Exploiting SSBR to treat cancer and inflammation

Question 1

How can DNA damage be targeted to treat cancer? (2)

Answer 1

• Induce more and more DNA damage in cancer cells to trigger cell death
• Challenge is how to target this to only cancerous cells and avoid healthy cells

Question 2

How do topoisomerase inhibitor chemotherapies work?

Answer 2

Trap the topoisomerase at the break site to prevent repair, therefore inducing large amounts of DNA damage

Question 3

What is synthetic lethality in terms of cancer treatment? (2)

Answer 3

• Exploitation of the genetic defect that the cancer cell is relying on for survival by combining the defect in an affected pathway with a pharmacologically induced defect in a compensatory pathway
• Will preferentially affect cancer cells as healthy cells won’t have the cancerous defect so will be able to compensate

Question 4

Which chemotherapies inhibit Top1? (2)

Answer 4

• Irinotecan
• Topotecan

Question 5

Which chemotherapies inhibit Top2? (2)

Answer 5

• Doxorubicin
• Etoposide

Question 6

How is induction of DNA damage targeted to cancerous cells? (2)

Answer 6

• Traditionally the only way was to rely on the fact that cancer cells replicate more rapidly than somatic cells
• Now methods exploit synthetic lethality to induce cell death which requires understanding of protein/pathway interactions

Question 7

What are DNA damage response (DDR) cancer signatures? (2)

Answer 7

• Certain cancers commonly have defects in certain DDR genes e.g. ovarian serous carcinoma commonly has mutated p53
• Shows which targets may be available for treating each cancer type

Question 8

What is the DDR cancer signature in breast cancer?

Answer 8

Defects in homologous recombination proteins

Question 9

What happens to a single strand break during DNA replication?

Answer 9

SSBs can become DSBs at the replication fork which require homologous recombination for repair

Question 10

What is homologous recombination? (2)

Answer 10

• Repair pathway for DSBs during S/G2 phase of the cell cycle
• Involves resection, strand invasion of a homologous template sequence and synthesis over the break site

Question 11

Which proteins are the main markers of homologous recombination (HR)? (2)

Answer 11

• RAD51
• BRCA1 and 2

Question 12

How could you treat cancer by exploiting the concept of synthetic lethality? (4)

Answer 12

• Persistent SSBs are converted to DSBs therefore if you inhibit SSBR all SSBs will convert to DSBs and be repaired by HR
• PARP would make a good target as it is upstream and essential in SSBR
• Knockdown isn’t easy for a therapy so inhibit pharmacologically
• HR deficient cancer cells (e.g. BRCA2 deficient breast cancer) can’t repair DSBs therefore inhibiting SSBR in these cells will lead to unrepaired damage and cell death but healthy cells will be unaffected as they can still do HR

Question 13

How can the synthetic lethality hypothesis be tested? (3)

Answer 13

• Inhibit PARP and observe increased RAD51 loci (marker for HR), suggesting increased HR in the absence of SSBR
• Inhibit PARP and observe increased γH2AX loci (marker of DSBs)
• Clonogenic survival assay shows that BRCA2 deficient cells are highly sensitive to PARP inhibitors and don’t survive

Question 14

What is a clonogenic survival assay?

Answer 14

An in vitro method to determine cells’ survival/proliferation potential by forming colonies in response to cancer drugs

Question 15

What type of cells are sensitive to PARP inhibitors? (2)

Answer 15

• BRCA2 -/- sensitive
• BRCA2 +/- INsensitive which is critical for targeting only the BRCA2 null tumour cells and no the BRCA2 +/- somatic healthy cells

Question 16

What is an example of a PARP inhibitor?

Answer 16

Olaparib

Question 17

What are the benefits of olaparib? (3)

Answer 17

• Highly specific
• No need for any other DNA damaging agents
• PARP knockout mice are healthy

Question 18

How do cancer cells develop resistance to therapy? (3)

Answer 18

• Switching off the target (e.g. Topo poisons, PARP inhibitors)
• Upregulating the primary repair mechanism (e.g. finding a way to still do SSBR without PARP)
• Upregulating a parallel repair mechanism (e.g. NHEJ instead of HR)

Question 19

What is cGAS? (2)

Answer 19

• A cytosolic sensor of dsDNA which is meant to sense pathogenic dsDNA
• Binds to dsDNA (self or pathogenic) and sets off a signalling cascade involving STING and ISGs, resulting in inflammation

Question 20

What is STING?

Answer 20

Stimulator of interferon genes

Question 21

What are ISGs?

Answer 21

Interferon stimulated genes

Question 22

How does self DNA end up in the cytoplasm?

Answer 22

DNA damage (endogenous/induced) can cause chromosome fragility leading to formation of micronuclei during mitosis

Question 23

What is AGS? (5)

Answer 23

• Aicardi-Goutieres syndrome
• Caused by build up of ribose contamination in DNA due to RNase H2 defect
• Mainly affects the brain, immune system and skin
• Results in encephalitis (inflammation of brain lining) and chilblains caused by inflammation of small blood vessels
• Mimic of congenital infection so often misdiagnosed

Question 24

How does RNase H2 defect cause autoinflammation? (3)

Answer 24

• Build up of ribose in the DNA causes fragile lagging chromosomes during mitosis, resulting in micronuclei
• Therefore RNase H2 negative cells have increased numbers of micronuclei
• Immunofluorescence shows a positive correlation between cGAS activation and γH2AX expression in micronuclei, resulting in inflammation via the cGAS pathway

Question 25

What is the function of RNase H2?

Answer 25

Removal of ribose contamination in DNA

Question 26

How does induced DNA damage cause autoinflammation? (3)

Answer 26

- Induce DNA damage with ionising radiation - Identified cells with γH2AX foci and split these into with or without micronuclei - Did single cell RNA sequencing and found upregulation of ISGs in cells with micronuclei compared to those without, resulting in inflammation via the cGAS pathway

Question 27

How do micronuclei cause autoinflammation? (3)

Answer 27

- DNA damage induces micronuclei - Micronuclei induce cGAS activation, STING activation, ISG activation and inflammation - Clear link between DNA damage and autoinflammation

Question 28

What are the clinical implications of the cGAS/STING pathway and autoinflammation? (2)

Answer 28

- Neurological disorders e.g. AGS: leakage of nuclear and mitochondrial DNA can activate cGAS-STING causing autoinflammation in the nervous system - Cancer: micronuclei frequently form in cancer cells so cGAS is often activated by this route during neoplastic transformation, leading to cGAS and STING-dependent tumour-suppressive immune responses

Question 29

How could cGAS-STING be used to treat cancer? (2)

Answer 29

- There may be selection pressure during cancer evolution to inactivate cGAS-STING signalling - As cGAS-STING is frequently inactivated in tumours, this pathway could be a target for reactivation with immunotherapy

Question 30

How is genome instability linked to innate immune responses?

Answer 30

cGAS senses ruptured micronuclei and activates a signalling cascade resulting in autoinflammation

Question 31

How can synthetic lethality be used with RNase H2? (2)

Answer 31

- RNase H2 defect leads to ribose remaining in the DNA which is then cleaved by Top1 - This cleavage product is a PARP substrate so this is a target for synthetic lethality using PARP inhibitors in RNase H2 deficient cancers