Marwa W. Aljardali

Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To reset the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls polysome assembly, translation, and stress granule dynamics in ovarian cancer cells.

Objectives: Mono(ADP-ribosyl)ation (MARylation), a post translational modification of proteins, is emerging as an important regulator of the biology of cancer cells. PARP7 (TiPARP), a mono (ADP-ribosyl) transferase (MART), MARylates its substrate α-tubulin in ovarian cancer cells, promoting destabilization of microtubules, cell growth, and migration. Recent development of RBN-2397, a potent inhibitor that selectively acts on PARP7, has provided a new tool for exploring the role of PARP7 catalytic activity in biological processes. In this study, we investigated the role of PARP7 catalytic activity in the regulation of ovarian cancer cell biology via MARylation of α-tubulin. Methods: Ovarian cancer cell lines (OVCAR4, OVCAR3) were treated with RBN-2397 and paclitaxel, both separately and in combination. Western blotting and immunoprecipitation confirmed the effects of RBN-2397 on α-tubulin MARylation and stabilization. Cell proliferation and migration were assessed, and α-tubulin stabilization was quantified using immunofluorescent imaging. RNA-sequencing was performed to assess the effects on gene expression changes. Results: RBN-2397 inhibited PARP7 activity, decreasing α-tubulin MARylation, leading to its stabilization, and reducing cancer cell proliferation and migration. The addition of paclitaxel further enhanced these effects, highlighting a synergistic interaction between the two drugs. Mutating the site of PARP7-mediated MARylation on α-tubulin similarly resulted in microtubule stabilization and decreased cell migration in the presence of paclitaxel. Conclusions: This study demonstrates that targeting PARP7 with RBN-2397, particularly in combination with paclitaxel, offers an effective strategy for inhibiting aggressive ovarian cancer cell phenotypes. Our findings underscore the potential of combining PARP7 inhibitors with established chemotherapeutics to enhance treatment efficacy in ovarian cancer.

PARP1 inhibition prevents ribosome biogenesis and decreases protein translation. Treatment with niraparib significantly decreases rRNA subunit (18S) production in endometrial (A) and ovarian (B) cancer cells as assayed by qPCR. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different; Student t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Puromycin incorporation is diminished with niraparib treatment in endometrial (C) and ovarian (D) cancer cells as assayed by Western blotting. E and F, Quantification of puromycin incorporation assay from C and D, respectively. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different; Student t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

PARP1 inhibition prevents ribosome biogenesis by inhibiting rRNA subunit production

PARP1 inhibition prevents ribosome biogenesis by inhibiting rRNA subunit production

DDX21 nucleolar localization associates with PARP1 expression and decreased survival outcomes. A and B, Representative immunohistochemical staining of PARP1 and DDX21 from endometrial (A) and ovarian (B) cancer commercial tissue microarrays (left) and box plots showing quantification of nucleolar DDX21 intensity in low versus high PARP1 endometrial (A) and ovarian (B) cancer samples (right). Student t test; ****, P < 0.0001. B, Representative IHC images from advanced stage endometrioid endometrial cancer patient samples showing low versus high nucleolar DDX21 staining by IHC. Kaplan–Meier curves showing high nucleolar localization of DDX21 is significantly associated with decreased OS (P-value = 0.0155; D) and PFS (P-value = 0.0092; E) in patients with advanced stage endometrioid endometrial cancer. Total n = 39 (number of patients with low DDX21 nucleolar staining = 14; number of patients with high DDX21 nucleolar staining = 25). log-rank (Mantel–Cox) test; *, P < 0.05; **, P < 0.01.

Niraparib inhibits ADPRylation and disrupts the nucleolar localization of DDX21 in endometrial and ovarian cancer cells. Inhibition of PARP1 catalytic activity in endometrial cancer cell lines (Ishikawa, HEC-1-A, KLE; A) and ovarian cancer cell lines (OVCAR3, OVCAR4, HCC5012; B) by niraparib promotes the redistribution of DDX21 from the nucleolus to the nucleoplasm but does not affect the nucleolar localization of NOP58 as assayed by immunofluorescent staining. C, Quantification of the results from experiments shown in A and B showing the proportion of cells that have disrupted nucleoplasmic localization of DDX21. Each bar represents the mean + SEM; n = 3 (endometrial), n = 6 (ovarian). Bars marked with asterisks are significantly different; Student t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, ADPRylation (PAR) is inhibited in endometrial and ovarian cancer cells treated with niraparib. PARP1 and DDX21 protein expression levels are not affected by niraparib treatment as assayed by Western blotting. E, Quantification of PAR levels from experiments shown in D for endometrial and ovarian cancer cell lines with and without niraparib treatment. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different; Student t test; *, P < 0.05; **, P < 0.01.

Niraparib decreases cell growth in endometrial and ovarian cancer cell lines

Endometrial and ovarian cancer cell lines are HR-proficient

Demographic data of 39 advanced stage endometrioid endometrial cancer patients