Abstract 3P
Background
Ovarian clear cell carcinoma (OCCC) is characterised by a high prevalence of ARID1A mutations and chemotherapy resistance. We previously found that loss of ARID1A causes ATR inhibitor (ATRi) sensitivity, which led to the phase II ATARI clinical trial (NCT04065269). We aim to identify novel genetic determinants of ATRi response in ARID1A-mutant OCCC.
Methods
A genome wide CRISPR knockout (CRISPRn) screen was performed in OCCC TOV21G cells. CRISPR prime gene-editing was used to introduce PPP2R1A p.R183 mutations. In vitro and in vivo ATRi sensitivity was assessed in PPP2R1A isogenic models. Cell cycle analysis was performed via flow cytometry. Phospho-proteomic profiling was performed using mass spectrometry.
Results
A CRISPRn screen in ARID1A mutant OCCC cells identified protein phosphatase 2 (PP2A) complex subunits as ATRi response genes. In OCCC, PPP2R1A missense mutations cause amino acid substitutions at residue p.R183. Characterisation of a cohort of OCCC primary tumours revealed a higher prevalence of structural subunit (PPP2R1A) mutations than previously reported (50%) which frequently co-occurred with ARID1A mutations. ARID1A-mutant OCCC cells with heterozygous PPP2R1A p.R183P or p.R183W mutations displayed enhanced ATRi sensitivity in vitro and in vivo. The most profound cell-cycle defect caused by PPP2R1A missense mutation was an ATRi-induced reduction in active S phase. ATRi exposure in PPP2R1A mutants increased 53BP1 bodies, a mark of residual DNA damage in mitosis. Phospho-proteomic profiling of PPP2R1A mutant OCCC cells revealed the selective increase in phosphorylation of Lysine Deficient Protein Kinase 1 (WNK1) following ATRi exposure, as well as increased phosphorylation of the WNK1 substrate Oxidative Stress Response Kinase 1 (OSR1). Depletion of WNK1 rescued ATRi sensitivity and S phase defects in PPP2R1A mutant cells suggesting a novel role for this kinase.
Conclusions
The ability of PPP2R1A missense mutations to enhance ATRi sensitivity in tumours cells with pre-existing ARID1A mutations suggests that the co-occurrence of these mutations may be better predictors of ATRi sensitivity than either mutation alone. Increased phosphorylation of WNK1 may drive ATRi sensitivity in PPP2R1A mutant cells.
Legal entity responsible for the study
The authors.
Funding
Cancer Research UK.
Disclosure
S. Banerjee: Financial Interests, Institutional, Research Grant: AstraZeneca; Financial Interests, Institutional, Research Grant: GlaxoSmithKline; Financial Interests, Personal, Advisory Board: Amgen; Financial Interests, Personal, Advisory Board: Genmabs; Financial Interests, Personal, Advisory Board: GlaxoSmithKline; Financial Interests, Personal, Advisory Board: Immunogen; Financial Interests, Personal, Advisory Board: MSD; Financial Interests, Personal, Advisory Board: Merck Sereno; Financial Interests, Personal, Advisory Board: Mersana; Financial Interests, Personal, Advisory Board: Oxcerna; Financial Interests, Personal, Advisory Board: Seagen; Financial Interests, Personal, Advisory Board: Shattuk Labs; Financial Interests, Personal, Invited Speaker: Amgen; Financial Interests, Personal, Invited Speaker: AstraZeneca; Financial Interests, Personal, Invited Speaker: Immunogen; Financial Interests, Personal, Invited Speaker: MSD; Financial Interests, Personal, Invited Speaker: Mersana; Financial Interests, Personal, Invited Speaker: Pfizer; Financial Interests, Personal, Invited Speaker: Roche. C. Lord: Financial Interests, Institutional, Research Grant: AstraZeneca; Financial Interests, Institutional, Research Grant: Merck; Financial Interests, Institutional, Research Grant: KGaG; Financial Interests, Institutional, Research Grant: Artios; Financial Interests, Personal, Advisory Board: Syncona; Financial Interests, Personal, Advisory Board: Sun Pharma; Financial Interests, Personal, Advisory Board: Gerson Lehrman; Financial Interests, Personal, Advisory Board: Merck; Financial Interests, Personal, Advisory Board: Vertex; Financial Interests, Personal, Advisory Board: AstraZeneca; Financial Interests, Personal, Advisory Board: Dark Blue Therapeutics; Financial Interests, Personal, Stocks/Shares: Tango; Financial Interests, Personal, Stocks/Shares: Ovibio; Financial Interests, Personal, Stocks/Shares: Enedra Tx; Financial Interests, Personal, Stocks/Shares: Hysplex; Financial Interests, Personal, Stocks/Shares: Tesselate. All other authors have declared no conflicts of interest.