Apoptosis Lecture Notes

Apoptosis

Learning Outcomes

• Understand the importance of apoptosis in animal development and function.
• Explain the intrinsic and extrinsic pathways that trigger apoptosis.
• Know the three key classes of Bcl2 family proteins and their roles.
• Understand how apoptosis dysregulation can occur in cancer, using Bcl2 in follicular lymphoma as an example, and how it can be treated.

Types of Cell Death

• Not all cell deaths are the same.
• Apoptotic cells have an intact plasma membrane, but the chromatin is distorted and concentrated at the margin of the nucleus.
• Necrotic cells appear to have exploded.

Apoptosis (Programmed Cell Death)

• Apoptosis, or programmed cell death (PCD), is a normal process that eliminates unwanted cells.
• It is critical in embryonic development (e.g., removing skin between digits) and removing damaged cells.
• An intracellular proteolytic cascade is activated, leading to:
  • Cell shrinkage and condensation.
  • Dissolution of the nuclear envelope.
  • Condensation and fragmentation of chromatin.
  • Collapse of the cytoskeleton.
  • Engulfment and destruction of the cell by phagocytosis (e.g., by a macrophage).
• No leakage of cell contents occurs, avoiding an inflammatory reaction.

Cell Survival Depends on Extracellular Signals

• Cells require survival factors to avoid apoptosis.
• Neurotrophin trophic factors provide survival signals for neurons.
• Experiment:
  • Wild-type mice express NGF (nerve growth factor) and neurotrophin-3 (NT-3).
  • Knockout mice without NGF or its TrkA receptor lack nociceptive neurons.
  • Mice without NT-3 or its TrkC receptor lack proprioceptive neurons.

Apoptosis is Mediated by a Caspase Cascade

• An apoptotic signal triggers the assembly of an adaptor-protein complex, which recruits further monomers that dimerise, activating each other.
• Initiator caspases (8 and 9) cleave and activate executor caspases (3, 6, and 7), which coordinate the apoptosis program.
• Executor caspases cleave multiple targets, including:
  • Nuclear lamin (by caspase 6).
  • Phospholipid transfer proteins, resulting in the exposure of phosphatidylserine (“eat me” signal).
• Caspases have a cysteine at their active site and cleave at aspartic acid sites.
• They exist as inactive soluble monomers in the cytosol.

Apoptosis Acts via the Intrinsic or Extrinsic Pathways

• Two main activation pathways activate initiator caspases:
  • Extrinsic pathway: signaled from outside the cell by death receptors (e.g., TNF family) activated by Fas ligands.
  • Intrinsic (mitochondrial) pathway: signaled from mitochondria inside the cell.
• The extrinsic pathway may need to recruit the intrinsic pathway to overcome caspase inhibitors.

The Extrinsic Pathway

• Tumor necrosis factor (TNF) interacts with cells through TNF receptors (Fas death receptors), which are homotrimeric and bind trimeric Fas ligands.
• Binding clusters the receptors, exposing death domains that bind and cluster FADD (Fas-associated death domain).
• Clustered FADD recruits inactive caspase-8, which oligomerizes, forming a death-inducing signaling complex (DISC).
• Activated caspase-8 cleaves itself to form mature caspase-8 dimers, which activate downstream executioner caspases.

The Intrinsic or Mitochondrial Pathway

• Activated from within the cell (e.g., developmental signals, DNA damage).
• Depends on the release of cytochrome c from the mitochondrial intermembrane space into the cytosol.
• Cytochrome c binds to Apaf1 (apoptotic protease activating factor 1), causing it to bind deoxy-ATP and oligomerize into a heptamer.
• The heptamer recruits inactive caspase-9 monomers, forming an apoptosome.
• Caspase-9 is activated by dimerization within the apoptosome and activates downstream executioner caspases.

Bcl2 Proteins are Critical Regulators of Apoptosis

• The BH3 domain is shared by all Bcl2 family members and mediates direct interactions between pro- and anti-apoptotic members.
• Three structural and functional classes of mammalian Bcl2 family proteins regulate Mitochondrial Outer Membrane Permeabilisation (MOMP):
  1. Anti-apoptotic (e.g., Bcl2) inhibit apoptosis by preventing MOMP.
  2. Pro-apoptotic effectors (e.g., Bax, Bak) directly induce MOMP.
  3. BH3-only proteins promote apoptosis by regulating the other two classes (e.g., Bim, Puma, Noxa).

How pro- and anti-apoptotic Bcl2 family proteins act

• Anti-apoptotic proteins Bcl2 and BclxL prevent MOMP by binding to the BH3 domains of Bak and Bax.
• Pro-apoptotic effectors Bak and Bax trigger MOMP by aggregating into oligomers in the outer mitochondrial membrane, allowing cytochrome c to escape.

Summary of the extrinsic and intrinsic pathways

• Caspase-8 is the key link between the extrinsic and intrinsic pathways.
• If apoptosis fails, the necroptosis pathway can be activated, triggered by RIPK1 kinase.
• This leads to oligomer formation in the plasma membrane, destabilizing it and inducing cell swelling and rupture.

How survival factors inhibit apoptosis

• Survival factors can stimulate transcription of anti-apoptotic Bcl2 family proteins (e.g., Bcl2 or BclxL).
• They can activate RTKs and Akt/PKB, which phosphorylates and inactivates the pro-apoptotic BH3-only protein Bad.
• Unphosphorylated Bad promotes apoptosis by binding to and inhibiting anti-apoptotic Bcl2 proteins.
• Phosphorylated Bad dissociates, freeing Bcl2 to suppress apoptosis.
• Akt can also suppress apoptosis by phosphorylating and inactivating transcription regulatory proteins that stimulate the transcription of genes encoding proteins that promote apoptosis, such as the BH3-only protein Bim.

Summary of pathway integration regulating MOMP

• Integration of multiple signaling pathways regulate MOMP and apoptosis:
  1. Growth factor inhibition of apoptosis.
  2. Absence of growth/survival factor promotes apoptosis.
  3. DNA damage or UV stimulates apoptosis.

Apoptosis is triggered by a variety of pathways

• Apoptosis can be activated by direct signaling or activation of DNA damage pathways.
• It can also occur if critical survival factors are withdrawn.

Excess or insufficient apoptosis can cause disease

• Heart attack and stroke can lead to cell death by necrosis, followed by apoptosis in the affected area.
• Mutations in the Fas receptor or ligand prevent normal lymphocyte death, leading to autoimmunity and lymphomas.
• Decreased apoptosis rates contribute to many cancers.

Dysregulated apoptosis and cancer

• Resistance to apoptosis is a hallmark of cancer.
• Mechanisms of evasion include:
  1. Disrupted balance of pro- and anti-apoptotic proteins.
  2. Reduced caspase function.
  3. Impaired death receptor signaling.

Tumour cells are frequently resistant to Fas-induced apoptosis

• Mechanisms include:
  • Decreased receptor expression.
  • Expression of soluble Fas.
  • Increased expression of c-FLIP.
  • Decreased expression of caspase-8.
  • Increased expression of Bcl-2 and/or decreased Bax/Bad/Bak.
  • Defects in signaling and gene expression.

Dysregulated apoptosis and haematopoietic cancers

• A disrupted balance of pro- and anti-apoptotic proteins plays a role in leukemias and lymphomas.
• In chronic lymphocytic leukemia (CLL), malignant cells have an anti-apoptotic phenotype.
• The BCL2 gene was identified due to the t(14; 18) chromosome translocation, present in $>90\%$ of follicular B cell lymphomas, resulting in Bcl2 overexpression.
• Mcl1 is another commonly expressed pro-survival protein in hematological malignancies.

Therapy: Venetoclax inhibits Bcl2

• Drugs have been developed to block the anti-apoptotic proteins Bcl2 and BclXL by binding to the BH3-binding groove, stimulating the intrinsic pathway.
• Venetoclax binds to Bcl2 and is used in chronic lymphocytic leukemia.

Bcl2 and Mcl1 inhibitors for haematological cancers

• BAX/BAK are essential to drive MOMP and are required for the action of venetoclax.
• MCL1 inhibition could be effective for multiple myeloma (MM) since MCL1 is the predominant survival protein.
• Some subtypes of multiple myeloma are also highly susceptible to BCL2 inhibition.
• The degree of AML dependence on BCL2 or MCL1 varies.
• Overall, both BCL2 and MCL1 appear to play prominent prosurvival roles in most AML cases.

Cancer therapeutic approaches targeting apoptosis

• Examples:
  • Targeting death receptor ligands (DRL)
  • Targeting growth factor receptors
  • BH3 mimetics
  • Targeting ER stress
  • Inhibiting MDM2
  • Targeting IAPs
  • Caseinolytic protease P (ClpP)

Key points

• Apoptosis eliminates unwanted cells without triggering inflammation. Extracellular survival factors inhibit apoptosis, and DNA damage can trigger it.
• Apoptosis relies on a proteolytic cascade mediated by initiator (caspase 8 & 9) and executor caspases (3, 6 & 7), activated by extrinsic or intrinsic pathways.
• Extrinsic pathway: Fas ligands bind Fas death receptors, clustering FADD proteins and caspase-8 to form the DISC.
• Intrinsic pathway: Cytochrome c is released into the cytosol, binding Apaf1, which oligomerizes and recruits caspase-9 to form the apoptosome.
• Bcl2 family proteins regulate MOMP: anti-apoptotic (Bcl2, Bclxl) prevent MOMP; pro-apoptotic (Bax, Bak) induce MOMP; BH3-only proteins are regulators (e.g., Bim, Puma, Noxa).
• Cancer is often caused by disrupted balance, reduced caspase function, or impaired death receptor signaling, common in leukemias and lymphomas (e.g., Bcl2 translocation in follicular lymphoma).
• Venetoclax blocks Bcl2, treating chronic lymphocytic leukemia.