PARP16 is a Druggable Regulator of Ribosome MARylation and Protein Homeostasis in Ovarian Cancer Cells

Abstract Cytosolic NAD□ synthesis supports ovarian cancer growth by enabling PARP16-dependent mono(ADP-ribosyl)ation (MARylation) of ribosomal proteins, thereby fine-tuning translation and maintaining protein homeostasis. While genetic depletion of PARP16 disrupts ribosome MARylation and impairs tumor cell growth, the therapeutic potential of pharmacologic PARP16 inhibition in this pathway remains unexplored. Here, we characterized the effects of DB008, a tool compound that functions as a selective inhibitor of PARP16, in ovarian cancer cells. Biochemical analyses demonstrated that PARP16 undergoes NAD□-dependent auto-MARylation and that NMNAT-2 supplies NAD□ to support this activity. DB008 potently inhibited PARP16 auto-MARylation in vitro. In ovarian cancer cells, DB008 engaged PARP16, reduced its MARylation, and decreased ribosome-associated MARylation. Consistent with PARP16 depletion, DB008 enhanced global protein synthesis, increased protein aggregation, and suppressed cell growth and anchorage-independent colony formation. CRISPR-mediated deletion of the PARP16 gene in ovarian cancer cells abolished the effects of DB008 on translation, protein aggregation, and proliferation, demonstrating on-target activity. Moreover, cells expressing a PARP16 mutant resistant to DB008 were unaffected by inhibitor treatment, further confirming that the cellular effects of DB008 require on-target inhibition. Finally, DB008 significantly inhibited tumor growth in OVCAR3 xenografts, with on-target engagement of PARP16 in the xenograft tumors. Collectively, these findings establish PARP16 as a druggable regulator of ribosome MARylation and protein homeostasis in ovarian cancer and provide pharmacologic proof-of-concept that disrupting ribosomal MARylation impairs tumor growth. Competing Interest Statement W.L. Kraus is a founder, consultant, and Scientific Advisory Board member for ARase Therapeutics, Inc. W.L. Kraus is also a co-holder of U.S. Patent 9,599,606 covering the ADP-ribose detection reagent used herein, which has been licensed to and is sold by EMD Millipore. Footnotes ↵6 Lead Contact / Address correspondence to: W. Lee Kraus, Ph.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-8511, Phone: 214-648-2388 | Fax: 214-648-0383, E-mail: Lee.Kraus{at}utsouthwestern.edu Data Availability All data and reagents presented within this article are available upon request.