Note for: Biomarker-Guided Development of DNA Repair Inhibitors

Note for: Biomarker-Guided Development of DNA Repair Inhibitors

doi: 10.1016/j.molcel.2020.04.035

Key theme;

• Drug targeting DNA damage response exploit the synthetic lethality

• Targeting the cells which are defective in DNA repair using PARPi

• Another selective agents for DNA repair -- >

• ATR

• CHK1

• WEE1

• ATM

• cNHEJ

• Alt EJ

• Review biomarkers which aid physician to decide when to use these inhibitors

• (in the old day) Oncologist used drugs targeting DDR which has cytotoxic property

• Alkylating agent temozolomide -- > repair by MGMT

• Cisplatin -- > repair by NER and ssDNA repair

• Bleomycin -- > cleave DNA and generating DNA double strand break

• Topo-I and II inhibitor -- > causing ssDNA break and dbDNA break, respectively

• DSB

• V(D)J recombination

• High error by cNHEJ -- > diversity of T-cell receptors and antibodies

• cNHEJ repair

• leading to characteristic DNA damage lesions

• large deletion

• loss of hetrozygosity

• telomeric allelic imbalance

• HR deficiency

• Genomic scar

• Trio of three proteins

• MRE11-RAD50-NBS1 (MRN complex)

• Recognize DSB

• Inhibitor of ATM and RAD51 – currently being developed

• PARPi

• Targeting BRCA1/2 defective

• Backup pathway -- > BER

• DSB

• HR relies on the availability of 3’ overhang

• 53BP1+shieldin complex (SHLD1-3, REV7) -- > block further resection by nuclease

• Thus, it can promote NHEJ

• 53BP1 and BRCA1 -- > play opposing role

• Inhibitor toward TRIP3 (amplified in many BRCA1-deficienct tumor) -- > might be useful to prevent PARPi resistant

PARP resistance

• Intrinsic -- > naturally resistant

• Extrinsic -- > acquired resistant

Alt-EJ dependent on PARP1

• DSB -- > MSN and PARP1 recognizes break (requires 2-20 bp homologous DNA sequence) -- > Pol theta mediates repairing

• In HR proficient -- > alt NHEJ rarely occur -- > in HR deficiency -- > tumor seems to relied on alt NHEJ

• Thus, Possibly -- > Pol theta is a good target for HR deficiency

Single-strand annealing

• Is DSB repair

• Not similar to alt-NHEJ

• Single strand annealing is inhibited by RAD51 and require RAD52 to recombines homologous ssDNA

Mode of mechanism

1. Relying on synthetic lethality, ssDNA could not be repaired due to PARP1i -- > causing accumulation of DSB during cell division -- > lack of HR -- > forcing the cell to repair through cNHEJ -- > toxic

2. Treating with the 3rd gen -- > reveal the toxicity correlates to PARP trapping ability at DNA damage site -- > PARP-DNA complex is lethal in HR defect

Inhibition of Pol-theta dependent altEJ

Pol-theta

• Has RAD51 binding site, ATPase, and helicase activity

• Pol-theta can remove RAD51 from DNA during repairing process

• If RAD51 cannot be removed – it becomes toxic to the cell -- > it disrupt repairing process

• Inhibition of Pol-theta -- > good for both 

1. HR-deficient cells  by inhibiting alt-NHEJ

2. cell acquired PARPi resistance via HR restoration

Mechanism of resistance in HR-deficient tumors

1. efflux of PARPi  -- > upregulation of drug-efflux transporters

2. Mutation of PARP which disrupt DNA binding (like PARP(-/-))

3. Restoring HR repair

4. Re-establishing replication fork stability 

Observation

• Patients who resist to Pt-based therapy -- > also correlate with resist PARPi

• But not in the case of cisplatin resistant

Predictive and pharmacodynamic biomarker for DDR inhibitor drug development

• Mostly detecting mutation in genes

• BRCA1/2

• PALB2

• RAD51C

• RAD51D

• Few on methylation state

Signature 3

• Genomic mutational signature – signature 3 -- > associated with HR repair deficiency -- > still having some patients showed no clear mutation in BRCA1, BRCA2 or PALB2.

• Requirement for WES or WGS -- > identifying by signature 3 (programming to analyze the HR deficiency profile) -- > but it is not widely available

• Clinical-grade targeted sequencing panels -- > being developed to detect signature 3

Possibility if acquired resistance to PARPi through genetic and non-genetic mechanism -- > highlight the need for functional biomarkers -- > determine HR proficiency -- > immunofluorescence-based RAD51 assay

RAD51 foci formation

• Critical step for HR pathway

• Can differentiate between HR-proficient cancers and HR-deficient cancer

• Using irradiated live tumor cells -- > predictive of HR-deficient breast cancer

• Live cell is normally not available -- > attempt to perform RAD51 assay on formalin fixed samples

Platinum sensitivity – correlate with PARP sensitivity -- > observation from ovarian cancer

Progress on developing integrative genomic assays predict HRD 

• Myriad genetics -- > HRD assay -- > based characteristic genomic findings -- > LOH, telomeric allelic imbalance (TAI), and large-scale state transition (LST)

• Foundation Medicine T5 NGS assay -- > assess mutation status of 30 HR genes -- > calculate percentage of LOH

• These two have been used to predict the PARPi (niraparib and rucaparib) sensitivity in large clinical trials, phase 3 -- > ovarian cancer

• Fail to identify all platinum-sensitivity patients who who benefited from PARPi

• Thus -- > Pt-sensitivity can be used as surrogate marker for PARPi responsiveness in ov ca

Pharmacodynamic assessment

PARP inhibitor + Pol-theta -- > cause more RAD51 foci formation and becoming toxic to the cell

PARP inhibitor + IDH1/2 (mutation) -- > cause more sensitivity to PARP

Two major acquired PARPi resistant

1. Restoration of HR

2. Stability at the replication forks

Inhibition of ATR/CHK1/WEE1 pw -- > cause complication -- > myelosuppression -- > might be hard when combining with PARPi

Strategies designed to increase replicative stress

• Cancer has inherent degree of endogenous replicative stress

• Top I and II inhibitors are thought to be a good strategic approach

• Increasing the replicative stress by decreasing dNTP through the use of hydroxyurea and gemcitabine

 

Combination of PARPi with cytotoxic agents

• Topo-I cleavage cpx is stabilized by PARP1

• Complication occurs when perform combination; full dose chemotherapy could not be done easily -- > overlapping myelosuppression

• Combination could also increase normal tissue becoming toxic

• Normal tissue vs cancer tissue -- > given same activity -- > no advantage can be achieved when combining with cytotoxic agent

• Cancer cell

• Have greater baseline deficiency in DDR

• More damage than normal cell in general sense

• Using this different to increase therapeutic index

• By doing this -- > we can observe the DDR defective profile in cancer

Combination of DDR inhibitors with immunotherapy

• There is a successfulness story regarding of MMR -- > more mistakes -- > generate neoantigen -- > good for immunotherapy

• Not all defective DNA repair pw can give rise to neoantigen which later provokes good immune response

• But, DNA damage can induce the expression of PDL1 -- > increase the amounts of targets which will be recognized by PDL1i

Conclusion

• Besides the successfulness of PARP1i on BRCA1/2 in OVCA, Breast and pancreatic cancer

• More drugs targeting DNA damage and repair are gradually entering clinical phase

• ATM

• ATR

• CHK1

• DNAPK

• WEE1

• Things to concern when making the combination with DDR

• Optimal clinical setting

• Dose intensity

• Drug scheduleing

• Predictive biomarkers to guide appropriate drugs in clinical trial to initiate DDR inhibitors -- > PD biomarkers should be incorporated to reveal whether DDRi precisely hit the target.

• More understanding of resistant regarding on DDR alone or combination to prevent acquired mutation or use it for reversing the resistant