be created using nanobodies against different targets.
We can also fuse nanobodies to a protein like serum albumin to increase their half-life by preventing degradation. This is a proprietary domain.
BI 836 880 (Ablynx pharma) is an inhibitor of tumour angiogenesis targeting both VEGF and angiopoietin II messengers. It is also linked to a proprietary domain.
There are no approved nanobodies yet, but clinical trials are still undergoing.
Using the immune system
The immune system is under-active in cancer, so there is an interest in using antibodies to activate the immune system and direct cells to the tumour site.
Natural killer cells are part of tumour surveillance. They look for and kill tumour cells, and also clear old cells.
T-killer cells kill damaged cells, like cancerous or infected cells. They release cytotoxins like protease enzymes and lead to cell apoptosis.
Many tumour cells “hide” from the immune system by upregulating immune checkpoint molecules such as PD-L1/PD-1 and CTLA-4 - e.g. KRAS mutations upregulate PD-L1.
These checkpoint molecules interact with their corresponding ligands on T cells (e.g. PD-1), which results in T cell exhaustion and suppression of the immune response.
This mechanism is important in normal cells to prevent immune-mediated cytotoxicity, but cancer cells use this interaction to escape immune attack.
Some biologics act as “immune checkpoint” inhibitors.
Antibodies such as Pembrolizumab, Nivolumab and Spartalizumab can be made to bind to the PD-1 receptor on T cells, preventing PD-L1 binding, so T cells can mount an immune response against cancer cells.
Pembrolizumab is used in triple-negative breast cancer tumours. These are harder to treat and often associated with poorer outcomes, resulting in an aggressive cancer phenotype. The cancer contains none of the common receptors in breast cancers - estrogen receptors (ER), progesterone receptors (PR), or human epidermal growth factor receptors (HER2) (i.e triple negative)
Atezolizumab and Durvalumab have a similar mechanism of action, but they bind to PD-L1 on cancer cells instead.
CDX-1140 is an agonist antibody at CD40, a receptor expressed on antigen presenting cells (APCs). This activates T cells and can drive T cell dependent tumour regression.
TRAIL Receptor Agonists (TRAs)
Antibodies have been trialled as agonists for the death receptor (DR) for tumour apoptosis.
TRAIL Receptor Agonists (TRAs) activate DRs to stimulate apoptosis, which is impaired in cancer.
Activation of death receptors (DR) on tumour cells initiates pro-apoptotic signalling, leading to cell death. Antibodies cross-linking 2 DRs mimic activation by the ligand.
Conatumumab is a DR5-directed antibody.
Recombinant TRAIL peptides are being tested - recombinant form of the endogenous ligand used to activate the receptor.
Bifunctional antibodies
An example of bifunctional antibodies is BiTE: Bispecific T-cell Engager.
These bind to CD3 on T cells as well as a cancer-specific protein to direct T cells to the cancer cells.
Vepsitamab is a bifunctional antibody that binds to MUC17 on tumour cells and CD3 on T cells.
- Mucins are proteins making up the mucus in the GIT.
- Mucin17 (MUC17), a membrane protein in epithelial cells, is overexpressed in 50% of gastric cancers.
Blinatumomab is used clinically for the treatment of acute lymphoblastic leukemia (ALL). It binds to CD3 on T cells and CD19 on B cells, allowing the targeting of T cells to malignant B cells.
Trifunctional Antibodies - Catumaxomab
Catumaxomab is a trifunctional antibody used in epithelial cell adhesion molecule (EpCAM) positive cancers.
EpCAM is expressed on malignant epithelial cells in cancers such as ovarian, colorectal and gastric cancers.
As well as binding to EpCAM, it also binds to CD3 antigen on T-cells and to Fcγ receptors (FcγRs) on NK cells or macrophages, bringing two immune cells in close proximity to the tumour.
These effector cells then induce tumour cell killing through various mechanisms like antibody-dependent cellular cytotoxicity and phagocytosis.
No longer licensed for use as the drug company went bankrupt, but other companies are trying to resurrect trifunctional antibodies.
Antibody-Drug Conjugates (ADCs)
Antibody-drug conjugates are a type of targeted cancer therapy that combines the specificity of monoclonal antibodies with the cytotoxic potency of chemotherapy drugs.
A cytotoxic drug is linked to an antibody.
Cytotoxic drugs normally attack all rapidly-dividing cells, which causes side effects - e.g. GI effects due to rapidly dividing cells in the gut. This is decreased with the use of ADCs.
Ado-trastuzumab emtansine (T-DM1)
Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate used for breast cancer.
Trastuzumab (Herceptin) is a monoclonal antibody against HER2 receptors. This is linked to emtansine (DM1), a cytotoxic drug that binds to tubulin and prevents microtubule formation, a process involved in cell proliferation. This leads to mitotic arrest and apoptosis.
The antibody targets the cytotoxic drug to HER2 positive cells, reducing off-target side effects.
The T-DM1 complex internalises into the cancer cell and is transported to lysosomes. The linker degrades and releases DM1, which allows cytotoxic effects to occur once in the cancer cell.
Trastuzumab also further contributes to the therapeutic effect as it inhibits HER2 signalling and mediates antibody-dependent cellular cytotoxicity.
Inotuzumab ozogamicin
Inotuzumab ozogamicin is an antibody-drug conjugate used in the treatment of acute lymphoblastic leukaemia (ALL).
Inotuzumab is a monoclonal antibody that binds CD22 on the surface of B cells.
This is linked to ozogamicin, a type of calicheamicin.
Once Inotuzumab binds CD22, the antibody-drug complex gets internalised through receptor-mediated endocytosis.
The drug is released as the linker connecting it to the antibody is cleaved and the antibody gets degraded.
Ozogamicin is a potent DNA-damaging agent derived from calicheamicin. It binds to DNA in the nucleus, causing double-stranded breaks, inhibiting cell proliferation and ultimately leading to cell death.
