Note: Cancer TARGETases: DSB repair as a pharmacological target

Note: Cancer TARGETases: DSB repair as a pharmacological target

(doi: 10.1016/j.pharmthera.2016.02.007)

DNA is not just the plain sequences which contain ATCG but we have to consider how it form the secondary structure within the strand, how it folds with the histone --> these are related to replication as well as DNA repair process. Some studies show that heterochromatin (tight) and euchromatin (loose) has been repaired by different repair mechanisms.

DNA repair, esp. DSB is good targets since it can sensitize the cancer cell to the conventional treatment.

1. over-expression

2. synthetic lethality partner (which relied on DSB repair)

This paper wants to review the suggested "targets" for the enzymes (ending with "ases") in the DSB pw.

"TARGETase"

1. Kinases (adding the phosphate group)

2. Phosphatase (removing the phosphate group)

3. Nucleases (cutting the DNA strand at the phospho diester bond)

4. Helicase (unwinding the H-bond)

5. Core recombination (a group of protein which involving in  recombination -- finding the homology)

6. DNA polymerase (filling in the gap)

Defective in DNA repair;

1. accumulation of mutations

2. mutation of "driver" genes + "passenger" genes

3. tumor formation - progression - resistant

The synthetic sickness or lethal (SSL) strategy, whereby mutation in one DNA repair pathway makes cancer cells addictive to an alternate (repair) pathway.

Many of the DDR pathways nevertheless have several steps in common: a) sensing the DNA lesion and coordination with other cellular processes, b) accumulation of repair factors at the damage site, c) actual repair, and d) signal termination.

The authors mentioned that cancer cells are relied on HR for survival since they often experience replication stress (due to the fast replication) and HR is used to repair the lesion at replication forks. 

On the other hands, NHEJ is used in the cancers which resists to the treatment. Therefore, both pws are attractive to develop the inhibitors.

The author mentioned that kinase is well-study but nuclease and helicase are understudied.

Kinases;

Two related kinase families, phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinase-related protein kinases (PIKKs), play substantial roles in the DDR pathway.

ATM, ATR and DNA-PKs are all part of the kinases. 700 proteins have been involved by these proteins! These kinases have been shown to be responsible for modification of approximately 700 proteins in response to DNA damage

Both ATM and ATR or their downstream kinases could be used in several therapeutic backgrounds:

1) to disrupt checkpoint in HR-deficient tumor cells,

2) to sensitize chemo- or radio-resistant tumors, and

3) to override checkpoint in

p53-mutant tumors that have lost their protective ability

inhibition of kinases is well tolerated in normal cells,making them suitable candidates for both combinational and single-agent therapies.

Inhibitors of various kinases;

1. adenosine triphosphate (ATP)-competitive inhibitors recognizing active conformation

2. kinase inhibitors recognizing inactive conformation

3. allosteric site of kinase

4. covalently link (irreversible one) in the active site of kinase

ATM (PIKK family) can be activated by MRN complexes, replication stress, interstrand crosslinks, hypotonic stress, crosstalk with ATR kinase.

Play switching role between apoptosis (p53) and cell cycle arrest (chk1,2)

Therefore, ATM is tumor suppressor genes (in cancer, loss of function mutations and downregulation) - no point to target ATM but finding the redundancy of DDRs besides ATM mutation would more suitable (synthetic lethality partner of ATM).

On the other hand, ATM could be the synthetic lethality partners of the other gene (like PolB).

Therefore, ATM is tumor suppressor genes (in cancer, loss of function mutations and downregulation) - no point to target ATM but finding the redundancy of DDRs besides ATM mutation would more suitable (synthetic lethality partner of ATM).

On the other hand, ATM could be the synthetic lethality partners of the other gene (like PolB).

ATM and FA gene play compensatory roles in genome maintaining.

ATM could be up-regulated in cancer bc of oncogene-induced replication stress (like oncogene induced senescent) could trigger the DNA repair which is important to maintain the cancer feature. Therefore, inhibiting the ATM could prevent the DNA repair, initiate cell-cycle arrest or apoptosis.

Sound to me if the particular protein playing role as switching -  therefore, it can play role as tumor suppressor or oncogene.

None of the ATM inhibitors, so far, are not in the clinical trials - since it can sensitize the normal cell to radiation.

ATM and ATR -- important for responding the replication stress which will be occurred during the S-phase (cancer divide pretty fast therefore tend to undergo S-phase more often), however some cells divide fast too, like hair follicle).

ATR inhibition can be used as a single agent for treatment of cancers with particular genetic background and especially might find application in the near future in relation to cells undergoing replication stress.

Activated DNA-PKcs phosphorylates many components of the NHEJ and V(D)J pathway, but these modifications are dispensable for DSB repair. An exception is autophosphorylation, which promotes release of DNA-PKcs, regulates nucleolytic processing of DNA ends, and thus also regulates the initial step of HR.

Really depending on the context;

Lower of DNA-PK and Ku70 -- causing the proliferation defect as well as increased apoptosis in Hela. However, DNA-PKs are upregulated in many cancers, gastric, colorectal, and nasopharyngeal cancers, CLL and others!

It has been shown that ICL-treated cancer has been linked to overexpression of DNA-PKs --> therefore, it might be suitable for cisplatin-resistant cancer.

To date, small-molecule inhibitors targeting the ATP binding site have been the most successful (for DNA-PK).

Chk1 and Chk2 are both downstream substrate of ATR and ATM, respectively (being activated by phosphorylation).

Chk2 acts throughout the cell cycle in an ATM dependent manner, links cell cycle response mainly through p53, and responds to DSBs. Chk1 also directly regulates several components of HR, including bloom syndrome helicase (BLM) and RAD51 during replication fork stalling or other perturbed conditions.

Direct targets of Chk2 include p53 and BRCA1.

Are there any ML model that can predict the poor pharmacokinetics?

Despite progress in preclinical and clinical phases, there is still room for developing more selective inhibitors with improved bioavailability (what kind of factor that determine bioavailability then?).

although si/shRNA-mediated screens might reveal new targets in the near future. Apart from effectiveness in a p53-deficient background, Chk2 deficiency might be an advantage for using PARP inhibitors, as Chk2 phosphorylates BRCA1 and so will result in HR impairment.

Taken together, the current research indicates that the selection of a successful therapeutic strategy largely depends upon particular genetic backgrounds, thus pointing to future personalized medicine.

CDKs are effector kinases governing cell cycle progression and transcription regulation and which tightly regulate HR --> another good candidate to develop the inhibitor.

It is believed that CDKs guide the decision by which cells choose between NHEJ and HR.

The role of phosphatases in the DDR pathway represents an unexplored yet emerging area.

Targeting phosphatases might therefore have a therapeutic value but also pose substantial challenges because of their extensive and dynamic roles, which still remain to be outlined.

Likewise, several other phosphatases that have regulatory roles in cancer are constantly being explored and these might open up new avenues in the field of cancer research.

Nucleases comprise a class of enzymes capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acid molecules.

Play an essential role in various DNA repair pathways from processing the damaged nucleotide and extensive resection of the DNA ends to resolution of various DNA intermediates.

MRE11 exhibits 3′ exonuclease and endonuclease activities, both of which are activated by RAD50 and NBS1.

Nuclease activity of MRE11 has been shown to be essential for the removal of covalently attached SPO11 from the DSB ends during meiosis and possibly the removal of covalent TOP1 (topoisomerase 1)-DNA and TOP2-DNA intermediates, and that could account for the resistance of cancer cells to topoisomerase poisons.

MRE11 inhibitors characterized to date, however, are not so potent, have off-target effects, and must be optimized before they can be used in preclinical and clinical trials.

DNA replication helicase/nuclease 2 (DNA2) is a highly conserved multifunctional enzyme involved in various aspects of DNA metabolism including DNA replication and repair, and maintenance of telomeric and ribosomal DNA. (never knew this enzyme).

DNA2 is responsible for the DNA end resection involved in the HR pathway.

Because current knowledge about genetic interactions of DNA2 inhuman cells is limited, to identify a synthetic lethal network of DNA2 in human cells would be beneficial in treating cancers with a particular genetic makeup.

Since EXO1 expression levels are increased in several cancers and promote their survival, targeting its nuclease activity through small-molecule inhibitors provides a clear therapeutic target in combating cancers.

There currently are no potent small-molecule inhibitors targeting MUS81, and their development would have potential benefits in basic as well as clinical setups.

Uncontrolled GEN1 activity in somatic cells is potentially harmful, since untimely HJ resolution can result in gross chromosomal rearrangements, higher frequencies of crossovers associated with loss of heterozygosity, and cancer susceptibility.

Therefore, many research groups have focused on developing potent FEN1 inhibitors.

Helicase - use energy obtained from ATP hydrolysis to separate complementary strands of nucleic acid duplexes.

Conserved helicase domains make them suitably druggable targets in anticancer therapy.

BLM inhibitors could be employed through two strategies. The first would be to target DNA binding, as several structural domains contribute to BLM DNA binding preferences for HJs or D-loops.

The second strategy would be to target the ATPase domain.

Furthermore, to fully explore the therapeutic potential of WRN, small-molecule inhibitors could serve as tools for unraveling WRN-mediated synthetic lethal interactions as a potent cancer therapy.

Meanwhile, overexpression of WRN in cancer cells confirms its potential as a direct target in cancer therapy.

The structure of the WRN exonuclease domain was determined on an atomic level providing a useful clue for future drug development.

Targeting WRN therefore displays very great potential in cancer therapy, although many of the avenues remain unexplored.

Other RecQ helicases play various roles in DNA repair, replication, and recombination. Because these helicases have not been sufficiently characterized on a structural or biochemical level compared to WRN or BLM, their therapeutic potential remains to be explored.

Future research is needed generally to investigate the druggability and biological consequences of inhibiting these helicases.

Other helicases;

1. PCNA-associated recombination

2. RTEL1 (regulator of telomere elongation helicase 1)

3.PIF1

Although all of the aforementioned helicases can be considered potential targets for anticancer therapy, their druggability, and exact biological roles should be considered and further characterized in order to progress potentially interesting new treatment strategies into clinical practice.

The key roles of these proteins in HR make them prominent for specific targeting of HR-proficient tumors. On the other hand, most of these factors, excluding RAD51, lack any apparent enzymatic activity, which makes their targeting difficult.

Alterations in many core recombination proteins are associated with tumor development and genomic instability. On the other hand, certain tumor types show strong HR dependence.

Several RAD51 ligands that are capable of inhibiting its biochemical activities have already been identified. Among natural compounds, caffeine was shown to interfere with RAD51-mediated homology search by displacing RAD51 from ssDNA at higher concentrations.

Exogenous expression of BRC repeats, which have been shown to disrupt RAD51–BRCA2 interaction, sensitizes cells to IR and decreases the number of IR-induced RAD51 foci, thus prompting the development of synthetic peptides mimicking BRC motifs interfering with RAD51 filament.

Although RAD51 inhibition seems to offer a very promising approach in treating different types of cancer, no direct RAD51 inhibitors are currently in clinical trials. It should be noted that the low druggability of RAD51 creates a huge obstacle in the development of RAD51 inhibitors.

In addition, BRCA2 is a thermodynamically unstable protein with a high rate of proteasomal degradation. Inasmuch as BRCA2 is an HSP90 client protein, inhibition of HSP90 has been shown to result in BRCA2 degradation.

Local induction of hyperthermia has been shown to cause BRCAness, HR deficiency, and sensitivity to PARP inhibition by destabilizing BRCA2 (thermolabile, I think) - An HSP90 inhibitor further enhanced this temperature-induced effect --> HSP helps BRCA2 to fold properly under the stress like temperature change.

Depletion of RAD52 has been shown to be synthetically lethal with BRCA1/2-deficiency. This observation points to great potential as an alternative to PARP1 inhibition in cancer cells showing BRCAness.

when a lesion is present in the DNA strand it can be bypassed TLS that is catalyzed by translesion DNA polymerases. TLS polymerases play a role in various DNA repair pathways, including HR and ICL repair.

PCNA is involved in mediating many important DNA maintenance pathways, however, and so the toxicity of potential inhibitors has to be taken into account.

Although they may be difficult to drug due to conserved structural features and overlapping roles, as new approaches for drug development are being employed and more insight into the biological functions of DNA polymerases is obtained, their therapeutic potential can be gradually unveiled and explored much further.

TARGER besides the enzymes involved in DRR-DSB repair!

PTM from protein-protein interaction;

1. Phosphorylation

2. Ubiquitinylation

3. Sumoylation

4. Methylation

5. Acetylation

6. Neddylation

7. PARylation

Now if we are talking about epigenetic;

1. Histone modifications

2. Chromatin remodeling

Small molecules will be the forefront as they mentioned in this paper. The bottleneck of developing of these small molecules are the techniques to clarify the complexity and robustness of human proteome as well as a good biomarker which can be used to justify the treatments.

  

Not the cancer itself but the stromal cells (nanny cells I would say) which provide the perfect microenvironments to support the cancer growth also becoming another targets which we should focus!