PXD067891

PXD067891 is an original dataset announced via ProteomeXchange.

Title	PARP Inhibitor Sensitivity in BRCA-Proficient ovarian cancer cells is regulated by the MSH6–CHAF1A axis: 2. ADP-Ribosylation
Description	Ovarian cancer is the leading cause of gynecologic cancer-related deaths, with high-grade serous ovarian cancer (HGSOC) being the most common and lethal subtype. While BRCA1/2 mutations are established biomarkers predicting sensitivity to PARP inhibitors (PARPis), a subset of patients with BRCA-proficient HGSOC also respond to PARPi therapy. However, the molecular mechanisms driving PARPi sensitivity in BRCA-proficient tumors remain poorly understood. We hypothesized that the composition of the PARP1 protein complex and PARylation-mediated signaling contribute to PARPi sensitivity or resistance in BRCA-proficient HGSOC. Viability screening of BRCA-proficient ovarian cancer cell lines identified PARPi-sensitive and PARPi-resistant lines. Chemical proteomics using rucaparib revealed enrichment of PARP1, PARP2, and associated binding partners, with notably higher MSH6 levels in sensitive lines. Co-immunoprecipitation further uncovered distinct PARP1–MSH6–PARP2 complex abundance between sensitive and resistant cells. Targeting of MSH6 via CRISPR and siRNA conferred rucaparib resistance, particularly in sensitive lines. To explore downstream signaling, we performed ADP-ribosylation proteomics using clickable NAD⁺ analogs, revealing distinct PARylation profiles between sensitive and resistant lines. CHAF1A, a known MSH6 interactor and PARP1 substrate, was enriched in PARPi-sensitive cells. MSH6 knockdown led to increased CHAF1A expression in both sensitive and resistant lines, regardless of rucaparib treatment. Importantly, CHAF1A silencing significantly impaired cell viability, especially in A2780 cells, and suppressed mTOR signaling, suggesting that CHAF1A acts downstream of MSH6 to regulate the mTOR axis. Furthermore, co-treatment with mTORC1 inhibitors enhanced the cellular effects of rucaparib in resistant cells, underscoring the therapeutic potential of targeting downstream mTOR effectors to overcome intrinsic resistance. In conclusion, this study identifies a novel PARP1–MSH6–CHAF1A axis that regulates PARPi sensitivity via mTOR signaling in BRCA-proficient ovarian cancer. By integrating chemical proteomics and ADP-ribosylation analysis, we delineate the interplay between PARP1 complex composition and signaling dynamics, highlighting MSH6 and CHAF1A as critical modulators of PARPi response and potential biomarkers to enhance therapeutic efficacy in BRCA-proficient HGSOC. Three tandem mass tag experiments were performed with the same experimental design to analyze three biological replicates. They are described with reporter ion, treatment (DMSO or 10 micromolar Rucaparib), and cell line: 126: DMSO A2780 127N: Rucaparib A2780 127C: DMSO Caov3 128N: Rucaparib Caov3 128C: DMSO OV CAR8 129N: Rucaparib OVCAR8 129C: DMSO SKOV3 130N: Rucaparib SKOV3 130C: DMSO OVCA432 131N: Rucaparib OVCA432 131C: DMSO HEYA8 132N: Rucaparib HEYA8 134N: DMSO Control Pool
HostingRepository	PRIDE
AnnounceDate	2026-08-21
AnnouncementXML	Submission_2026-08-21_10:27:21.308.xml
DigitalObjectIdentifier	https://doi.org/10.6019/PXD067891
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	John Koomen
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2025-08-29 19:41:06	ID requested
⏵ 1	2026-08-21 10:27:21	announced

10.6019/PXD067891;

submitter keyword: Ovarian Cancer, PARP Inhibitor,ADP-Ribosylation

Uwe Rix
contact affiliation	Drug Discovery Moffitt Cancer Center Tampa, FL, USA
contact email	uwe.rix@moffitt.org
lab head
John Koomen
contact affiliation	Moffitt Cancer Center
contact email	john.koomen@moffitt.org
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD067891
     1. Label: PRIDE project
     2. Name: PARP Inhibitor Sensitivity in BRCA-Proficient ovarian cancer cells is regulated by the MSH6–CHAF1A axis: 2. ADP-Ribosylation