Lecture 10 - Genetic Mutations and Signalling Pathways

How do signalling pathways convey information?

• Protein-protein interactions

• Enzymatic activity

• Protein/amino acid modifications

• Up- or down-regulation

• Temporal control

• Spatial control

• Pathway abundance

What is intercellular signalling?

• Communication between different cells to coordinate responses

• Cells send and receive signals (messenger molecules)

What is autocrine signalling?

A cell signals itself by producing a signal that binds to its own receptors

What is paracrine signalling?

Signals affect nearby cells, like growth factors

What is endocrine signalling?

Signals (hormones) travel through the bloodstream to distant cells

What is juxtacrine (contact-dependent) signalling?

It requires direct cell-to-cell contact

What is intracellular signalling?

Communication inside a cell, often triggered by an intercellular signal binding to a receptor and the signal is transduced into a cellular response

What are receptors in intracellular signalling?

Receptors detect signals and can be located on the cell surface or inside the cell

What are second messengers?

Molecules like cAMP, Ca²⁺, and IP₃ that transmit the signal internally

What are signalling cascades?

Sequences of proteins (often kinases) that amplify and transmit the signal

What are transcription factors/effectors?

Final proteins that change gene expression, metabolism, or cell behaviour

What are mutations?

Permanent alterations in the DNA sequence that are a source of genetic diversity but can also lead to disease

Give an example of a mutation

The GAG → GTG mutation in the β-globin gene causes sickle cell anemia

What are some methods for detecting mutations?

PCR, Sanger sequencing, NGS, FISH, or immunochemistry

What can mutations do to proteins?

Mutations can alter protein folding or stability, modify enzyme activity or binding affinity, or disrupt signalling cascade dynamics

What is a missense mutation?

A single nucleotide change that alters one amino acid in the protein

What happens when a missense mutation occurs?

The protein is made, but its function may be altered (gain-of-function or loss-of-function)

Give an example of a missense mutation

KRAS G12D → Glycine (G) at position 12 is replaced by Aspartic acid (D), often activating KRAS and leading to uncontrolled cell growth (oncogenic)

What is a nonsense mutation?

A single nucleotide change that converts a codon into a stop codon

What happens when a nonsense mutation occurs?

The protein is truncated and usually non-functional

Give an example of a nonsense mutation

TP53 R213X → Arginine (R) at position 213 becomes a stop codon (X), producing a shortened p53 protein, often unable to suppress tumours

What is a frameshift mutation?

Insertion or deletion of nucleotides not in multiples of 3, shifting the reading frame

What happens as a result of a frameshift mutation?

It alters all downstream amino acids and usually creates a premature stop codon, producing a nonfunctional protein

Give an example of a frameshift mutation

BRCA1 insertion can cause a truncated BRCA1 protein, impairing DNA repair and increasing cancer risk

What is copy number variation?

Sections of the genome are duplicated or deleted, changing the number of copies of a gene

What are the effects of copy number variation?

• Amplification → over-expression of the gene

• Deletion → loss of gene function

Give an example of copy number variation.

HER2 amplification → extra copies cause overproduction of the HER2 receptor, leading to uncontrolled cell growth

What causes uncontrolled cell growth?

It occurs when mutations activate growth pathways or remove growth restraints

What happens when there is no cell growth or cell death?

It occurs when mutations block essential growth or survival functions

What is the RAS–MAPK signalling pathway involved in?

Growth and proliferation

What does the PI3K–AKT pathway regulate?

Survival and metabolism

What is the function of the Wnt/β-catenin pathway?

Cell fate control

What role does p53 play in cellular processes?

Genome integrity maintenance

How does the RAS-MAPK pathway begin?

A growth factor binds to a receptor tyrosine kinase (RTK) on the cell membraneWhat happens after a growth factor binds to the RTK?

What happens after a growth factor binds to the RTK?

Binding causes the RTK to dimerise and become phosphorylated on tyrosine residues, which activates the receptor

What does Grb2 do after the RTK is phosphorylated?

Grb2 binds to the phosphorylated RTK and recruits SOS, a guanine nucleotide exchange factor (GEF)

How does SOS connect the activated receptor to Ras?

SOS connects the activated receptor to Ras, a small GTPase

What is the difference between inactive and active Ras?

Inactive Ras is bound to GDP (Ras–GDP), and active Ras is bound to GTP (Ras–GTP)

How does SOS activate Ras?

SOS causes Ras to release GDP and bind GTP, switching Ras to its active form

What happens after Ras is activated?

Activated Ras triggers a phosphorylation cascade, starting with Raf (MAPKKK)

What is the next step after Raf is activated?

Raf phosphorylates MEK (MAPKK), which then phosphorylates ERK (MAPK)

What does activated ERK do?

Activated ERK moves into the nucleus and phosphorylates transcription factors (TFs)

What is the outcome of the RAS-MAPK signalling pathway under normal conditions?

Leads to controlled cell division

What type of protein is KRAS?

KRAS is a small GTPase that acts as a molecular switch

What is the inactive form of KRAS?

KRAS–GDP (KRAS bound to GDP) is inactive

What is the active form of KRAS?

KRAS–GTP (KRAS bound to GTP) is active

How is KRAS normally activated?

KRAS is activated when a growth factor binds to a receptor tyrosine kinase (RTK)

How does KRAS turn itself off?

After signalling, KRAS hydrolyses GTP to GDP, switching itself off

What is the G12D mutation in KRAS?

The G12D mutation replaces Glycine (G) at position 12 with Aspartic acid (D)

What effect does the G12D mutation have on KRAS?

The G12D mutation prevents KRAS from efficiently hydrolysing GTP and blocks interaction with GTPase-activating proteins (GAPs)

What is the result of the G12D mutation?

KRAS remains GTP-bound and active, leading to continuous activation of the RAS-MAPK pathway

What is the consequence of constitutively active KRAS?

Constitutively active KRAS continuously activates the RAS-MAPK pathway, making KRAS oncogenic and driving tumour formation

What are some common mutations in codon 12 of RAS?

G12C, G12D, G12V, G12R, G12S

What effect do mutations at codon 12 have on RAS?

They block GTP hydrolysis, causing constitutive activation of RAS

What are some common mutations in codon 13 of RAS?

G13D, G13C, G13R

What effect do mutations at codon 13 have on RAS?

They impair GAP-mediated inactivation, leading to constitutive activation

What happens with mutations at codon 61 of RAS?

They reduce intrinsic GTPase activity, prolonging the active GTP-bound state

What are some common mutations in codon 61 of RAS?

Q61H, Q61L, Q61R

Where is Glycine 12 located in KRAS?

Glycine 12 is located in the P-loop (phosphate binding loop), which is a flexible region crucial for coordinating the GTP molecule’s phosphate groups

What happens structurally when Glycine is replaced by Aspartate at position 12?

The single change from the small, non-polar glycine to the larger, negatively charged aspartate introduces a substantial steric and electrosteric clash

What is the functional consequence of the G12D mutation in KRAS?

The aspartate side chain physically interferes with the binding and catalytic action of GTPase Activating Proteins (GAPs)

Why are GAPs important for KRAS function?

GAPs are necessary to stimulate GTP hydrolysis, turning off the activation of KRAS

What happens when the GAP mechanism is blocked by the G12D mutation?

By blocking the turn-off mechanism, the KRAS protein becomes perpetually locked in its active, GTP-bound state

What are common KRAS activating mutations and their associated cancers?

• G12D, G12V, G12R

• Associated cancers: Pancreatic (PDAC), Colorectal (CRC), Lung (NSCLC)

• Frequency:

  • Pancreatic (~90%), CRC (~45%), Lung (~30%)

• Clinical significance: The most frequently mutated oncogene, driving aggressive tumour growth

What is the KRAS G12C mutation and its associated cancers?

• Mutation: G12C

• Associated cancers: Lung Adenocarcinoma, Colorectal

• Frequency:

  • Lung NSCLC (~13%), CRC (~3%)

• Clinical significance: Targetable by specific KRAS G12C inhibitors (e.g., Sotorasib, Adagrasib)

What are common NRAS activating mutations and their associated cancers?

• Mutations: Q61K, Q61R, Q61L

• Associated cancers: Melanoma, Acute Myeloid Leukemia (AML), Thyroid

• Frequency:

  • Melanoma (~15-20%), AML (~10%)

• Clinical significance: Activates pathway independent of EGFR; associated with poor prognosis in melanoma

What are common HRAS activating mutations and their associated cancers?

• Mutations: G12, G13, Q61

• Associated cancers: Head & Neck (HNSCC), Bladder, Thyroid

• Frequency:

  • HNSCC (~5%), Bladder (~5%)

• Clinical significance: Targetable by Farnesyltransferase inhibitors (e.g., Tipifarnib) in HNSCC

What is the BRAF V600E mutation and its associated cancers?

• Mutation: V600E

• Associated cancers: Melanoma, Thyroid (Papillary), Colorectal, Lung

• Frequency:

  • Melanoma (~50%), Thyroid (~45%), CRC (~10%)

• Clinical significance: Strong activator of MEK/ERK. Highly sensitive to BRAF/MEK inhibitors (e.g., Vemurafenib, Dabrafenib) in Melanoma/Lung

What are non-V600 BRAF mutations and their associated cancers?

• Mutation: Non-V600 (Class II/III)

• Associated cancers: Lung (NSCLC), Colorectal

• Frequency:

  • Lung (~2-4%)

• Clinical significance: Often ras-independent or require dimerization; less sensitive to classical BRAF inhibitors

What is the clinical impact of NF1 loss-of-function mutations?

• Mutations: Nonsense, Frameshift

• Associated cancers: Neurofibromas, Glioblastoma, Lung, Melanoma

• Frequency:

  • MPNST (>50%), Glioblastoma (~10-15%)

• Clinical significance: Loss of negative regulation (GAP activity) leads to sustained RAS activation

What is the clinical impact of PTPN11 activating mutations?

• Mutations: Exon 3 / Exon 13

• Associated cancers: JMML (Leukemia), Solid tumors (rare)

• Frequency:

  • JMML (~35%)

• Clinical significance: Encodes SHP2 phosphatase; promotes RAS signaling

What is the on target resistance mechanism for KRAS G12C inhibitors (Sotorasib/Adagrasib) in lung cancer (NSCLC)?

Acquired KRAS mutations (e.g., Y96D, G13D, A59) prevent the drug from binding to the G12C pocket

What is the off target resistance mechanism for KRAS G12C inhibitors (Sotorasib/Adagrasib) in lung cancer (NSCLC)?

MET Amplification → tumor amplifies the MET gene to bypass the blocked KRAS signaling

What is the phenotypic resistance mechanism for KRAS G12C inhibitors (Sotorasib/Adagrasib) in lung cancer (NSCLC)?

Phenotypic Squamous Transformation → lung adenocarcinoma cells transform into squamous cell carcinoma, which relies on different signaling drivers

What is the on target resistance mechanism for BRAF inhibitors (Vemurafenib/Dabrafenib) in melanoma?

BRAF splice variants (e.g., p61-BRAF) form dimers that are resistant to first-generation BRAF inhibitors

What is the off target resistance mechanism for BRAF inhibitors (Vemurafenib/Dabrafenib) in melanoma?

NRAS Mutations → new mutations in NRAS (e.g., Q61) reactivate the pathway from upstream

What is the phenotypic resistance mechanism for BRAF inhibitors (Vemurafenib/Dabrafenib) in melanoma?

MEK1/2 Mutations → mutations in MEK (e.g., C121S) keep the pathway active even when BRAF is blocked

What is the adaptive feedback resistance mechanism for BRAF inhibitors (Vemurafenib) in colorectal cancer (CRC)?

EGFR Reactivation → in colorectal cancer, blocking BRAF causes a rapid feedback loop that activates EGFR, rendering monotherapy ineffective

What happens when growth factors bind to receptor tyrosine kinases (RTKs) on the cell membrane?

The RTKs become activated and recruit PI3K

What does PI3K do in the PI3K-AKT-mTORC1 pathway?

PI3K phosphorylates PIP2 to form PIP3 in the plasma membrane

What role does PIP3 play in the PI3K-AKT-mTORC1 pathway?

PIP3 acts as a docking site for proteins with PH domains, including PDK1 and AKT

How does AKT get activated?

AKT is brought to the membrane for activation:

• PDK1 phosphorylates AKT at one site

• mTORC2 phosphorylates AKT at a second site

• Fully phosphorylated AKT is active

What is the role of PTEN in the PI3K-AKT-mTORC1 pathway?

PTEN converts PIP3 back to PIP2, removing the membrane docking signal for AKT and acting as a brake on the pathway

How does AKT regulate TSC1/TSC2 in the pathway?

Active AKT phosphorylates and inhibits TSC1/TSC2, releasing inhibition of Rheb

What is the role of Rheb in the PI3K-AKT-mTORC1 pathway?

Rheb-GTP activates mTORC

What does activated mTORC1 do?

mTORC1 phosphorylates:

• S6K → increases ribosome biogenesis

• 4E-BP1 → releases eIF4E to initiate translation

What are the overall outcomes of the PI3K-AKT-mTORC1 pathway?

• Increased protein synthesis

• Increased cell growth

• Increased cell proliferation

What does PTEN stand for?

Phosphatase and Tensin Homolog

What type of gene is PTEN?

Tumour suppressor

What is the function of PTEN?

Encodes a phosphatase enzyme that removes a phosphate from PIP3, converting it back to PIP2, negatively regulating the PI3K–AKT pathway

What happens when PTEN function is lost?

PIP3 accumulates unchecked, causing AKT to remain continuously active even without growth factor signals

What are the consequences of persistent AKT activation due to PTEN loss?

• Increased protein synthesis

• Enhanced cell survival (resisting apoptosis)

• Uncontrolled cell growth and proliferation

Why is PTEN loss considered oncogenic?

Because it leads to continuous AKT activation, promoting uncontrolled cell growth and survival

What does PI3K (Phosphoinositide 3-kinase) do?

PI3K is an enzyme that phosphorylates PIP2 to generate PIP3

What is the role of PIP3 in the cell?

PIP3 recruits and activates AKT, promoting cell growth, survival, and metabolism

Where does PI3K act in the cell signaling pathway?

PI3K acts downstream of receptor tyrosine kinases (RTKs) in growth signaling

What is the common activating (gain-of-function) mutation in PI3K?

The H1047R mutation in the catalytic subunit (p110α) of PI3K, where histidine (H) is replaced by arginine (R) at amino acid 1047

How does the H1047R mutation affect PI3K?

It enhances the kinase activity of PI3K, leading to constitutive activation

What are the consequences of constitutive PI3K activation?

• Enhanced cell growth and proliferation

• Increased survival and resistance to apoptosis

• Metabolic changes favoring tumor progression

How does the gain-of-function mutation in PI3K impact cellular controls?

It disrupts normal cellular controls on proliferation and survival