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Updated: Apr 27, 2026
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling
David B Rosen, Ling Y Leung, Brent Louie
1Research, Nodality Inc,, 170 Harbor Way, Suite 200, South San Francisco, CA 94080, USA. rachael.hawtin@Nodality.com.
Background:
Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity.
Methods:
Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates.
Results:
Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples.
Conclusions:
SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.
Insights
Single cell network profiling (SCNP) quantifies DNA damage response (DDR) pathways, aiding in identifying cancer risks and drug resistance mechanisms. This method accurately characterizes homologous recombination repair (HRR) deficiencies in clinical samples.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Homologous recombination repair (HRR) pathway deficiencies are critical in cancer predisposition and treatment.
- Accurate methods are needed to functionally characterize these deficiencies for risk assessment and understanding drug resistance.
Purpose of the Study:
- To develop and apply quantitative methods for functionally characterizing DNA damage response (DDR) pathways.
- To identify patients at risk for cancer and understand drug resistance/sensitivity mechanisms.
Main Methods:
- Flow cytometry-based single cell network profiling (SCNP) was employed.
- Drug-induced activation of DDR proteins was measured in cell lines with HRR mutations and primary acute myeloid leukemia (AML) samples.
- Both non-homologous end joining (NHEJ) and HRR pathways were assessed using specific protein readouts and normalized for proliferation.
Main Results:
- Etoposide induced DNA damage and DDR protein activation in primary AML and ATM+/+ cells, but not ATM-/- cells.
- PARPi treatment distinguished HRR-deficient/impaired cell lines from proficient ones based on p-H2AX induction.
- SCNP applied to AML samples revealed heterogeneous repair pathway activities, including predominant NHEJ in some cases.
Conclusions:
- SCNP successfully identified functional DDR readouts for both NHEJ and HRR pathways.
- The assay can identify cells with BRCA1 haploinsufficiency and characterize differential DDR pathway functionality in clinical samples.
