DNA damage

What are the sources of DNA damage and have cells adapted to this?

DNA damage threatens genomic DNA. It can come from endogenous places like replication, stress, ROS or exogenous places like UV, chemotherapies and environmental chemicals. It occurs frequently in every cell, every day but cells have adapted to dealing with it very rapidly.

What are the types of DNA damage?

There are several distinct DNA lesions: base mismatches, single-stranded breaks, adducts, intrastrand crosslinks, interstrand crosslinks and double-stranded breaks (most cytotoxic). Mismatches are repaired with mismatch repair (MMR), SSBs are repair with base excision repair (BER), adducts/intrastrand crosslinks are repaired with nucleotide excision repair, and the last two are repaired with homologous recombination or non-homologous end joining (and other pathways).

What is the DNA damage response?

DNA damage repair is collectively known as the DNA damage response (DDR). Initially, lesions are sensed by sensor proteins, which recruit mediator proteins upon finding damage. This amplifies the signal, enabling it to impact various downstream cellular processes like stopping the cell cycle, DNA repair, chromatin changes, gene expression and cell fate (apoptosis or senescence). This is done via effector proteins. Overall, this means that in healthy cells there is usually a low mutational burden.

Not all repair happens at the same time, depending on how the damage presents itself with the presence of homologous templates. For example DSBs activate different repair and signaling pathways depending on the cell cycle stages. If it occurs in G1, the kinase ATM activates the CHK2/p53 pathway which prevents progression into S phase. If it occurs at S/G2, the kinase ATR is central to DSB repair and CHK1/CDC25/WEE1 signalling which prevents progression. 100s of proteins are involved in coordinating DDR pathways in time (cell cycle) and space (chromatin/nuclear architecture).

What is homologous recombination and non-homologous end joining?

Homologous recombination occurs in S/G2 and is the most accurate as it uses homologous sequence as a template. This is because the sister chromatid is present at this checkpoint, since DNA duplication has already occurred. BRCA1-2, RAD51 (and others) are crucial. NHEJ can be used from G1-G2, it is fast but can be mutagenic since no homologous template is used. There is a fluidity between different lesions, in particular, between SSBs and DSBs - for example, if SSBs is not repaired and undergoes DNA replication, it results in a single-ended DSB that needs to be repaired by homologous repair. 

What processes is DNA damage essential?

Not all damage is bad. For instance, it can lead to the formation of gametes through meiosis. Namely, it requires recombination to take place - DNA damage and repair via homologous recombination promotes pairing of paternal and maternal chromosomes, required for segregation of homologous chromosomes. Another example is in the immune system via VDJ gene recombination that generates Ab diversity and specificity during B cell development. DBS induction and repair facilitate random arrangement of variable, diverse and joining genes that make up the antigen-recognising region of Abs. Also, class switch recombination is key for generating different classes of Ab, which is also induced by DSB and repair. Finally, it is important in neuroplasticity, in which neuronal activity induces DNA damage, and DNA damage and repair alters neuronal transmission (cycle). SSB repair by BER modulates synaptic transmission, and DNA repair modulates plasticity by altering synaptic connections and transmission. 

What diseases arise from DDR genes?

DDS is important for health. Many mutations in DDR genes are incompatible with life, whereas others cause hereditary syndromes and predispositions. For example, ATM kinase mutations can cause ataxia telangiectasia, FANC gene mutations lead to Fanconi anaemia, NSB1 mutations lead to Nijmegen breakage syndrome, WRN helicase mutations leads to Werner syndrome (premature ageing) etc. All of these have predisposition to cancer as a common factor.

Why is DNA damage good and bad in cancer?

DNA damage in cancer is a double-edged sword. It is one of the hallmarks; genome instability and mutation. On one hand, the increased mutational burden leads to loss of tumour suppressors and oncogene activation which drives tumorigenesis. On the other hand, too much genome instability in rapidly proliferating cancer cells leads to replication stress. Radio-/chemotherapies can tip the balance, leading to mutational overload and cell death - these are still quite untargeted as it affects normal proliferating cells, which is why they have severe side effects.

What problems are there with targeted therapies?

There is a need for targeted therapies with less side effects. This is challenging as there are several questions that need to be answered; what makes cancer cells different? How can the differences be targeted selectively? And how can patients be stratified in a way that they will respond to the identified treatments?

How are cancer mutations diverse?

The genomes of many cancers have been sequenced to identify differences to normal cells, with many alterations being identified. However, cancers are often highly heterogeneous. For example, in serous ovarian cancer, there is not one single defect that holds true for all patients. Some patients have germline mutations (predisposition), meaning it is present from the gamete and is found in all cells of the body and can be inherited by offspring. For somatic mutations, they are not hereditary.

What types of cancers can be targeted and by what process?

There are subsets of cancers with shared features that can be targeted. For example, serous ovarian cancers have a high % of patients being BRCA deficient, meaning they can be selectively targeted with PARP inhibitors such as olaparib. The selective targeting is facilitated by synthetic lethality.

What is synthetic lethality?

In cells, there are genes that do not exist in isolation. Inhibiting one gene is not harmful, but when both are targeted, it is synthetically lethal so the cell dies. However, since cancer mutations can inactivate certain genes, only 1 gene needs to be inactivated. For example, gene A is BRCA which gets inactivated in cancer, and gene B is PARP which gets inhibited pharmacologically. 

What does synthetic lethality mean for normal people and carriers?

SSBs convert to DSBs which need to be repaired with HR. This requires BRCA which is not found in the cancer cells, but is in normal cells. Germline BRCA1-2 mutations increase the risk for breast and ovarian cancer dramatically. For carriers, they have a recessive mutation meaning BRCA function is retained. In cancer, the mutational rate is high so this can lead to a loss of heterozygosity and therefore BRCA inactivation of the second chromosome. Normal and carriers are PARPi resistant, but cancer is PARPi sensitive, since PARP is required for SSB repair. If they are not repaired then, there is more conversion into DSBs etc.

What are the biomarker tests used for cancer?

 Patients are identified by biomarker tests like DNA sequencing. However, other genes besides BRCA may also lead to HR deficiency, and BRCA inactivation by epigenetic silencing (promoter hypermethylation) is not picked up.

Next generation sequencing-based in vitro diagnostic tests using DNA from formalin-fixed-paraffin embedded (FFPE) tumour tissue specimens assess the status of BRCA genes and determine the genomic instability score (GIS). This is based on loss of heterozygosity, telomeric allelic imbalance and large-scale transitions.

Functional assays are also being developed which use proteins to be recruited to genes to determine whether they are functional. For example, the RAD51 immunofluorescence test recruits RAD5 to the implicated gene - no binding means HR deficient, RAD51 binding is HR proficient. This indicates the predicted response to drugs. 

What are the characteristics required for a good biomarker test?

Biomarker tests need to be suitable for the clinic;

• Ease of sample extraction/prep (FFPE is feasible rather than live tissues).

• Rapid (simple and streamlined).

• Reliable.

Why is resistance a challenge and what is being done to overcome this?

Resistance mechanisms are also a major challenge. For example, partial reactivation of BRCA genes via reversion mutations restore parts of the open reading frames, promoter switching through chromosomal translocation (BRCA promoter hypermethylation), and inactivation of genes that cause synthetic viability (ie: genes that limit steps required for HR) + other underlying mechanisms. This makes them resistant to olaparib. 

This means that there is an effort to discover other synthetic lethal relationships to give options for treatments. One scheme is large-scale lethality screens using CRISPR/Cas9 screens. This, combined with studying underlying mechanisms is powerful in identifying these relationships. For example, ATM deficiency is common in many sporadic cancers. This can be synthetically lethal with inhibitors of several DNA repair enzymes such as PARP, TOP1, DNA-PKcs, ATR and POLQ. Also, microsatellite instability is common in uterine, colorectal and stomach cancers, and is caused by mutations in MLH1, and MSH2, 3, 6. It is synthetically lethal with Werner inhibitors. Further clinical testing, development of inhibitors and fundamental discovery work is still required to optimise patient stratification and benefit.