title: Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP).
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.
authors: David B
authors: DB
authors: Rosen
authors: Ling Y
authors: LY
authors: Leung
authors: Brent
authors: B
authors: Louie
authors: James A
authors: JA
authors: Cordeiro
authors: Andrew
authors: A
authors: Conroy
authors: Iuliana
authors: I
authors: Shapira
authors: Scott Z
authors: SZ
authors: Fields
authors: Alessandra
authors: A
authors: Cesano
authors: Research, Nodality Inc,, 170 Harbor Way, Suite 200, South San Francisco, CA 94080, USA. rachael.hawtin@Nodality.com.
authors: Rachael E
authors: RE
authors: Hawtin
mesh_terms: D000328:Adult; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D019313:BRCA1 Protein / Q000378:metabolism; D024682:BRCA2 Protein / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002648:Child; D056751:Cyclin A2 / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D057895:Haploinsufficiency / Q000187:drug effects; D006657:Histones / Q000378:metabolism; D059765:Homologous Recombination / Q000187:drug effects; D006801:Humans; D009153:Mutagens / Q000633:toxicity; D010766:Phosphorylation / Q000187:drug effects; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*; D000077204:Temozolomide
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein; D056751:Cyclin A2; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; D009153:Mutagens; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D005047:Etoposide; D003606:Dacarbazine; D011065:Poly(ADP-ribose) Polymerases; D064007:Ataxia Telangiectasia Mutated Proteins; D000077204:Temozolomide