Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP)
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RESEARCH
Open Access
Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP) David B Rosen1, Ling Y Leung1, Brent Louie1, James A Cordeiro1, Andrew Conroy1, Iuliana Shapira3, Scott Z Fields3, Alessandra Cesano2 and Rachael E Hawtin1*
Abstract 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. Keywords: Genomic instability, DNA damage repair, Cell cycle, Homologous recombination, Non-homologous end joining, BRCA, ATM, PARP, Multiparameter flow cytometry, FACS
Background The DNA damage response (DDR), through the action of sensors, transducers and effectors, orchestrates the appropriate recognition of DNA damage and the repair and resolution of stalled DNA replication [1]. This process is coordinated through complex interplay between cell cycle, apoptosis, and cell survival signaling. The two major * Correspondence: [email protected] 1 Research, Nodality Inc., 170 Harbor Way, Suite 200, South San Francisco, CA 94080, USA Full list of author information is available at the end of the article
mechanisms of DNA double strand break (DSB) repair are homologous recombination repair and non-homologous end joining
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